Академический Документы
Профессиональный Документы
Культура Документы
Antimicrobial Peptides
Discovery, Design and Novel Therapeutic
Strategies
Edited by
Eppley Institute
University of Nebraska Medical Center
Omaha, Nebraska, USA
Advances in Molecular and
Cellular Microbiology
Through the application of molecular and cellular microbiology we now recognize the
diversity and dominance of microbial life forms that exist in all environments on our
planet. These microbes have many important planetary roles, but for humans a major
problem is their ability to colonize our tissues and cause disease. The same techniques
of molecular and cellular microbiology have been applied to the problems of human
and animal infection since the 1990s and have proved to be immensely powerful tools
in elucidating how microorganisms cause human pathology. This series has the aim of
providing information on the advances that have been made in the application of
molecular and cellular microbiology to specific organisms and the diseases they cause.
The series is edited by researchers active in the application of molecular and cellular
microbiology to human disease states. Each volume focuses on a particular aspect of
infectious disease and will enable graduate students and researchers to keep up with
the rapidly diversifying literature in current microbiological research.
Series Editors
Microbial Metabolomics
Edited by Silas Villas-Bas and Katya Ruggiero
Earlier titles in the series are available from Cambridge University Press (www.cup.cam.ac.uk).
CABI is a trading name of CAB International
A catalogue record for this book is available from the British Library,
London, UK.
RS431.P37A575 2010
615.1--dc22
2010016796
Contributors xi
Preface xiii
Introduction xv
Michael Zaslo
v
vi Contents
Part II: Expanding the Peptide Space: Prediction Methods, Design Strategies and
Peptidomimetics
10 Lung Infection: Shifting the Equilibrium Towards the Free and Active Form
of Human LL-37 and the Design of Alternative Antimicrobial Agents 169
Paul A. Janmey and Robert Bucki
10.1 Introduction 169
10.1.1 Electrostatic properties of LL-37 169
10.1.2 Interaction of polyvalent cations with linear polyelectrolytes 170
10.2 Production of Anionic Polyelectrolytes by Host and Microbial Sources 171
10.3 Sequestration and Inactivation of LL-37 by DNA and F-actin 172
10.4 Releasing LL-37 from Polyelectrolyte Bundles 172
10.4.1 Severing polymers with DNase, gelsolin and alginase 173
10.4.2 Destabilizing bundles with small multivalent anions 174
10.5 Designing Antimicrobial Agents Resistant to Inactivation by Polyelectrolytes 174
10.5.1 Cationic steroids: minimizing and redistributing charge 175
10.5.2 Increasing the hydrophobicity of antimicrobial agents 175
10.6 Possible Therapeutic Use 175
10.6.1 Selective use in some body fluids and not others 175
10.6.2 Stimulation of host cells 176
10.7 Conclusion 176
Contents ix
12 Fine Tuning Host Responses in the Face of Infection: Emerging Roles and Clinical
Applications of Host Defence Peptides 195
Matthew L. Mayer, Donna M. Easton and Robert E.W. Hancock
12.1 Mammalian Host Defence (Antimicrobial) Peptides 196
12.1.1 Defensins 196
12.1.2 Cathelicidins 197
12.2 The Role of Endogenous Host Defence Peptides in the Response to Infection 198
12.2.1 Natriuretic peptides 201
12.3 Potential Therapeutic Uses beyond Anti-infective Activity 202
12.3.1 Adjuvant potential 204
12.3.2 Wound-healing activity 205
12.4 Recent Clinical Advances in Therapeutic Application of Host Defence
Peptides 206
12.5 Innate Defence Regulators as Anti-infective Therapeutics 208
12.6 Rational Design of Immunomodulatory Peptides 209
12.7 Limitations and Challenges 209
12.8 Conclusions 211
Index 221
This page intentionally left blank
Contributors
Bitton, Ari J., Departments of Physiology and Medicine, McGill University, McIntyre Building,
Room 1112, 3655 Drummond St, Montreal, QC, Canada, H3G 1Y6
Bucki, Robert, Institute for Medicine and Engineering, University of Pennsylvania, 1010
Vagelos Laboratories, 3340 Smith Walk, Philadelphia, PA 19104, USA
Cho, Ju Hyun, Department of Biology, Research Institute of Life Science, Gyeongsang National
University, Jinju 660-701, Korea
Cotter, Paul D., Moorepark Food Research Centre, Teagasc, Moorepark, Fermoy, Cork, Ireland.
E-mail: paul.cotter@teagasc.ie
Craik, David J., Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD
4072, Australia. E-mail: d.craik@imb.uq.edu.au
Easton, Donna M., Centre for Microbial Diseases and Immunity Research, 2259 Lower Mall
Research Station, University of British Columbia, Vancouver, BC, Canada, V6T 1Z4
Epand, Raquel F., Department of Biochemistry and Biomedical Sciences, McMaster University,
Hamilton, ON, Canada, L8N 3Z5
Epand, Richard M., Department of Biochemistry and Biomedical Sciences, McMaster
University, Hamilton, ON, Canada, L8N 3Z5. E-mail: epand@mcmaster.ca
Hancock, Robert E.W., Department of Microbiology and Immunology, University of British
Columbia, Room 232, 2259 Lower Mall Research Station, Vancouver, BC, Canada, V6T 1Z4.
E-mail: bob@cmdr.ubc.ca
Healy, Brian, Department of Microbiology, University College Cork, Cork, Ireland
Hill, Colin, Alimentary Pharmabiotic Centre, Cork, Ireland. E-mail: c.hill@ucc.ie
Janmey, Paul A., Institute for Medicine and Engineering, University of Pennsylvania, 1010
Vagelos Laboratories, 3340 Smith Walk, Philadelphia, PA 19104, USA. E-mail: janmey@mail.
med.upenn.edu
Kaas, Quentin, Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD
4072, Australia
Kim, Sun Chang, Department of Biological Sciences, Korea Advanced Institute of Science and
Technology, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea. E-mail:
sunkim@kaist.ac.kr
Li, Xia, Eppley Institute, University of Nebraska Medical Center, 986805 Nebraska Medical
Center, Omaha, NE 68198, USA
Mayer, Matthew L., Centre for Microbial Diseases and Immunity Research, 2259 Lower Mall
Research Station, University of British Columbia, Vancouver, BC, Canada, V6T 1Z4
xi
xii Contributors
The significance and benefits of naturally occurring antimicrobial peptides to human health
have recently begun to be appreciated. As eector molecules of the innate immune system,
they are capable of rapidly eliminating invading pathogens to keep us healthy; as signalling
molecules, they elegantly modulate the adaptive immune system to trigger other physiological
actions. The purpose of this book is to provide a comprehensive account on current antimicrobial
peptide research in two major directions. The first direction delineates the classic path for
peptide development, ranging through identification, design, structure and mode of action
studies. The second direction describes novel strategies for developing peptide therapeutics
based on our knowledge of host defence antimicrobial peptides discovered in living
organisms.
The book commences with a vivid and inspiring introduction contributed by Professor
Michael Zaslo, a distinguished forerunner in the field. Part I provides an overview of
nomenclature, classification and bioinformatic analysis of antimicrobial peptides from bacteria,
plants and animals. Subsequently, lantibiotics from bacteria and cyclotides from plants are
presented. These unique peptide templates with multiple sulfur-mediated bridges are resistant
to proteases, rendering them attractive for engineering new compounds for food preservation,
pest control and curing diseases. Part II discusses database-aided peptide prediction and
design methods, synthetic combinatorial libraries and peptide mimetics that expand the
conformational space of natural antimicrobial peptides. These approaches also allow for the
optimization of the desired properties and therapeutic index of the peptide analogues or
mimicries. An in-depth understanding of the mode of action of these peptides is essential for
drug development. As a consequence, Part III covers the biophysical and structural
characterization of antimicrobial peptides and their complexes. While many peptides (e.g.
magainin and protegrin-1) target bacterial membranes, apidaecin, buforin II, PR-39 and others
can cross bacterial membranes and associate with internal targets such as heat-shock proteins
and nucleic acids. Structural determination sheds light on how such peptides recognize
membrane or non-membrane targets at atomic resolution, and provides the basis for structure-
based peptide design. Finally, Part IV focuses on novel strategies for developing peptide-based
therapies. These include liberating LL-37 from the inactive bound state in infected lungs,
enhancing the expression of human cathelicidin and human -defensin-2 by vitamin D, and
stimulating immune responses to bacterial invasion by applying peptide analogues that may
or may not kill bacteria directly.
Antimicrobial Peptides: Discovery, Design and Novel Therapeutic Strategies has been written,
and anonymously reviewed, by an enthusiastic group of recognized investigators in the field.
xiii
xiv Preface
As the editor, I thank all of the authors and reviewers for their contributions. In particular, I am
grateful to Professor Zaslo for his excellent introduction and Professors Richard M. Epand,
Amram Mor and Donnatella Barra, who recruited the anonymous reviewers for the chapters
written by the editor and his co-authors. This book is directed to graduate students, postdoctoral
fellows, research faculty, principal investigators, educators, clinicians and all others who are
interested in the education, research, development and applications of antimicrobial
peptides.
Guangshun Wang, PhD
Introduction
Michael Zasloff
xv
xvi M. Zasloff
evidence of inflammation or infection. This ah-ha moment was followed by a realization that
the wound on the abdomen of the frog could only heal if a potent antimicrobial mechanism
existed in the skin of that animal, since these aquatic frogs were healing without diculty in
tanks that were densely populated with microbes.
Within several months I discovered the presence of antimicrobial peptides in the skin of
Xenopus laevis, the first being magainin. Some years earlier Hans Boman and colleagues had
described the remarkable cecropins in the silk-moth pupa, and Bob Lehrer and colleagues had
characterized the defensins in mammalian phagocytic cells. With my observation, three clear
examples of the existence of antimicrobial peptides in animals had been described, each
operating in a dierent physiological context.
With the help of many colleagues I began to search the tissues of every animal I could study,
especially those tissues that seemed to be protected by mysterious antimicrobial defences. In
addition, I wanted to better understand how antimicrobial peptides were used in humans to
maintain health, and how their failure to function properly might cause disease. At about the
same time I founded Magainin Pharmaceuticals. As a physician scientist, I was thrilled by the
opportunity to have the chance to bring a discovery made at the bench to the clinic.
In the beginning, most of us were amazed by the antimicrobial properties of these naturally
occurring, simple, short amphipathic peptides. They could rapidly kill practically every species
of bacteria and many species of fungi, inactivate viruses and lyse protozoa. I can recall a
dramatic moment when, following the addition of a solution of magainin into the abdominal
cavity of a mouse filled with Ehrlich ascites tumour cells, I withdrew some ascites and could
see that the peptide had killed most of the tumour cells within minutes of administration.
Furthermore, the simplicity of the design of the -helical peptides and the simplicity of the
Merrifield method of peptide synthesis fostered an explosion of structureactivity studies. In
addition, studies of animals and plants led to the discovery of an enormous diversity of
naturally occurring antimicrobial peptides.
An understanding of the mechanism of action of antimicrobial peptides has evolved over
time. As amphipathic, cationic peptides, it was apparent from the start that antimicrobial
peptides targeted the membranes of the microbes they killed, a conclusion consistent with
studies utilizing model membranes. Precisely how a particular peptide strikes the fatal blow in
a particular microbial target remains the subject of continued and active investigation.
We continue to learn more about how antimicrobial peptides participate in defence against
microbes and permit multicellular organisms to live in balance with microbes. In general, most
healthy epithelial surfaces exposed to microbes express an array of antimicrobial peptides, and
a dtente of sorts is maintained between the microbes that utilize that surface as a niche and
the epithelium that is being inhabited. Upon injury to the epithelium, batteries of dierent
antimicrobial peptides are generally launched. Of great interest are the details of the scenarios
that characterize the responses of various organs to specific microbes and the settings in which
they fail to unfold normally. I believe that the existence of microbial sensors, such as the Toll-
related receptors, permits this system of defence to mould an antimicrobial response that is far
more microbe-specific than might have been otherwise imagined.
We have come to learn that antimicrobial peptides, initially discovered on the basis of their
antibiotic activity, exhibit other biological properties in the context of injury and infection that
promote recovery and healing. The cathelicidins can stimulate epithelial growth and promote
angiogenesis. Several of the mammalian defensins and cathelicidins can attract cells of the
immune system such as macrophages, neutrophils and dendritic cells. In the context of injury,
as the process of repair unfolds in time, batteries of antimicrobial peptides are successively
expressed, protecting the healing surface in a temporally appropriate context and calling into
the healing environment immune cells that complement the process. The relatively low potency
of the chemokine activity of most antimicrobial peptides (micromolar) ensures that only those
cells within close range of the site of injury will be called to action, as opposed to the more
systemic signalling that occurs with the release of classical cytokines.
Introduction xvii
The discovery that antimicrobial peptides are inducible has opened up the possibility of
new therapeutic opportunities. The remarkable discovery that the human cathelicidin gene is
induced by vitamin D, and the association of certain bacterial and viral illnesses with vitamin
D deficiency, has vast implications with respect to public health. Clinical studies suggesting
that vitamin D supplementation can reduce the incidence of tuberculosis, upper respiratory
infections and influenza A have been reported and many other trials are underway. The
discovery that simple nutrients and metabolites, such as butyrate and isoleucine, can induce
epithelial antimicrobial peptides is being evaluated in studies using the oral administration of
these substances in the treatment of bacterial and viral dysentery in humans.
Abnormal regulation of the expression of antimicrobial peptides has been linked in recent
years to several human diseases. Conditions such as eczema and Crohns disease are associated
with the depressed expression of specific antimicrobial peptides. In diseases such as cystic
fibrosis, the milieu (the ionic strength of the airway surface fluid) in which the antimicrobial
peptide is normally designed to operate is disturbed. In these conditions the failure of
antimicrobial peptides to protect the epithelial barrier forces the body to support the defence
of the barrier by mounting a secondary inflammatory response that creates the havoc of
disease. In diseases such as psoriasis, however, an unexplained over-expression of epithelial
antimicrobial peptides appears to excite the inflammatory arm of the adaptive immune system,
creating the skin lesions that characterize this disease.
Although humans have one cathelicidin gene, we have numerous copies of the defensin
family locus, resulting in diering levels of expression of both the - and -defensins between
individuals. How these genetic dierences play out in health and disease is of great interest.
The development of antimicrobial peptides as human therapeutics remains a challenge.
Early on it became clear that peptides such as magainin, although active in the test tube, exhibit
a poor therapeutic index when evaluated in the setting of an infected animal. Most of the
antimicrobial peptides discovered in nature seem to have this characteristic. Unfortunately, we
do not know how to re-engineer a peptide to improve its pharmacological activity in such a
way that would make it a more eective therapeutic. Certain naturally occurring molecules
that exhibit the ecacy and safety in animals expected for a therapeutic such as plectasin
from a fungal organism have, however, been discovered. Surely the insights molecules such
as these will provide will help advance the development of antimicrobial peptides as drugs. In
addition, remarkable non-peptide antimicrobial molecules have been created that mimic the
activity of antimicrobial peptides. These synthetic molecules exhibit an attractive therapeutic
index, and can be synthesized inexpensively. In other words, it is very likely that antimicrobial
peptides, be they of natural or synthetic origin, will surely join the armamentarium of anti-
infective agents in the coming years.
Many of the contributors to this book have been responsible for establishing the foundations
of what we now know about antimicrobial peptides. I hope the knowledge that is shared here
in this book will stimulate others to make new discoveries and further advance this exciting
and important field.
This page intentionally left blank
1
A Database View of Naturally
Occurring Antimicrobial Peptides:
Nomenclature, Classification and Amino
Acid Sequence Analysis
Abstract
The nomenclature and classification of naturally occurring antimicrobial peptides (AMPs) are complex
and have not been fully standardized. The Antimicrobial Peptide Database (http://aps.unmc.edu/AP/
main.php) is a useful tool that facilitates peptide naming and classification. The names of AMPs are
normally derived from peptide properties, source species or a combination of both. In the database,
AMPs are classified based on source organisms (protozoa, bacteria, archaea, fungi, plants and animals),
biological activities (antibacterial, antifungal, antiviral, antiparasitic, spermicidal and insecticidal),
peptide features (charge, length, hydrophobic residue content, chemical modification and three-
dimensional structure), binding targets (membranes and non-membranes) and mechanisms of action of
the peptides. This database also enables bioinformatic analysis of AMPs that reveals amino acid
composition signatures as well as frequently occurring residues.
All organisms possess specialized defence 1981; Selsted et al., 1985; Giovannini et al.,
systems tailored for survival in a variety of 1987; Zaslo, 1987) stimulated research on
environments. Antimicrobial peptides AMPs and led to the isolation and character-
(AMPs) bridge this diversity by functioning ization of hundreds of new peptides.
as key components of defence systems. In Meanwhile, the global problem of antibiotic
invertebrates, AMPs are the major defence resistance further fuelled this research field
molecules of innate immunity. In vertebrates, with a hope of identifying new therapeutics.
AMPs serve not only as eectors in innate The rapid increase in the number of
immunity, but also as modulators for adap- AMPs demands a more ecient data regis-
tive immune systems (Shai, 2002; Tossi and tration and management method. In the past
Sandri, 2002; Zaslo, 2002; Boman, 2003; several years, 13 databases have been built
Brogden et al., 2005; Zanetti, 2005; Hancock for AMPs (Table 1.1). These databases facili-
and Sahl, 2006; Amiche et al., 2008; Conlon, tate information management, annotation,
2008). The identification of cecropins, retrieval and peptide analysis. This chapter
magainins and defensins in insects, amphib- focuses on peptide nomenclature, classifica-
ians and humans in the 1980s (Steiner et al., tion and sequence analysis based on the
Chapter editor: Donatella Barra.
* Corresponding author.
CAB International 2010. Antimicrobial Peptides: Discovery, Design and Novel
Therapeutic Strategies (ed. G. Wang) 1
2 G. Wang et al.
updated version of the Antimicrobial Peptide precursor. For some AMPs such as human
Database (APD), which contains AMPs with LL-37, both the precursor protein and mature
fewer than 100 amino acid residues (Wang peptide were registered. The database had
and Wang, 2004; Wang et al., 2009). also collected 45 antimicrobial proteins. In
addition, this database tabulated the AMP
literature (19972004) alphabetically based on
the last name of the first author. Only AMPs
1.1 Database Scope and Overview from prokaryotes were excluded (Tossi and
Sandri, 2002). In 2002, a database for synthetic
The major databases for AMPs shown in antibiotic peptides (SAPD) was also estab-
Table 1.1 can be classified into three lished (Wade and Englund, 2002). Since regis-
categories: (i) general databases for natural tration is needed, SAPD is not a freely
AMPs (AMSDb, ANTIMIC, APD and accessible tool.
CAMP); (ii) specialized databases for natural In 2004, three additional databases were
AMPs (Peptaibol, PenBase, Cybase, simultaneously reported in the database
BACTIBASE, Defensins and PhytAMP); and issue of the journal Nucleic Acids Research.
(iii) specialized databases for non-natural The Peptaibol database, originally created in
AMPs (SAPD and RAPD). In the following 1997, contains 307 peptides isolated from soil
paragraphs, we briefly describe the functions fungi (Whitmore and Wallace, 2004). As the
of each database in a chronological order. peptaibol name implies, such peptides (<20
The first version of the Antimicrobial residues) are rich in non-standard amino
Sequences Database (AMSDb) (approxi- acids such as -aminoisobutyric acid,
mately 300 entries) was built by an under- isovaleric acid, ethyl norvaline and the imino
graduate as a part of thesis work and placed acid hydroxyproline, which are represented
online in 1997 (Alex Tossi, personal communi- by the non-amino acid letters U, J, Z and O,
cation, 2006). The information was extracted respectively. This is an important feature, as
from Swiss-Prot and kept in the same data other databases have collected peptides with
format. According to the AMSDb, the number the standard 20 amino acids or their deriva-
of AMPs nearly doubled between 1994 and tives. Also published are two general data-
2001. As of 2004, this database hosted 895 bases (APD and ANTIMIC), which cover
peptides from eukaryotes, including both AMPs from a variety of biological sources.
precursor and mature peptide sequences. These two databases, both created by gradu-
Among them, 278 entries contained the word ate students as part of their master degree
Database View of Naturally Occurring AMPs 3
theses, contain registered information as well the peptides collected in AMSDb and Swiss-
as some useful tools for AMP research. Prot. After a recent update, BACTIBASE now
Unfortunately, ANTIMIC (Brahmachary et hosts 177 bacteriocins. The Defensins data-
al., 2004) became inaccessible several years base provides detailed information for vari-
ago. The first version of the APD (Wang and ous defensins as well as related literature,
Wang, 2004) collected mature AMPs primar- including patents. In 2008, a specialized data-
ily from natural sources, ranging from proto- base for recombinant AMPs (RAPD) was also
zoa to bacteria, archaea, fungi, plants and built to document peptide expression, host,
animals, including humans. Gene-encoded carrier, cleavage method and yield (Li and
AMPs that are post-translationally modified Chen, 2008). In 2009, another specialized
are also collected, as are a few AMPs synthe- database was established for plant AMPs
sized by multienzyme systems. Some (PhytAMP) (Hammami et al., 2009).
synthetic peptides of particular interest are Currently, PhytAMP contains 271 peptide
included, too. At present, the following entries. In January 2010, CAMP appeared
polypeptides are not collected: (i) propep- (Thomas et al., 2010). This database has
tides or precursors of AMPs; (ii) genome- collected 1192 peptides with known activi-
predicted or isolated peptides, the ties, 1589 peptides from patents and 1084
antimicrobial activities of which have not predicted AMPs, the antimicrobial activities
been experimentally validated; (iii) peptides of which have not been validated. The data-
that have been found to be inactive against base also incorporates a few prediction tools.
microbes; and (iv) antimicrobial proteins For raw data and additional information,
with greater than 100 amino acid residues. users can consult the original articles, Swiss-
These boundaries merely reflect the current Prot, the Protein Data Bank and PubMed.
status of the database, rather than set restric-
tions for future database expansion. The first
version of the APD collected 525 mature and 1.2 Nomenclature of Antimicrobial
active peptides. Among them, 498 were Peptides
annotated to have antibacterial activities, 155
antifungal activities, 28 antiviral activities Peptide name search is a common feature of
and 18 anticancer activities. The APD all the databases listed in Table 1.1. A unique
provides interactive interfaces for peptide aspect of the name search of the updated
search, prediction and design. It also APD (Wang et al., 2009) is that it allows for
provides statistical data for a selected group multiple-word searches, thereby increasing
of peptides or all peptides in the database. search accuracy and flexibility. Up to three
The database has since been updated and search words can be entered into the search
expanded significantly. By March 2010, when boxes. The output decreases with increases
this chapter was written, there were 1528 in the number of search words. For example,
peptide entries in the APD. we found 179 AMPs when defensin was
Subsequently, several other databases, searched; 12 entries appeared when both
mostly for special types of AMPs, were built. defensin and human were searched; and
While PenBase is dedicated to shrimp AMPs six human -defensins appeared when
(Gueguen et al., 2006), Cybase is a specialized defensin, human and alpha were all
database for cyclic polypeptides from searched. Note that other Greek letters such
bacteria, plants and animals (Mulvenna et al., as , and are represented in the APD as
2006). Some cyclic polypeptides such as plant beta, gamma and delta, respectively. A study
cyclotides have been found to be antimicro- of the AMP names collected in the APD
bial or human immunodeficiency virus (HIV) reveals a complex picture. Various methods
inhibitory (Chapter 3). In 2007, AMPer (Fjell have been employed to name a newly identi-
et al., 2007), BACTIBASE (Hammami et al., fied peptide. These methods fall into three
2007) and Defensins Knowledgebase (Seebah categories: (i) peptide-based method; (ii)
et al., 2007) were also established. AMPer is source-based method; and (iii) source and
an AMP prediction tool developed based on peptide combined method.
4 G. Wang et al.
Both Amiche et al. (2008) and Conlon 1.3 Annotation and Classification of
(2008) proposed a consistent nomenclature Antimicrobial Peptides
for amphibian AMPs. This led to the
re-naming of some peptides (Amiche et al., 1.3.1 Source organisms and peptide
2008). To facilitate the transition from the old family classification
names to the recommended names, the APD
has also collected both old and new names,
Source organisms and their taxonomy
with the preferred name appearing first. It is
suggested that the name of the first published AMPs in the APD are classified based on
AMP in this genus takes priority. their source organisms. The classic five-
Orthologous peptides from the same genus kingdom system proposed by Robert H.
and dierent species can be distinguished by Whittaker in 1969 has been adopted. The five
capitalizing the first letter of the species kingdoms are Prokaryotae (bacteria and
name. Two capitalized letters should be used archaea), Protista (protozoa and algae), Fungi
if the same letter has been used by another (fungi), Plantae (plants) and Animalia
species (Conlon, 2008). (animals). This classification was enabled in
The nomenclature of AMPs is further the database by appending key words such
complicated by the discovery of antimicro- as bacteria, plants or animals behind
bial activities of polypeptides, which were peptide names. For example, partial informa-
initially identified and named based on other tion in the name field for peptide entry
biological functions or medical significances. AP01228 is represented as: Microcin V (MccV,
For example, plant-isolated kalata B1 was (old name) Colicin V, ColV; class 2a microcins,
used as a uterotonic agent to facilitate child- bacteriocins, Gram-negative bacteria).
birth in Congo. Its name is derived from a The above text indicates that microcin V
native medicine, kalata-kalata (Gran, 1973). (abbreviated as MccV), once called colicin V
The discovery of -core in cysteine-contain- (or ColV), is a class IIa microcin (one type of
ing AMPs such as defensins led to testing the bacteriocin) synthesized by a Gram-negative
antimicrobial activity of brazzein (Young and bacterium. This format allows users to
Yeaman, 2004), which is a well-known sweet- retrieve AMPs from a specific kingdom. The
tasting protein (Hellekant and Danilova, number of peptides in dierent kingdoms is
2005). The peptide name is derived from the given in Fig. 1.1. These numbers were
African plant Pentadiplandra brazzeana. It has obtained by database query using search
been demonstrated that some neuropeptides words such as plants and fungii in the
and hormones exhibit antimicrobial activity name field. Note that the use of plants and
and can participate in host defence as well fungii for search gave more accurate results,
(Brogden et al., 2005). These peptides were since occasionally the names of some AMPs
initially named in a dierent biological contain plant or fungi. Human AMPs are
context. Likewise, chemokines such as annotated as an independent group, currently
CXCL14 also display wide-spectrum anti- with 54 entries isolated from saliva, skin,
bacterial activity (Maerki et al., 2009). eyes, liver and other organs. Furthermore,
Chemokines have their own naming rules prokaryotic AMPs have been split into two
and are grouped based on the number and groups: bacteria and archaea. This separation
arrangement of conserved N-terminal enables a calculation of AMPs from the three
cysteine motifs: C, CC, CXC and CX3C, domains proposed by Carl R. Woese in the
where X is a non-conserved residue (Cole et 1970s: bacteria, archaea and eukarya (Fig.
al., 2001). Interestingly, some known AMPs 1.1A). There are 118, two and 1369 peptides
such as human defensins and cathelicidin from these domains, respectively. We
LL-37 are known to have chemotactic eects conclude that AMPs have been identified in
(Murakami et al., 2004; Taylor et al., 2008). all life domains or kingdoms.
Thus, chemotaxis is an important property of Figure 1.1A shows that 71.3% of peptides
AMPs that links the innate and adaptive in the APD originate from animals. The
immune systems (Zaslo, 2002). diversity of animal AMPs requires further
6 G. Wang et al.
Fish 50
Peptide family classification is enabled, and
(B)
Amphibians 592 can be searched, in the name field of the
Vertebrates Reptiles 8 APD. Bacterial AMPs have a general name of
Birds 27 bacteriocins. Initially, those isolated from
Animals Gram-positive bacteria were divided into
Insects 166
Spiders 24
four families based on the original classifica-
Invertebrates tion scheme of Klaenhammer (1993). A fifth
Molluscs 6
Worms 19 group was proposed by Kemperman et al.
Crustaceans 11
(2003). Class I peptides are extensively
Fig. 1.1. (A) The number of antimicrobial peptides modified after translation and are referred to
from a variety of kingdoms. (B) The number of as lantibiotics. Examples are nisins, ericins
AMPs from selected animal families. Data from the and lacticins. Lantibiotics are complex and
Antimicrobial Peptide Database analysed in
have been further classified (see Chapter 2).
February 2010. (Total AMP number: 1528.)
Peptides in class II are composed of normal
amino acids without chemical modifications.
classification. Broadly, they belong to either Class III bacteriocins are peptides conjugated
invertebrates or vertebrates. For inverte- to lipid or other moieties, which have not
brates, source names such as insects, spiders, been isolated to date. Class IV contains large
molluscs, worms and crustaceans are bacteriocins. In the current APD, there are no
included behind the peptide names; for verte- members of class III or IV bacteriocins from
brates, fish, amphibians, reptiles, birds and Gram-positive bacteria. Finally, class V
mammals are used as additional classifiers. contains circular AMPs where the N- and
The number of AMPs from select animal C-termini of the polypeptide chain are
groups currently annotated in the database is connected by a peptide bond. Enterocin
provided in Fig. 1.1B. In particular, amphib- AS-48, gassericin A, circularicin A and clos-
ian peptides account for 38.7% of total AMPs, ticin 574 are circular bacteriocins.
representing a rich source of AMP discovery. Cotter et al. (2005a) have simplified the
Likewise, the APD has collected 166 AMPs classification scheme for bacteriocins from
from insects. Gram-positive bacteria. In this approach,
The source organism for an AMP is not classes I and II (above) are maintained. While
an issue as long as the organism under inves- the original class III is removed, class IV is
tigation is pure. However, problems may re-assigned as a new class III for bacterio-
occur when the organism is so large that a lysins larger than 10 kDa. In addition, there
microbial species or parasite also lives within are subclasses in class II. While pediocin-like
it. For example, cecropin P1 was initially peptide bacteriocins (e.g. leucocin A and
thought to be isolated from pigs. Later, it was divercin V41) are placed in class IIa, class IIb
found that this peptide originated from a contains bacteriocins with two independent
large round worm (nematode) living in pigs polypeptide chains. Plantaricin JK and
(Andersson et al., 2003). The origin of an lactocin 705 are examples of class IIb
AMP can be more complex in the parasite members. The above circular class V bacteri-
host system. For example, a parasitic tick ocins are re-assigned as class IIc. The remain-
generates AMPs in its gut by enzyme diges- ing linear non-pediocin peptides are
tion of the haemoglobin protein from cattle combined into class IId (e.g. entericin Q and
or rabbit blood (Fogaa et al., 1999; Nakajima MR10). We have adopted this modified
et al., 2003). In these cases, it is proper to use approach (summarized in Table 1.2) because
Database View of Naturally Occurring AMPs 7
names may be absent in the original articles. particular AMP, the net charge can vary
Secondly, some peptides are not included slightly depending on the nature of chemical
due to a lack of activity data. modification. For example, the charge
Scientists have been curious as to why increases by 1 when the C-terminus of the
frogs generate such a large number of peptide is amidated. Evidently, the majority
peptides. One possibility is that multiple (88.6%) are cationic AMPs with an average
peptides are created for dierent functions net charge of +4.4 per peptide. Figure 1.2
that are not yet fully understood. Another shows the number of AMPs as a function of
possibility is that the shear multiplicity of net charge. The distribution for all AMPs
environmental microbes requires a variety of (Fig. 1.2A) is relatively smooth with a peak at
host defence peptides. Furthermore, some +3. For bacterial AMPs (Fig. 1.2B), two peaks
peptides can work together to achieve opti- exist at +2 and +4, while the peaks shift
mal protection. Significantly, some peptides slightly to 0 and +2 for plant AMPs (Fig.
are ineective when evaluated alone, but 1.2C). The amphibian AMPs appear to have a
become antibacterial when evaluated in more clustered net charge distribution, with
combination (Li et al., 2007). Amphibian the majority in the range of +1 to +4 (Fig.
AMPs that show a synergistic eect include 1.2D). There are a few outliers not depicted
magainin 2 and PGLa (Matsuzaki et al., 1998; in these plots. Oncorhyncin II and OaBac11
Strandberg et al., 2009); magainin 2 and (Anderson and Yu, 2003; Fernandes et al.,
cecropin A (Cirioni et al., 2008); and tempo- 2004) are the most positive peptides with a
rins A, B and L (Rosenfeld et al., 2006). Note net charge of +30. In amphibians, the most
that temporins A, B and L were found to positively charged AMP is buforin II with a
show a synergistic eect in both antimicro- net charge of +13. Current research is primar-
bial and antiendotoxin (lipopolysaccharide) ily focused on cationic AMPs, with a favour-
activities. Some bacteriocins such as entero- able assumption that they can selectively
cin L50 (Cintas et al., 1998) and lichenicidin target negatively charged bacterial
(Begley et al., 2009) comprise two independ- membranes. The identification of anionic
ent peptide chains, which display higher peptides, however, has further expanded the
activity when combined in a 1:1 ratio. AMP spectrum. The two most negatively
Peptide synergistic information can now be charged peptides contain a net charge of 6.
searched through the additional informa- While the anionic peptide SAAP (sequence
tion field by using synerg or via the name DDDDDD) requires Zn2+ to be antibacterial
field by entering the JJsn key. By March (Brogden et al., 1996), naegleriapore B is a
2010, the APD had collected 15 such syner- pore-forming peptide from a parasitic proto-
gistic pairs. zoon (Herbst et al., 2002). Anionic AMPs have
also been found in other sources such as
amphibians and humans (Schittek et al., 2001;
Lai et al., 2002). Anionic dermcidin acts using
a dierent mechanism from cationic LL-37
1.3.3 Classification based on peptide
(Senyrek et al., 2009).
characteristics
AMPs can also be classified based on
their length. In the APD and this book,
Peptide charge, length and hydrophobic
peptides are defined as containing fewer
residue content
than 100 amino acid residues. The amino
In the database search interface of the APD, acid sequence of an AMP, in the single-letter
there are search icons for peptide charge, amino acid code (e.g. C, G and R), can be
length and percentage of hydrophobic resi- entered in part or full into the search box of
dues. Based on the charge, AMPs can be the sequence field. This is the most accurate
classified into cationic and anionic peptides. method of discovering whether a particular
Of a total of 1528 AMPs, 1355 peptides have peptide has been registered in the APD. The
a net positive charge, 95 have a net charge of majority of AMPs (98.4%) are listed with a
zero and 78 have a net negative charge. For a complete amino acid sequence. Peptides with
10 G. Wang et al.
Number of AMPs
20
Number of AMPs
200
150
100 10
50
0 0
4 3 2 1 0 +1 +2 +3 +4 +5 +6 +7 +8 +9 +10
4 3 2 1 0 +1 +2 +3 +4 +5 +6 +7 +8 +9 +10
Net charge
Net charge
(C) (D)
40 160
Number of AMPs
Number of AMPs
30 120
20 80
10 40
0 0
4 3 2 1 0 +1 +2 +3 +4 +5 +6 +7 +8 +9 +10 4 3 2 1 0 +1 +2 +3 +4 +5 +6 +7 +8 +9 +10
Net charge Net charge
Fig. 1.2. Distribution of antimicrobial peptides in the Antimicrobial Peptide Database versus net charge
from (A) all sources, (B) bacteria, (C) plants and (D) amphibians. The total number of peptides in each
kingdom is provided in Fig. 1.1.
incomplete amino acid sequences can be by Kyte and Doolittle (1982). For all AMPs,
searched in the name field by using BWQ. the peak is located at 4150% (labelled as
The database also allows searching for AMPs 50%) (Fig. 1.4A). Out of 118 bacterial AMPs
in a defined length range, such as 1120 or (Fig. 1.4B), 56 have a hydrophobic residue
2130 amino acids. The number of AMPs as a content between 31 and 40%, and 110 out of
function of peptide length is plotted in Fig. 217 plant AMPs (Fig. 1.4C) are within the
1.3. When all of the 1528 AMPs in the data- hydrophobic residue content of 3140%.
base (Fig. 1.3A) are analysed, the distribution Nearly all amphibian AMPs (Fig. 1.4D) have
plot gives a peak at 30 (i.e. 2130 residues). a hydrophobic content between 41 and 70%.
In both bacteria (Fig. 1.3B) and plants (Fig. The higher hydrophobic content in this
1.3C), most of the AMPs have 2150 residues, animal family is related to a high content of
whereas 97.3% of amphibian AMPs have alanine residues. Three AMPs from bacteria
1140 residues (Fig. 1.3D). The shortest show hydrophobic contents even greater
peptides collected in the database contain than 80%. They are gramicidins that form
only five residues, whereas the longest transmembrane ionic pores. These unusual
peptides contain fewer than 100 residues due AMPs dier from the majority of the
to the peptide definition. peptides collected in the APD in that they are
AMPs can also be classified based on synthesized by a non-ribosome multienzyme
their hydrophobic residue content. A distri- system.
bution of the AMPs in the database versus
hydrophobic residue content (hydrophobic
residues divided by total residues) is
Chemical modifications of AMPs
presented in Fig. 1.4. In the APD, the follow-
ing residues are defined as hydrophobic: Antimicrobial peptides can also be classified
isoleucine, valine, leucine, phenylalanine, based on the types of chemical modifica-
cysteine, methionine, alanine and tryp- tions. This is the case with bacteriocins from
tophan. This was obtained by expanding the Gram-positive bacteria (Table 1.2). Post-
original set of hydrophobic residues defined translational modifications play a critical
Database View of Naturally Occurring AMPs 11
(A) (B)
600
30
Number of AMPs
Number of AMPs
500
400
20
300
200
10
100
0 0
10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100
Peptide length Peptide length
(C) (D)
120 300
Number of AMPs
Number of AMPs
80 200
40 100
0 0
10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100
Peptide length Peptide length
Fig. 1.3. Distribution of antimicrobial peptides in the Antimicrobial Peptide Database versus peptide
length from (A) all sources, (B) bacteria, (C) plants and (D) amphibians. The total number of peptides in
each kingdom is provided in Fig. 1.1. Each column represents the number of the peptides in a range
defined between the two adjacent numbers. For example, 20 means 1120.
Number of AMPs
50
400 40
300 30
200 20
100 10
0 0
10 20 30 40 50 60 70 80 >80 10 20 30 40 50 60 70 80 >80
Hydrophobic residues (%) Hydrophobic residues (%)
(C) (D)
120 250
Number of AMPs
Number of AMPs
100 200
80
150
60
100
40
20 50
0 0
10 20 30 40 50 60 70 80 >80 10 20 30 40 50 60 70 80 >80
Hydrophobic residues (%) Hydrophobic residues (%)
Fig. 1.4. Distribution of antimicrobial peptides in the Antimicrobial Peptide Database versus hydrophobic
residue (%) from (A) all sources, (B) bacteria, (C) plants and (D) amphibians. The total number of
peptides in each kingdom is provided in Fig. 1.1. Each column represents the number of the peptides in a
range defined between the two adjacent numbers. For example, 20 means 1120.
12 G. Wang et al.
ring structure is realized between the back- provide unique tools for peptide engineering.
bone amide of residue glycine 1 and the side Cotter et al. (2005b) found an enzyme that
chain of glutamic acid 8 (Rosengren et al., converts a dehydrated l-serine to d-alanine.
2003). In addition, some amino acid residues Such enzymes may be harnessed to incorpo-
also form a ring structure. N-terminal rate d-amino acids into bacterially expressed
glutamine residues can spontaneously polypeptides. The discovery of the broad
cyclize to become pyroglutamates. This substrate specificity of the nisin modification
N-terminal blocking makes peptide sequenc- enzymes (Rink et al., 2005) may open the door
ing by Edman degradation ineective. The to enzyme-mediated introduction of thioether
APD has collected 12 such peptides (XXQ in rings into a peptide template for required
Table 1.5). They have been isolated from biological activity or structural stability.
plants, spiders, insects, amphibians and
reptiles, revealing another general modifying
mechanism in nature.
Three-dimensional structures of antimicrobial
d-amino acids were first identified in
peptides
natural AMPs from amphibians (Simmaco et
al., 2009). They have also been found in Protein structures have been classified into ,
bacteriocins (Cotter et al., 2005b). The APD , and + families (Murzin et al., 1995).
lists seven d-amino-acid-containing peptides Proteins in the family are composed of
(Table 1.5). However, the identification and primarily -helices, while those in the
characterization of d-amino acids requires family consist of primarily -strands. In the
elegant techniques. Kawai et al. (2004) family, -helices and -strands are inter-
reported that gassericin A and reutericin 6 spersed. However, the -helices and -strands
are cyclic bacteriocins with an identical in the + family are largely segregated. We
amino acid sequence diering only in the have adopted this classification scheme for
number of d-alanines. A recent revisit of AMPs with two modifications. First, the
these two AMPs demonstrated that they are family contains all AMPs that have both
identical and there are no d-amino acids and structures, regardless of whether they
(Arakawa et al., 2010). This work indicates are segregated. Secondly, dierent from the
the importance of purifying natural peptides scheme of Murzin et al. (1995), we define a
to homogeneity before a full biochemical and non- family that includes all AMPs that
biophysical characterization is made. form neither nor structures. Thus, we
Some AMPs may be chemically modi- classify AMP structures into four families: ,
fied in multiple ways. For instance, the , and non-. Representatives from the
sequence of styelin D from the sea squirt is four structural families are provided in Fig.
extensively modified, including halogenation 1.5. Magainin, an -helical AMP (Fig. 1.5A), is
(XXH) of tryptophan 2 and hydroxylation a typical member of the family. Lactoferricin
(XXK) of arginine, lysine and tyrosine resi- B (Fig. 1.5B), with a pair of -strands, is used
dues (detailed in the database). Such modifi- as a representative of the family. Heliomicin,
cations might be essential for the peptide to with both -helices and -strands (e.g. in Fig.
remain active even at high salt concentra- 1.5C), is a member of the family. Finally,
tions. Indeed, the native peptide is more indolicidin, with neither nor structures
active than a synthetic unmodified peptide (Fig. 1.5D), is a representative member in the
(Taylor et al., 2000). The current version of non- family. These four types of peptide
the APD allows a simultaneous search for structures (, , and non-) can be
three types of chemical modifications (Table searched in the structure search field by
1.5) in the name field. When XXA (amida- choosing helix, beta, combined helix and
tion), XXP (phosphorylation) and XXS (sulfa- beta and rich in amino acids, respectively.
tion) were searched, only chrombacin Although the word rich includes all
appeared. peptides rich in certain amino acids, they
Understanding the mechanism of chemi- may or may not form a non- structure.
cal modification of natural AMPs may When the terms rich and NMR (nuclear
14 G. Wang et al.
Table 1.7. Keys for the search of binding partners or targets of AMPs.a
Number of
Key Binding partner peptides Examples
BBBh2o Oligomers in water 6 hBD-3; LL-37
BBBm Oligomers in membranes 1 Protegrin-1
BBII Metal ions (e.g. Zn2+) 12 Histatin 5, hepcidin 25
BBW Bacterial cell wall precursors (e.g. lipid II) 3 Mersacidin, lactocin S
BBL Lipopolysaccharides 26 Temporin L, LL-37
BBr Bacterial cell surface receptor (protein) 1 SMAP-29, hRNase 7
BBMm Inner membranes (e.g. lipid bilayers) 48 Magainins, LL-37
BBN Nucleic acids (DNA/RNA) 5 Buforin II, indolicidin
BBP Proteins (inside cells) 16 Drosocin, pyrrhocoricin
BBS Carbohydrates 21 HNP1, AFP1, Ac-AMP2, RTD-1
a Some of the keys were originally published in Wang et al. (2009). A key search can be conducted in the name field.
4
1.4 Amino Acid Sequence Analysis of
Antimicrobial Peptides 0
I V L F C M AW G P T S Y Q N E D H K R
peptides isolated from frogs of the subfamily Cole, A.M., Ganz, T., Liese, A.M., Burdick, M.D.,
Phyllomedusinae. Peptides 29, 20742082. Liu, L. and Strieter, R.M. (2001) Cutting edge:
Anderson, R.C. and Yu, P.L. (2003) Isolation and IFN-inducible ELR CXC chemokines display
characterisation of proline/arginine-rich defensin-like antimicrobial activity. Journal of
cathelicidin peptides from ovine neutrophils. Immunology 167, 623627.
Biochemical and Biophysical Research Conlon, J.M. (2008) Reflections on a systematic
Communications 312, 11391146. nomenclature for antimicrobial peptides from
Andersson, M., Boman, A. and Boman, H.G. (2003) the skins of frogs of the family Ranidae. Peptides
Ascaris nematodes from pig and human make 29, 18151819.
three antibacterial peptides: isolation of cecropin Conlon, J.M., Kolodziejek, J. and Nowotny, N.
P1 and two ASABF peptides. Cellular and (2009) Antimicrobial peptides from the skins of
Molecular Life Sciences 60, 599606. North American frogs. Biochimica et Biophysica
Arakawa, K., Kawai, Y., Ito, Y., Nakamura, K., Chujo, Acta 1788, 15561563.
T., Nishimura, J., Kitazawa, H. and Saito, T.
Cotter, P.D., Hill, C. and Ross, R.P. (2005a)
(2010) HPLC purification and re-evaluation of
Bacteriocins: developing innate immunity for
chemical identity of two circular bacteriocins,
food. Nature Reviews. Microbiology 3, 777
gassericin A and reutericin 6. Letters in Applied
788.
Microbiology 50, 406411.
Cotter, P.D., OConnor, P.M., Draper, L.A., Lawton,
Begley, M. Cotter, P.D., Hill, C. and Ross, R.P. (2009)
Identification of a novel two-peptide lantibiotic, E.M., Deegan, L.H., Hill, C. and Ross, R.P.
lichenicidin, following rational genome mining (2005b) Posttranslational conversion of
L-serines to D-alanines is vital for optimal
for LanM proteins. Applied and Environmental
Microbiology 75, 54515460. production and activity of the lantibiotic lacticin
Boman, H.G. (2003) Antibacterial peptides: basic 3147. Proceedings of the National Academy of
facts and emerging concepts. Journal of Internal Sciences of the USA 102, 1858418589.
Medicine 254, 197215. Dennison, S.R., Harris, F., Bhatt, T., Singh, J. and
Brahmachary, M., Krishnan, S.P., Koh, J.L., Khan, Phoenix, D.A. (2009) The effect of C-terminal
A.M., Seah, S.H., Tan, T.W., Brusic, V. and Bajic, amidation on the efficacy and selectivity of
V.B. (2004) ANTIMIC: a database of antimicrobial antimicrobial and anticancer peptides. Molecular
sequences. Nucleic Acids Research 32 and Cellular Biochemistry 332, 4350.
(Database issue), D586D589. Dos Santos Cabrera, M.P., Arcisio-Miranda, M.,
Brogden, K.A., De Lucca, A.J., Bland, J. and Elliott, Broggio Costa, S.T., Konno, K., Ruggiero, J.R.,
S. (1996) Isolation of an ovine pulmonary Procopio, J. and RuggieroNeto, J. (2008) Study
surfactant-associated anionic peptide bac- of the mechanism of action of anoplin, a helical
tericidal for Pasteurella haemolytica. antimicrobial decapeptide with ion channel-like
Proceedings of the National Academy of activity, and the role of the amidated C-terminus.
Sciences of the USA 93, 412416. Journal of Peptide Science 14, 661669.
Brogden, K.A., Guthmiller, J.M., Salzet, M. and Duquesne, S., Destoumieux-Garzon, D., Peduzzi,
Zasloff, M. (2005) The nervous system and J. and Rebuffat, S. (2007) Microcins, gene-
innate immunity: the neuropeptide connection. encoded antibacterial peptides from
Nature Immunology 6, 558564. enterobacteria. Natural Product Reports 24,
Bulet, P. and Stocklin, R. (2005) Insect antimicrobial 708734.
peptides: structures, properties and gene Egorov, T.A., Odintsova, T.I., Pukhalsky, V.A. and
regulation. Protein and Peptide Letters 12, Grishin, E.V. (2005) Diversity of wheat anti-
311.
microbial peptides. Peptides 26, 20642073.
Cintas, L.M., Casaus, P., Holo, H., Hernandez, P.E.,
Fernandes, J.M., Molle, G., Kemp, G.D. and Smith,
Nes, I.F. and Hvarstein, L.S. (1998) Enterocins
V.J. (2004) Isolation and characterisation of
L50A and L50B, two novel bacteriocins from
oncorhyncin II, a histone H1-derived
Enterococcus faecium L50, are related to
antimicrobial peptide from skin secretions of
staphylococcal hemolysins. Journal of
Bacteriology 180, 19881994. rainbow trout, Oncorhynchus mykiss.
Cirioni, O., Silvestri, C., Ghiselli, R., Orlando, F., Developmental and Comparative Immunology
Riva, A., Mocchegiani, F., Chiodi, L., Castelletti, 28, 127138.
S., Gabrielli, E., Saba, V., Scalise, G. and Fjell, C.D., Hancock, R.E. and Cherkasov, A. (2007)
Giacometti, A. (2008) Protective effects of the AMPer: a database and an automated discovery
combination of alpha-helical antimicrobial tool for antimicrobial peptides. Bioinformatics
peptides and rifampicin in three rat models of 23, 11481155.
Pseudomonas aeruginosa infection. Journal of Fogaa, A.C., da Silva, P.I. Jr, Miranda, M.T.,
Antimicrobial Chemotherapy 62, 13321338. Bianchi, A.G., Miranda, A., Ribolla, P.E. and
Database View of Naturally Occurring AMPs 19
Matsuzaki, K., Mitani, Y., Akada, K.Y., Murase, O., Roush, R.F., Nolan, E.M., Lhr, F. and Walsh, C.T.
Yoneyama, S., Zasloff, M. and Miyajima, K. (2008) Maturation of an Escherichia coli
(1998) Mechanism of synergism between ribosomal peptide antibiotic by ATP-consuming
antimicrobial peptides magainin 2 and PGLa. N-P bond formation in microcin C7. Journal of
Biochemistry 37, 1514415153. the American Chemical Society 130,
Montalbn-Lpez, M., Spolaore, B., Pinato, O., 36033609.
Martnez-Bueno, M., Valdivia, E., Maqueda. M. Schittek, B., Hipfel, R., Sauer, B., Bauer, J.,
and Fontana, A. (2008) Characterization of Kalbacher, H., Stevanovic, S., Schirle, M.,
linear forms of the circular enterocin AS-48 Schroeder, K., Blin, N., Meier, F., Rassner, G.
obtained by limited proteolysis. FEBS Letters and Garbe, C. (2001) Dermcidin: a novel human
antibiotic peptide secreted by sweat glands.
582, 32373242.
Nature Immunology 2, 11331137.
Mulvenna, J.P., Wang, C. and Craik, D.J. (2006)
Seebah, S., Suresh, A., Zhuo, S., Choong, Y.H.,
CyBase: a database of cyclic protein sequence
Chua, H., Chuon, D., Beuerman, R. and Verma,
and structure. Nucleic Acids Research 34
C. (2007) Defensins knowledgebase: a manually
(Database issue), D192D194.
curated database and information source
Murakami, M., Lopez-Garcia, B., Braff, M., focused on the defensins family of antimicrobial
Dorschner, R.A. and Gallo, R.L. (2004) peptides. Nucleic Acids Research 35 (Database
Postsecretory processing generates multiple issue), D265D268.
cathelicidins for enhanced topical antimicrobial Selsted, M.E., Harwig, S.S., Ganz, T., Schilling,
defense. Journal of Immunology 172, J.W. and Lehrer, R.I. (1985) Primary structures
30703077. of three human neutrophil defensins. Journal of
Murzin, A.G., Brenner, S.E., Hubbard, T. and Clinical Investigation 76, 14361439.
Chothia, C. (1995) SCOP: a structural Senyrek, I., Paulmann, M., Sinnberg, T., Kalbacher,
classification of protein database for the H., Deeg, M., Gutsmann, T., Hermes, M., Kohler,
investigation of sequences and structures. T., Gtz, F., Wolz, C., Peschel, A. and Schittek,
Journal of Molecular Biology 247, 536540. B. (2009) Dermcidin-derived peptides show a
Nakajima, Y., Ogihara, K., Taylor, D. and Yamakawa, different mode of action than the cathelicidin
M. (2003) Antibacterial hemoglobin fragments LL-37 against Staphylococcus aureus.
from the midgut of the soft tick, Ornithodoros Antimicrobial Agents and Chemotherapy 53,
moubata (Acari: Argasidae). Journal of Medical 24992509.
Entomology 40, 7881. Shai, Y. (2002) Mode of action of membrane active
Otero-Gonzlez, A.J., Simas Magalhes, B., antimicrobial peptides. Biopolymers 66,
Garcia-Villarino, M., Lpez-Abarrategui, C., 234248.
Amaro Sousa, D., Campos Dias, S. and Luiz Simmaco, M., Kreil, G. and Barra, D. (2009)
Franco, O. (2010) Antimicrobial peptides from Bombinins, antimicrobial peptides from Bombina
marine invertebrates as a new frontier for species. Biochimica et Biophysica Acta 1788,
microbial infection control. FASEB Journal 24, 15511555.
Steiner, H., Hultmark, D., Engstrm, A., Bennich, H.
13201334.
and Boman, H.G. (1981) Sequence and
Rink, R., Kuipers, A., de Boef, E., Leenhouts, K.J.,
specificity of two antibacterial proteins involved
Driessen, A.J., Moll, G.N. and Kuipers, O.P.
in insect immunity. Nature 292, 246248.
(2005) Lantibiotic structures as guidelines for
Strandberg, E., Tremouilhac, P., Wadhwani, P. and
the design of peptides that can be modified by
Ulrich, A.S. (2009) Synergistic transmembrane
lantibiotic enzymes. Biochemistry 44, insertion of the heterodimeric PGLa/magainin 2
88738882. complex studied by solid-state NMR. Biochimica
Rosenfeld, Y., Barra, D., Simmaco, M., Shai, Y. and et Biophysica Acta 1788, 16671679.
Mangoni, M.L. (2006) A synergism between Taylor, K., Clarke, D.J., McCullough, B., Chin, W.,
temporins toward Gram-negative bacteria Seo, E., Yang, D., Oppenheim, J., Uhrin, D.,
overcomes resistance imposed by the Govan, J.R., Campopiano, D.J., MacMillan, D.,
lipopolysaccharide protective layer. Journal of Barran. P. and Dorin, J.R. (2008) Analysis and
Biological Chemistry 281, 2856528574. separation of residues important for the
Rosengren, K.J., Clark, R.J., Daly, N.L., Gransson, chemoattractant and antimicrobial activities of
U., Jones, A. and Craik, D.J. (2003) Microcin -defensin 3. Journal of Biological Chemistry
J25 has a threaded sidechain-to-backbone ring 283, 66316639.
structure and not a head-to-tail cyclized Taylor, S.W., Craig, A.G., Fischer, W.H., Park, M.
backbone. Journal of the American Chemical and Lehrer, R.I. (2000) Styelin D, an extensively
Society 125, 1246412474. modified antimicrobial peptide from ascidian
Database View of Naturally Occurring AMPs 21
Abstract
Since the 1925 discovery that strains of Escherichia coli can retard the growth of neighbouring bacteria, the
study of bacteriocins has continuously evolved. During the intervening period, a large and heterogeneous
collection of these antimicrobial peptides has been isolated from a myriad of sources and numerous
investigations have been carried out with a view to harnessing their potency. The most thoroughly
investigated class of bacteriocins, the lantibiotics, is the main focus of this review. These antimicrobial
peptides inhibit many human and animal pathogens. They have been the focus of considerable eorts to
maximize the potential of existing lantibiotics, identify new and better lantibiotics from nature and utilize
bioengineering-based approaches to further improve upon existing well-characterized compounds.
* Corresponding authors.
CAB International 2010. Antimicrobial Peptides: Discovery, Design and Novel
22 Therapeutic Strategies (ed. G. Wang)
Lantibiotic-related Research and Application 23
and (ii) class II bacteriocins, which are a form Lan and meLan from Dha and Dhb,
heterogeneous group of non-lanthionine respectively. Insight into the role of LanC
(Lan)-containing peptides. As a consequence was first provided by Meyer et al. (1995)
of their unusual structure, unique mechan- who, while working on the lantibiotic Pep5,
isms of action and potential as antimicrobials showed that with the removal of the pepC
for food, veterinary and clinical applications, gene, the peptide produced contained
the lantibiotics have received much attention dehydrated residues but not Lan or meLan
in recent years. They are the focus of this bridges. In addition to being essential for
chapter. converting the peptide to an active form, the
Lan and meLan structures are also believed
to contribute to protease resistance
(Kluskens et al., 2005). Unlike the type I
2.2 Biosynthesis and peptides, which are modified by two
Post-translational Modifications separate modification enzymes, type II
peptides such as lacticin 481 and mersacidin
The name lantibiotics is derived from are modified by a single protein referred to
Lan-containing antibiotics and reflects the as LanM (Willey and van der Donk, 2007).
feature that is shared by all of these peptides: Another variation on this theme arises as a
intra-molecular rings formed by the thioether consequence of the existence of two peptide
amino acids Lan and methylanthionine lantibiotics (e.g. lacticin 3147) that require
(meLan) (Sahl et al., 1995). Lantibiotic- the combined activity of two Lan- and
associated gene clusters can be located on meLan-containing peptides for optimal
chromosomes (including transposable activity. In most cases, the production of
elements) or plasmids. In addition to the lantibiotics of this kind relies on the presence
genes encoding a structural peptide(s) and of two LanM proteins, each of which is
the post-translational modification machinery responsible for the dehydration and
that acts thereon, other genes responsible for cyclization of its respective subunit
regulation, transport and immunity are (McAulie et al., 2000; Lawton et al., 2007b).
usually also present (Cotter et al., 2005b). The A third category of peptides, type III, is
following paragraphs summarize the steps not described in this review as those
involved in lantibiotic biosynthesis. identified to date, while possessing Lan and
The lantibiotic prepropeptide contains a meLan structures, lack antimicrobial activity
leader region, which is eventually cleaved, (Willey and van der Donk, 2007). Yet another
and a propeptide, which is modified to category, modified by a novel group of
become the active antimicrobial. The lantibiotic synthetases designated LanL, has
corresponding gene is generically designated recently been discovered (Goto et al., 2010). It
lanA. Lantibiotics can be further subdivided remains to be established if LanL-modified
according to the enzymes that catalyse Lan peptides possess antimicrobial activity, and
and meLan formation. Type I prepropeptides thus it has been suggested that the term
such as the prototypic lantibiotic nisin A lantipeptides be used to describe Lan-
undergo dehydration as a result of the containing peptides that lack antimicrobial
catalytic activity of the NisB protein activity (Goto et al., 2010). With respect to the
(generically referred to as LanB enzymes) lantibiotics, in addition to the LanB, LanC
(Karakas Sen et al., 1999). As a result, specific and LanM enzymes, a number of other
serine and threonine residues are enzymes have been associated with the
dehydrated to become the unique amino modification of specific peptides (Kupke and
acids 2,3-dehydroalanine (Dha) and Gotz, 1997; Peschel et al., 1997; Majer et al.,
2,3-dehydrobutyrine (Dhb), respectively. 2002; Cotter et al., 2005c).
Some or all of these new residues are then After modification, the peptide is
the subject of a LanC-catalysed reaction. transported via an ABC transport system to
This results in their interaction with the thiol the cell surface (Fath and Kolter, 1993) and
groups of cysteines within the peptide to the leader sequence is proteolytically
24 B. Healy et al.
Fig. 2.2. Some representative lantibiotic structures. Modified residues are shaded or dashed. Ala-S-Ala,
lanthionine (dashed); Abu-S-Ala, -methyllanthionine (light grey); Ala, D-alanine (grey dashed); Dha,
dehydroalanine (dark grey); Dhb, dehydrobutyrine (black, white text); Asp, -hydroxy-aspartate (grey,
white text); Lys-NH-Ala, lysinoalanine (black, grey text).
Lantibiotic-related Research and Application 27
2.6 Lantibiotics and Their Medical infection (Kruszewska et al., 2004) and nisin
Applications F, both alone and when used in combination
with lysozyme and lactoferrin, can
The possibility of using lantibiotics to control successfully treat respiratory tract MRSA
or treat multidrug-resistant forms of infections in mice (De Kwaadsteniet et al.,
pathogens such as Staphylococcus aureus, 2009). Trials investigating the use of
Enterococcus species and Clostridium dicile lantibiotics to control the microorganisms
has gained increased attention in recent years responsible for dental plaque, halitosis,
due to a number of positive results obtained strep throat (Hillman, 2002; Burton et al.,
by researchers in the field (for a 2006; Dierksen et al., 2007) and even bovine
comprehensive review, see Piper et al., mastitis (Ryan et al., 1999a; Twomey et al.,
2009a). In vitro, many lantibiotics, including 2000) have all been successful.
lacticin 3147, mutacin B-Ny266, nisin and
mutacin 1140, show activity against clinical
targets such as methicillin-resistant Staph. 2.7 Engineering of Lantibiotics
aureus (MRSA), vancomycin-resistant Entero-
coccus faecalis (VRE), penicillin-resistant Lantibiotics are gene encoded. Advantage
Pneumococcus, Propionibacterium acne, Strepto- can be taken of this trait to engineer novel
coccus mutans, Strep. pyogenes, Strep. variants of the parent peptide. Such variants
pneumoniae, C. dicile, Listeria and Bacillus have been used to study structurefunction
species (Severina et al., 1998; Galvin et al., relationships and, in some cases, engineering
1999; Mota-Meira et al., 2000; Brumfitt et al., strategies have led to the generation of
2002; Rea et al., 2007; Ghobrial et al., 2009; peptides with enhanced antimicrobial
Piper et al., 2009b). It is also interesting that activity. Lantibiotic engineering can take
both Pep5 and epidermin successfully inhibit place in vivo (i.e. by manipulating the original
the adhesion of staphylococcal cells to the producing strain or expressing the genes
surfaces of catheters (Fontana et al., 2006). It heterologously in an alternative host) or in
is important to note, however, that these vitro (i.e. by harnessing the activity of
represent just a selection of the studies that purified forms of the individual components
have highlighted the ecacy of lantibiotics of biosynthetic machinery outside of a host).
against Gram-positive clinical pathogens. It The application of engineering to lantibiotic
is anticipated that the number of studies in research commenced in 1992. Although
this area will continue to increase as a initial nisin-focused investigations did not
consequence of the further investigation of lead to the production of variants with
existing lantibiotic peptides and the enhanced activity (Kuipers et al., 1992), they
continued identification of new forms of clearly demonstrated the power of this
these antimicrobials. Two recent examples of technology. During the same year, the
note are the two-peptide lantibiotic extreme consequences of making single,
lichenicidin, which exhibits antimicrobial deliberate amino acid changes were
activity against MRSA and VRE strains demonstrated when it was established that a
(Begley et al., 2009), and microbisporicin, single residue change in subtilin resulted in a
which is active against MRSA, VRE and 57-fold increase in its biological and chemical
clinical streptococci (Castiglione et al., 2008). stability (Liu and Hansen, 1992). This
While the in vitro success of a technology has continued to be applied, and
chemotherapeutic agent does not always in 2006 the first alanine-scanning
correspond to in vivo ecacy, a number of mutagenesis of a lantibiotic, lacticin 3147,
studies have indicated that this may not be a was completed (Cotter et al., 2006a). In this
major failing of lantibiotics. It has been study, alanine (or glycine in cases where an
revealed that mutacin B-Ny266 can be as alanine was already present) was introduced
active as vancomycin against MRSA in vivo in place of the 59 amino acids, in turn, and
(Mota-Meira et al., 2005), mersacidin can be the impact on the antimicrobial activity of
employed to eradicate a nasal MRSA the associated producing strain was
Lantibiotic-related Research and Application 31
quantified. The data generated highlighted KFI and KSI (the letters indicate the amino
specific areas of the peptides in which subse- acids present at positions 4, 5 and 6),
quent site-specific mutagenesis approaches displayed increased antimicrobial activity
might be beneficial. This strategy was taken against a number of bacteria such as
a step further when site-saturation Leuconostoc mesenteroides, Lactobacillus
mutagenesis was employed to engineer both johnsonii and Lactococcus lactis. KFI, and
nukacin ISK-1 (Islam et al., 2009) and another variant, VFG, also inhibited the
mersacidin (Appleyard et al., 2009). Both outgrowth of B. subtilis 168 spores more
studies provided an in-depth insight into the eectively than the wild type.
structurefunction relationships within the It should be noted that these and other
respective peptides. In the case of nukacin (bio)engineering-related strategies have also
ISK-1, two variants displaying a twofold been used for a variety of other purposes,
increase in specific activity were identified such as increasing lantibiotic production
(Islam et al., 2009). (Cotter et al., 2006b; Heinzmann et al., 2006),
The use of engineering to study or introducing Lans into class II bacteriocins
improve nisin has also continued at pace. (Majchrzykiewicz et al., 2010) and even post-
Since the 1990s, this lantibiotic has been the translational modification of other bioactive
subject of a number of engineering-based peptides (Kuipers et al., 2004; Kluskens et al.,
strategies, which have employed site- 2009; Rink et al., 2010). In addition to these
directed, site-saturation and random approaches, the ever-improving ability of
mutagenesis. While various dierent regions chemists to generate lantibiotic-like peptides
of the peptide have been engineered, the through synthetic chemistry (Cobb and
N-terminal and hinge regions have received Vederas, 2007; Arnusch et al., 2008; Ross et al.,
the greatest attention. The benefits of 2010) is particularly exciting and has already
manipulating the hinge (consisting of Asn20- facilitated the creation of potent nisin
Met21-Lys22) have been particularly notable vancomycin hybrids (Arnusch et al., 2008).
(Yuan et al., 2004; Field et al., 2008). Yuan et al.
(2004) employed a site-directed approach
whereby either positively or negatively 2.8 Screening for New Lantibiotics
charged amino acids were introduced into
the hinge. These studies demonstrated that While scientists are continuing with their
specific changes (i.e. N20K and M21K) eorts to further improve known lantibiotics,
increased the activity of the peptide against there is still considerable merit attached to
Gram-negative bacteria such as Shigella, identifying new peptides. It has also become
Pseudomonas and Salmonella species. In the apparent in recent years that bioinformatics
case of Field et al. (2008), screening of a bank can be a very useful means of screening for
of random mutagenized nisin variants such novel lantibiotics. The availability of
revealed that a K22T variant displayed databases, including both general (NCBI)
enhanced activity against the mastitic patho- and dedicated (BAGEL and BACTIBASE)
gen Streptococcus agalactiae. This prompted systems (see Chapter 1, Table 1.1, for more)
the use of site-saturation mutagenesis for (de Jong et al., 2006; Hammami et al., 2007,
each of the individual hinge residues that, 2010), has facilitated the use of in silico
when coupled with a larger selection of approaches to lantibiotic screening. The
target strains, led to the identification of a two-peptide lantibiotic lichenicidin (Begley
number of peptides with enhanced activity et al., 2009) was identified using such an
against Strep. agalactiae, Staph. aureus and L. approach. Here, the highly conserved nature
monocytogenes. Yet another study, focusing on of the lanM gene was exploited to screen the
rings A and B at the N-terminal end of nisin ever-increasing number of bacterial genome
A, showed that the various activities of nisin sequences that are publicly available. Initial
A can be altered by changing the amino acid screening revealed 89 lanM genes, of which
arrangement in this region of the peptide 61 had not previously been associated with
(Rink et al., 2007). Two mutants, designated lantibiotic production. One of the potential
32 B. Healy et al.
novel lantibiotic producers identified, Bacillus therapeutics that can target the multidrug-
licheniformis ATCC 14580, was selected and resistant Gram-positive clinical pathogens,
from it lichenicidin was isolated (Begley et including the particularly problematic
al., 2009). A similar approach was previously pathogens MRSA, VRE and C. dicile. A
employed to identify another two-peptide number of these peptides are particularly
lantibiotic, haloduracin, which is produced attractive as a consequence of research that
by Bacillus halodurans C-125 (McClerren et al., has established that they have mechanisms
2006; Lawton et al., 2007a). Bioinformatics of action and target binding sites that are
has also been of considerable use when distinct from those of non-lantibiotics. This,
designing engineered lantibiotics and novel coupled with their high potency and
Lan-containing peptides. A study by Rink et generally non-cytotoxic nature, could lead to
al. (2005) used bioinformatics to predict the these compounds having clinical applications
impact of flanking amino acids on the in the future. The possibility of engineering
dehydration of serine and threonine residues, new and improved lantibiotics, producing
and subsequent Lan and meLan formation, novel chemotherapeutics through the fusion
in lantibiotic peptides. An in silico of lantibiotics with antibiotics, introducing
comparison of known lantibiotics found that lantibiotic-associated modifications into
the majority of modified serines and non-lantibiotics and chemically synthesizing
threonines were flanked by hydrophobic new lantibiotic-like peptides as well as the
residues. In silico models predicted the likely ongoing use of traditional and in silico
impact of specific residues on modification strategies to find novel compounds all
when located adjacent to hydroxyl-amino bring this potential to a new level. In
acid residues. The subsequent creation, and addition to these new markets, it should not
investigation, of these peptides validated this be forgotten that a lantibiotic, nisin, has been
theory. successfully employed by the food industry
The discovery of the lantibiotics micro- for over a half century. Other lantibiotics
bisporicin and planosporicin was achieved have the potential to be similarly employed
using a more traditional screening method and, as a consequence of the limited activity
(Castiglione et al., 2007, 2008). This method of nisin against certain target strains and
involved 120,000 broth extracts obtained by species, together with its poor activity at
fermenting 40,000 actinomycetes. The neutral pH, there are obvious niche-markets
microbial products were screened to assess that these can fill. The commercial potential
their activity against Staph. aureus before of lantibiotics and lantibiotic-related
selecting those that retained activity technology and the cutting-edge funda-
following exposure to a -lactamase cocktail mental science that underpins lantibiotic
or d-alanyl-d-alanine anity resin (i.e. they research will ensure that these peptides
were neither -lactams nor vancomycin-like continue to attract great attention in the
glycopeptides). Those that retained anti- coming years.
microbial activity after these steps were
selected for further investigation. This
strategy yielded 35 lantibiotics, of which five Acknowledgement
showed little or no similarity to any known
lantibiotics. The authors would like to thank Des Field
for contributing to figure preparation.
Altena, K., Guder, A., Cramer, C. and Bierbaum, G. Breukink, E., van Kraaij, C., Demel, R.A., Siezen,
(2000) Biosynthesis of the lantibiotic mersacidin: R.J., Kuipers, O.P. and de Kruijff, B. (1997) The
organization of a type B lantibiotic gene cluster. C-terminal region of nisin is responsible for the
Applied and Environmental Microbiology 66, initial interaction of nisin with the target
25652571. membrane. Biochemistry 36, 69686976.
Appleyard, A.N., Choi, S., Read, D. M., Lightfoot, Breukink, E., Wiedemann, I., van Kraaij, C., Kuipers,
A., Boakes, S., Hoffmann, A., Chopra, I., O.P., Sahl, H. and de Kruijff, B. (1999) Use of the
Bierbaum, G., Rudd, B. A., Dawson, M.J. and cell wall precursor lipid II by a pore-forming
Cortes, J. (2009) Dissecting structural and peptide antibiotic. Science 286, 23612364.
functional diversity of the lantibiotic mersacidin. Brotz, H., Bierbaum, G., Markus, A., Molitor, E. and
Chemistry & Biology 16, 490498. Sahl, H.G. (1995) Mode of action of the
Arnusch, C.J., Bonvin, A.M., Verel, A.M., Jansen, lantibiotic mersacidin: inhibition of peptidoglycan
W.T., Liskamp, R.M., de Kruijff, B., Pieters, R.J. biosynthesis via a novel mechanism?
and Breukink, E. (2008) The vancomycin- Antimicrobial Agents and Chemotherapy 39,
nisin(112) hybrid restores activity against 714719.
vancomycin resistant Enterococci. Biochemistry Brotz, H., Josten, M., Wiedemann, I., Schneider,
47, 1266112663. U., Gotz, F., Bierbaum, G. and Sahl, H.G. (1998)
Asaduzzaman, S.M., Nagao, J., Iida, H., Zendo, T., Role of lipid-bound peptidoglycan precursors in
Nakayama, J. and Sonomoto, K. (2009) Nukacin the formation of pores by nisin, epidermin and
ISK-1, a bacteriostatic lantibiotic. Antimicrobial other lantibiotics. Molecular Microbiology 30,
Agents and Chemotherapy 53, 35953598. 317327.
Begley, M., Cotter, P.D., Hill, C. and Ross, R.P. Brumfitt, W., Salton, M.R. and Hamilton-Miller, J.M.
(2009) Identification of a novel two-peptide (2002) Nisin, alone and combined with
lantibiotic, lichenicidin, following rational genome peptidoglycan-modulating antibiotics: activity
mining for LanM proteins. Applied and against methicillin-resistant Staphylococcus
Environmental Microbiology 75, 54515460. aureus and vancomycin-resistant enterococci.
Bierbaum, G. and Sahl, H.G. (1985) Induction of Journal of Antimicrobial Chemotherapy 50,
autolysis of staphylococci by the basic peptide 731734.
antibiotics Pep 5 and nisin and their influence Buchman, G.W., Banerjee, S. and Hansen, J.N.
on the activity of autolytic enzymes. Archives of (1988) Structure, expression, and evolution of a
Microbiology 141, 249254. gene encoding the precursor of nisin, a small
Bierbaum, G. and Sahl, H.G. (1987) Autolytic protein antibiotic. Journal of Biological Chemistry
system of Staphylococcus simulans 22: 263, 1626016266.
influence of cationic peptides on activity of Burton, J.P., Chilcott, C.N., Moore, C.J., Speiser, G.
N-acetylmuramoyl-L-alanine amidase. Journal and Tagg, J.R. (2006) A preliminary study of the
of Bacteriology 169, 54525458. effect of probiotic Streptococcus salivarius K12
Bierbaum, G., Reis, M., Szekat, C. and Sahl, H.G. on oral malodour parameters. Journal of Applied
(1994) Construction of an expression system for Microbiology 100, 754764.
engineering of the lantibiotic Pep5. Applied and Castiglione, F., Cavaletti, L., Losi, D., Lazzarini, A.,
Environmental Microbiology 60, 43324338. Carrano, L., Feroggio, M., Ciciliato, I., Corti, E.,
Bierbaum, G., Szekat, C., Josten, M., Heidrich, C., Candiani, G., Marinelli, F. and Selva, E. (2007)
Kempter, C., Jung, G. and Sahl, H.G. (1996) A novel lantibiotic acting on bacterial cell wall
Engineering of a novel thioether bridge and role synthesis produced by the uncommon
of modified residues in the lantibiotic Pep5. actinomycete Planomonospora sp. Biochemistry
Applied and Environmental Microbiology 62, 46, 58845895.
385392. Castiglione, F., Lazzarini, A., Carrano, L., Corti, E.,
Bonelli, R.R., Schneider, T., Sahl, H.G. and Ciciliato, I., Gastaldo, L., Candiani, P., Losi, D.,
Wiedemann, I. (2006) Insights into in vivo Marinelli, F., Selva, E. and Parenti, F. (2008)
activities of lantibiotics from gallidermin and Determining the structure and mode of action of
epidermin mode-of-action studies. Antimicrobial microbisporicin, a potent lantibiotic active
Agents and Chemotherapy 50, 14491457. against multiresistant pathogens. Chemistry &
Booth, M.C., Bogie, C.P., Sahl, H.G., Siezen, R.J., Biology 15, 2231.
Hatter, K.L. and Gilmore, M.S. (1996) Structural Chatterjee, C., Paul, M., Xie, L. and van der Donk,
analysis and proteolytic activation of W.A. (2005) Biosynthesis and mode of action of
Enterococcus faecalis cytolysin, a novel lantibiotics. Chemical Reviews 105, 633684.
lantibiotic. Molecular Microbiology 21, 1175 Chatterjee, S., Lad, S.J., Phansalkar, M.S., Rupp,
1184. R.H., Ganguli, B.N., Fehlhaber, H.W. and Kogler,
34 B. Healy et al.
H. (1992) Mersacidin, a new antibiotic from Dierksen, K.P., Moore, C.J., Inglis, M., Wescombe,
Bacillus. Fermentation, isolation, purification P.A. and Tagg, J.R. (2007) The effect of ingestion
and chemical characterization. Journal of of milk supplemented with salivaricin
Antibiotics 45, 832838. A-producing Streptococcus salivarius on the
Chen, P., Qi, F., Novak, J. and Caufield, P.W. (1999) bacteriocin-like inhibitory activity of streptococcal
The specific genes for lantibiotic mutacin II populations on the tongue. FEMS Microbiology
biosynthesis in Streptococcus mutans T8 are Ecology 59, 584591.
clustered and can be transferred en bloc. Dischinger, J., Josten, M., Szekat, C., Sahl, H.G.
Applied and Environmental Microbiology 65, and Bierbaum, G. (2009) Production of the novel
13561360. two-peptide lantibiotic lichenicidin by Bacillus
Cobb, S.L. and Vederas, J.C. (2007) A concise licheniformis DSM 13. PLoS One 4, e6788.
stereoselective synthesis of orthogonally Dougherty, B.A., Hill, C., Weidman, J.F., Richardson,
protected lanthionine and -methyllanthionine. D.R., Venter, J.C. and Ross, R.P. (1998)
Organic & Biomolecular Chemistry 5, 1031 Sequence and analysis of the 60 kb conjugative,
1038. bacteriocin-producing plasmid pMRC01 from
Cotter, P.D., Hill, C. and Ross, R.P. (2005a) Bacterial Lactococcus lactis DPC3147. Molecular
lantibiotics: strategies to improve therapeutic Microbiology 29, 10291038.
potential. Current Protein & Peptide Science 6, Draper, L.A., Ross, R.P., Hill, C. and Cotter, P.D.
6175. (2008) Lantibiotic immunity. Current Protein &
Cotter, P.D., Hill, C. and Ross, R.P. (2005b) Peptide Science 9, 3949.
Bacteriocins: developing innate immunity for Dufour, A., Hindre, T., Haras, D. and Le Pennec, J.P.
food. Nature Reviews. Microbiology 3, 777 (2007) The biology of lantibiotics from the
788. lacticin 481 group is coming of age. FEMS
Cotter, P.D., OConnor, P.M., Draper, L.A., Lawton, Microbiology Reviews 31, 134167.
E.M., Deegan, L.H., Hill, C. and Ross, R.P. Engelke, G., Gutowski-Eckel, Z., Kiesau, P.,
(2005c) Posttranslational conversion of L-serines Siegers, K., Hammelmann, M. and Entian, K.D.
to D-alanines is vital for optimal production and (1994) Regulation of nisin biosynthesis and
activity of the lantibiotic lacticin 3147. immunity in Lactococcus lactis 6F3. Applied and
Proceedings of the National Academy of Environmental Microbiology 60, 814825.
Sciences of the USA 102, 1858418589. Ersfeld-Dressen, H., Sahl, H.G. and Brandis, H.
Cotter, P.D., Deegan, L.H., Lawton, E.M., Draper, (1984) Plasmid involvement in production of
L.A., OConnor, P.M., Hill, C. and Ross, R.P. and immunity to the staphylococcin-like peptide
(2006a) Complete alanine scanning of the two- Pep 5. Journal of General Microbiology 130,
component lantibiotic lacticin 3147: generating 30293035.
a blueprint for rational drug design. Molecular Fath, M.J. and Kolter, R. (1993) ABC transporters:
Microbiology 62, 735747. bacterial exporters. Microbiol Reviews 57, 995
Cotter, P.D., Draper, L.A., Lawton, E.M., McAuliffe, 1017.
O., Hill, C. and Ross, R P. (2006b) Overproduction Field, D., Connor, P.M.O., Cotter, P.D., Hill, C. and
of wild-type and bioengineered derivatives of Ross, R.P. (2008) The generation of nisin
the lantibiotic lacticin 3147. Applied and variants with enhanced activity against specific
Environmental Microbiology 72, 44924496. Gram-positive pathogens. Molecular Micro-
de Jong, A., van Hijum, S.A., Bijlsma, J.J., Kok, J. biology 69, 218230.
and Kuipers, O.P. (2006) BAGEL: a web-based Fontana, M.B., de Bastos Mdo, C. and Brandelli, A.
bacteriocin genome mining tool. Nucleic Acids (2006) Bacteriocins Pep5 and epidermin inhibit
Research 34, W273-W279. Staphylococcus epidermidis adhesion to
De Kwaadsteniet, M., Doeschate, K.T. and Dicks, catheters. Current Microbiology 52, 350353.
L.M. (2009) Nisin F in the treatment of respiratory Fredenhagen, A., Fendrich, G., Marki, F., Marki, W.,
tract infections caused by Staphylococcus Gruner, J., Raschdorf, F. and Peter, H.H. (1990)
aureus. Letters in Applied Microbiology 48, Duramycins B and C, two new lanthionine
6570. containing antibiotics as inhibitors of
Deegan, L.H., Cotter, P.D., Hill, C. and Ross, P. phospholipase A2. Structural revision of
(2006) Bacteriocins: Biological tools for bio- duramycin and cinnamycin. Journal of Antibiotics
preservation and shelf-life extension. Inter- 43, 14031412.
national Dairy Journal 16, 10581071. Galvin, M., Hill, C. and Ross, R.P. (1999) Lacticin
Delves-Broughton, J., Blackburn, P., Evans, R.J. 3147 displays activity in buffer against Gram-
and Hugenholtz, J. (1996) Applications of the positive bacterial pathogens which appear
bacteriocin, nisin. Antonie Van Leeuwenhoek insensitive in standard plate assays. Letters in
69, 193202. Applied Microbiology 28, 355358.
Lantibiotic-related Research and Application 35
Ghobrial, O.G., Derendorf, H. and Hillman, J.D. cinnamycin, complexed with lysophos-
(2009) Pharmacodynamic activity of the phatidylethanolamine by 1H-NMR. Journal of
lantibiotic MU1140. International Journal of Biochemistry 119, 226230.
Antimicrobial Agents 33, 7074. Hsu, S.T., Breukink, E., Bierbaum, G., Sahl, H.G.,
Goto, Y., Li, B., Claesen, J., Shi, Y., Bibb, M.J. and de Kruijff, B., Kaptein, R., van Nuland, N.A. and
van der Donk, W.A. (2010) Discovery of unique Bonvin, A.M. (2003) NMR study of mersacidin
lanthionine synthetases reveals new mechanistic and lipid II interaction in dodecylphosphocholine
and evolutionary insights. PLoS Biology 8, micelles. Conformational changes are a key to
e1000339. antimicrobial activity. Journal of Biological
Gratia, A. (1925) Sur un remarquable exemple Chemistry 278, 1311013117.
dantagonisme entre deux souches de Hsu, S.T., Breukink, E., Tischenko, E., Lutters,
Colibacille. Comptes Rendus des Sances et M.A., de Kruijff, B., Kaptein, R., Bonvin, A.M.
Mmoires de la Socit de Biologie 93, 1040 and van Nuland, N.A. (2004) The nisinlipid II
1041. complex reveals a pyrophosphate cage that
Gross, E. and Morell, J.L. (1971) Structure of nisin. provides a blueprint for novel antibiotics. Nature
Journal of the American Chemical Society 93, Structural & Molecular Biology 11, 963967.
46344635. Hyink, O., Balakrishnan, M. and Tagg, J.R. (2005)
Guder, A., Schmitter, T., Wiedemann, I., Sahl, H.G. Streptococcus rattus strain BHT produces both
and Bierbaum, G. (2002) Role of the single a class I two-component lantibiotic and a class
regulator MrsR1 and the two-component system II bacteriocin. FEMS Microbiology Letters 252,
MrsR2/K2 in the regulation of mersacidin 235241.
production and immunity. Applied and Environ- Islam, M.R., Shioya, K., Nagao, J., Nishie, M.,
mental Microbiology 68, 106113. Jikuya, H., Zendo, T., Nakayama, J. and
Hammami, R., Zouhir, A., Ben Hamida, J. and Fliss, Sonomoto, K. (2009) Evaluation of essential
I. (2007) BACTIBASE: a new web-accessible and variable residues of nukacin ISK-1 by NNK
database for bacteriocin characterization. BMC
scanning. Molecular Microbiology 72, 1438
Microbiology 7, 89.
1447.
Hammami, R., Zouhir, A., Le Lay, C., Ben Hamida,
Jack, R., Benz, R., Tagg, J. and Sahl, H.G. (1994)
J. and Fliss, I. (2010) BACTIBASE second
The mode of action of SA-FF22, a lantibiotic
release: a database and tool platform for
isolated from Streptococcus pyogenes strain
bacteriocin characterization. BMC Microbiology
FF22. European Journal of Biochemistry 219,
10, 22.
699705.
Hasper, H.E., de Kruijff, B. and Breukink, E. (2004)
Jack, R.W., Tagg, J.R. and Ray, B. (1995)
Assembly and stability of nisinlipid II pores.
Bacteriocins of Gram-positive bacteria.
Biochemistry 43, 1156711575.
Microbiological Reviews 59, 171200.
Havarstein, L.S., Diep, D.B. and Nes, I.F. (1995) A
Jung, G. (1991) Lantibiotics ribosomally
family of bacteriocin ABC transporters carry out
synthesized biologically active polypeptides
proteolytic processing of their substrates
concomitant with export. Molecular Microbiology containing sulfide bridges and ,-
16, 229240. didehydroamino acids. Angewandte Chemie
Heinzmann, S., Entian, K.D. and Stein, T. (2006) (International ed. in English) 30, 10511192.
Engineering Bacillus subtilis ATCC 6633 for Kaletta, C., Entian, K.D., Kellner, R., Jung, G., Reis,
improved production of the lantibiotic subtilin. M. and Sahl, H.G. (1989) Pep5, a new lantibiotic:
Applied Microbiology and Biotechnology 69, structural gene isolation and prepeptide
532536. sequence. Archives of Microbiology 152,
Hillman, J.D. (2002) Genetically modified Strepto- 1619.
coccus mutans for the prevention of dental Kaletta, C., Entian, K.D. and Jung, G. (1991)
caries. Antonie Van Leeuwenhoek 82, 361 Prepeptide sequence of cinnamycin (Ro
366. 090198): the first structural gene of a
Holo, H., Jeknic, Z., Daeschel, M., Stevanovic, S. duramycin-type lantibiotic. European Journal of
and Nes, I.F. (2001) Plantaricin W from Biochemistry 199, 411415.
Lactobacillus plantarum belongs to a new family Karakas Sen, A., Narbad, A., Horn, N., Dodd, H.M.,
of two-peptide lantibiotics. Microbiology 147, Parr, A.J., Colquhoun, I. and Gasson, M.J.
643651. (1999) Post-translational modification of nisin.
Hosoda, K., Ohya, M., Kohno, T., Maeda, T., Endo, The involvement of NisB in the dehydration
S. and Wakamatsu, K. (1996) Structure deter- process. European Journal of Biochemistry 261,
mination of an immunopotentiator peptide, 524532.
36 B. Healy et al.
Karaya, K., Shimizu, T. and Taketo, A. (2001) New Kupke, T. and Gotz, F. (1997) The enethiolate anion
gene cluster for lantibiotic streptin possibly reaction products of EpiD. pKa value of the
involved in streptolysin S formation. Journal of enethiol side chain is lower than that of the thiol
Biochemistry 129, 769775. side chain of peptides. Journal of Biological
Kellner, R., Jung, G., Horner, T., Zahner, H., Schnell, Chemistry 272, 47594762.
N., Entian, K.D. and Gotz, F. (1988) Gallidermin: Lawton, E.M., Cotter, P.D., Hill, C. and Ross, R.P.
a new lanthionine-containing polypeptide (2007a) Identification of a novel two-peptide
antibiotic. European Journal of Biochemistry lantibiotic, haloduracin, produced by the
177, 5359. alkaliphile Bacillus halodurans C-125. FEMS
Kleerebezem, M. (2004) Quorum sensing control of Microbiology Letters 267, 6471.
lantibiotic production; nisin and subtilin Lawton, E.M., Ross, R.P., Hill, C. and Cotter, P.D.
autoregulate their own biosynthesis. Peptides (2007b) Two-peptide lantibiotics: a medical
25, 14051414. perspective. Mini Reviews in Medicinal
Klein, C., Kaletta, C. and Entian, K.D. (1993) Chemistry 7, 12361247.
Biosynthesis of the lantibiotic subtilin is Liu, W. and Hansen, J.N. (1992) Enhancement of
regulated by a histidine kinase/response the chemical and antimicrobial properties of
regulator system. Applied and Environmental subtilin by site-directed mutagenesis. Journal of
Microbiology 59, 296303. Biological Chemistry 267, 2507825085.
Kluskens, L.D., Kuipers, A., Rink, R., de Boef, E., Maffioli, S.I., Potenza, D., Vasile, F., De Matteo, M.,
Fekken, S., Driessen, A.J., Kuipers, O.P. and Sosio, M., Marsiglia, B., Rizzo, V., Scolastico, C.
Moll, G.N. (2005) Post-translational modification and Donadio, S. (2009) Structure revision of the
of therapeutic peptides by NisB, the dehydratase lantibiotic 97518. Journal of Natural Products
of the lantibiotic nisin. Biochemistry 44, 12827 72, 605607.
Majchrzykiewicz, J.A., Lubelski, J., Moll, G.N.,
12834.
Kuipers, A., Bijlsma, J.J., Kuipers, O.P. and Rink,
Kluskens, L.D., Nelemans, S.A., Rink, R., de Vries,
R. (2010) Production of a class II two-component
L., Meter-Arkema, A., Wang, Y., Walther, T.,
lantibiotic of Streptococcus pneumoniae using
Kuipers, A., Moll, G.N. and Haas, M. (2009)
the class I nisin synthetic machinery and leader
Angiotensin-(17) with thioether bridge: an
sequence. Antimicrobial Agents and
angiotensin-converting enzyme-resistant,
Chemotherapy 54, 14981505.
potent angiotensin-(17) analog. Journal of
Majer, F., Schmid, D.G., Altena, K., Bierbaum, G.
Pharmacology and Experimental Therapeutics
and Kupke, T. (2002) The flavoprotein MrsD
328, 849854.
catalyzes the oxidative decarboxylation reaction
Kordel, M., Schuller, F. and Sahl, H.G. (1989)
involved in formation of the peptidoglycan
Interaction of the pore forming-peptide
biosynthesis inhibitor mersacidin. Journal of
antibiotics Pep 5, nisin and subtilin with non-
Bacteriology 184, 12341243.
energized liposomes. FEBS Letters 244, McAuliffe, O., Hill, C. and Ross, R.P. (2000) Each
99102. peptide of the two-component lantibiotic lacticin
Kruszewska, D., Sahl, H.G., Bierbaum, G., Pag, U., 3147 requires a separate modification enzyme
Hynes, S.O. and Ljungh, A. (2004) Mersacidin for activity. Microbiology 146, 21472154.
eradicates methicillin-resistant Staphylococcus McAuliffe, O., OKeeffe, T., Hill, C. and Ross, R.P.
aureus (MRSA) in a mouse rhinitis model. (2001) Regulation of immunity to the two-
Journal of Antimicrobial Chemotherapy 54, component lantibiotic, lacticin 3147, by the
648653. transcriptional repressor LtnR. Molecular
Kuipers, A., de Boef, E., Rink, R., Fekken, S., Microbiology 39, 982993.
Kluskens, L.D., Driessen, A.J., Leenhouts, K., McClerren, A.L., Cooper, L.E., Quan, C., Thomas,
Kuipers, O.P. and Moll, G.N. (2004) NisT, the P.M., Kelleher, N.L. and van der Donk, W.A.
transporter of the lantibiotic nisin, can transport (2006) Discovery and in vitro biosynthesis of
fully modified, dehydrated, and unmodified haloduracin, a two-component lantibiotic.
prenisin and fusions of the leader peptide with Proceedings of the National Academy of
non-lantibiotic peptides. Journal of Biological Sciences of the USA 103, 1724317248.
Chemistry 279, 2217622182. Meyer, C., Bierbaum, G., Heidrich, C., Reis, M.,
Kuipers, O.P., Rollema, H.S., Yap, W.M., Boot, H.J., Suling, J., Iglesias-Wind, M.I., Kempter, C.,
Siezen, R.J. and de Vos, W.M. (1992) Molitor, E. and Sahl, H.G. (1995) Nucleotide
Engineering dehydrated amino acid residues in sequence of the lantibiotic Pep5 biosynthetic
the antimicrobial peptide nisin. Journal of gene cluster and functional analysis of PepP
Biological Chemistry 267, 2434024346. and PepC. Evidence for a role of PepC in
Lantibiotic-related Research and Application 37
thioether formation. European Journal of Paik, S.H., Chakicherla, A. and Hansen, J.N. (1998)
Biochemistry 232, 478489. Identification and characterization of the
Morgan, S.M., Galvin, M., Ross, R.P. and Hill, C. structural and transporter genes for, and the
(2001) Evaluation of a spray-dried lacticin 3147 chemical and biological properties of, sublancin
powder for the control of Listeria monocytogenes 168, a novel lantibiotic produced by Bacillus
and Bacillus cereus in a range of food systems. subtilis 168. Journal of Biological Chemistry
Letters in Applied Microbiology 33, 387391. 273, 2313423142.
Mortvedt, C.I., Nissen-Meyer, J., Sletten, K. and Peschel, A., Schnell, N., Hille, M., Entian, K.D. and
Nes, I.F. (1991) Purification and amino acid Gotz, F. (1997) Secretion of the lantibiotics
sequence of lactocin S, a bacteriocin produced epidermin and gallidermin: sequence analysis
by Lactobacillus sake L45. Applied and of the genes gdmT and gdmH, their influence
Environmental Microbiology 57, 18291834. on epidermin production and their regulation by
Mota-Meira, M., LaPointe, G., Lacroix, C. and EpiQ. Molecular & General Genetics 254, 312
Lavoie, M.C. (2000) MICs of mutacin B-Ny266, 318.
nisin A, vancomycin, and oxacillin against Piard, J.C., Muriana, P.M., Desmazeaud, M.J. and
bacterial pathogens. Antimicrobial Agents and Klaenhammer, T.R. (1992) Purification and
Chemotherapy 44, 2429. partial characterization of lacticin 481, a
Mota-Meira, M., Morency, H. and Lavoie, M.C. lanthionine-containing bacteriocin produced by
(2005) In vivo activity of mutacin B-Ny266. Lactococcus lactis subsp. lactis CNRZ 481.
Journal of Antimicrobial Chemotherapy 56, Applied and Environmental Microbiology 58,
869871. 279284.
Navaratna, M.A., Sahl, H.G. and Tagg, J.R. (1998) Piard, J.C., Kuipers, O.P., Rollema, H.S.,
Two-component anti-Staphylococcus aureus Desmazeaud, M.J. and de Vos, W.M. (1993)
lantibiotic activity produced by Staphylococcus Structure, organization, and expression of the
aureus C55. Applied and Environmental lct gene for lacticin 481, a novel lantibiotic
Microbiology 64, 48034808. produced by Lactococcus lactis. Journal of
Nissen-Meyer, J., Rogne, P., Oppegard, C., Haugen, Biological Chemistry 268, 1636116368.
H.S. and Kristiansen, P.E. (2009) Structure Piper, C., Cotter, P.D., Ross, R.P. and Hill, C. (2009a)
function relationships of the non-lanthionine- Discovery of medically significant lantibiotics.
containing peptide (class II) bacteriocins Current Drug Discovery Technologies 6, 118.
produced by Gram-positive bacteria. Current Piper, C., Draper, L.A., Cotter, P.D., Ross, R.P. and
Pharmaceutical Biotechnology 10, 1937. Hill, C. (2009b) A comparison of the activities of
Oman, T.J. and van der Donk, W.A. (2009) Insights lacticin 3147 and nisin against drug-resistant
into the mode of action of the two-peptide Staphylococcus aureus and Enterococcus
lantibiotic haloduracin. ACS Chemistry & Biology species. Journal of Antimicrobial Chemotherapy
4, 865874. 64, 546551.
OSullivan, L., Ross, R.P. and Hill, C. (2003) A Pridmore, D., Rekhif, N., Pittet, A.C., Suri, B. and
lacticin 481-producing adjunct culture increases Mollet, B. (1996) Variacin, a new lanthionine-
starter lysis while inhibiting nonstarter lactic containing bacteriocin produced by Micrococcus
acid bacteria proliferation during Cheddar varians: comparison to lacticin 481 of
cheese ripening. Journal of Applied Microbiology Lactococcus lactis. Applied and Environmental
95, 12351241. Microbiology 62, 17991802.
Ottenwalder, B., Kupke, T., Brecht, S., Gnau, V., Qiao, M. and Saris, P.E. (1996) Evidence for a role
Metzger, J., Jung, G. and Gotz, F. (1995) of NisT in transport of the lantibiotic nisin
Isolation and characterization of genetically produced by Lactococcus lactis N8. FEMS
engineered gallidermin and epidermin analogs. Microbiology Letters 144, 8993.
Applied and Environmental Microbiology 61, Rawlinson, E.L., Nes, I.F. and Skaugen, M. (2002)
38943903. LasX, a transcriptional regulator of the lactocin
Pag, U. and Sahl, H.G. (2002) Multiple activities in S biosynthetic genes in Lactobacillus sakei L45,
lantibiotics models for the design of novel acts both as an activator and a repressor.
antibiotics? Current Pharmaceutical Design 8, Biochimie 84, 559567.
815833. Rea, M.C., Clayton, E., OConnor, P.M., Shanahan,
Pag, U., Heidrich, C., Bierbaum, G. and Sahl, H.G. F., Kiely, B., Ross, R.P. and Hill, C. (2007)
(1999) Molecular analysis of expression of the Antimicrobial activity of lacticin 3147 against
lantibiotic Pep5 immunity phenotype. Applied clinical Clostridium difficile strains. Journal of
and Environmental Microbiology 65, 591598. Medical Microbiology 56, 940946.
38 B. Healy et al.
Riley, M.A. and Wertz, J.E. (2002) Bacteriocin Sahl, H.G., Ersfeld-Dressen, H., Bierbaum, G.,
diversity: ecological and evolutionary Josten, M., Kordel, M., Reis, M. and Schuller, F.
perspectives. Biochimie 84, 357364. (1987) Different mechanisms of insensitivity to
Rince, A., Dufour, A., Le Pogam, S., Thuault, D., the staphylococcin-like peptide Pep 5.
Bourgeois, C.M. and Le Pennec, J.P. (1994) Zentralblatt fr Bakteriologie, Mikrobiologie und
Cloning, expression, and nucleotide sequence Hygiene A 267, 173185.
of genes involved in production of lactococcin Sahl, H.G., Jack, R.W. and Bierbaum, G. (1995)
DR, a bacteriocin from Lactococcus lactis Biosynthesis and biological activities of
subsp. lactis. Applied and Environmental lantibiotics with unique post-translational
Microbiology 60, 16521657. modifications. European Journal of Biochemistry
Rink, R., Kuipers, A., de Boef, E., Leenhouts, K.J., 230, 827853.
Driessen, A.J., Moll, G.N. and Kuipers, O.P. Schnell, N., Engelke, G., Augustin, J., Rosenstein,
(2005) Lantibiotic structures as guidelines for R., Ungermann, V., Gotz, F. and Entian, K.D.
the design of peptides that can be modified by (1992) Analysis of genes involved in the
lantibiotic enzymes. Biochemistry 44, 8873 biosynthesis of lantibiotic epidermin. European
8882. Journal of Biochemistry 204, 5768.
Rink, R., Wierenga, J., Kuipers, A., Kluskens, L.D., Severina, E., Severin, A. and Tomasz, A. (1998)
Driessen, A.J., Kuipers, O.P. and Moll, G.N. Antibacterial efficacy of nisin against multidrug-
(2007) Dissection and modulation of the four resistant Gram-positive pathogens. Journal of
distinct activities of nisin by mutagenesis of Antimicrobial Chemotherapy 41, 341347.
rings A and B and by C-terminal truncation. Siezen, R.J. (1996) Subtilases: subtilisin-like serine
Applied and Environmental Microbiology 73, proteases. Advances in Experimental Medicine
58095816. and Biology 379, 7593.
Rink, R., Arkema-Meter, A., Baudoin, I., Post, E., Skaugen, M., Nissen-Meyer, J., Jung, G.,
Stevanovic, S., Sletten, K., Inger, C., Abildgaard,
Kuipers, A., Nelemans, S.A., Akanbi, M.H.J. and
M. and Nes, I.F. (1994) In vivo conversion of
Moll, G.N. (2010) To protect peptide
L-serine to D-alanine in a ribosomally synthesized
pharmaceuticals against peptidases. Journal of
polypeptide. Journal of Biological Chemistry
Pharmacological and Toxicological Methods 61,
269, 2718327185.
210218.
Skaugen, M., Abildgaard, C.I. and Nes, I.F. (1997)
Rogers, L.A. (1928) The inhibiting effect of
Organization and expression of a gene cluster
Streptococcus lactis on Lactobacillus bulgaricus.
involved in the biosynthesis of the lantibiotic
Journal of Bacteriology 16, 321325.
lactocin S. Molecular & General Genetics 253,
Ross, A.C., Liu, H., Pattabiriman, V.R. and Vederas,
674686.
J.C. (2010) Synthesis of the lantibiotic lactocin S
Stock, A.M., Robinson, V.L. and Goudreau, P.N.
using peptide cyclizations on solid phase.
(2000) Two-component signal transduction.
Journal of the American Chemical Society 132,
Annual Review of Biochemistry 69, 183215.
462463. Tagg, J.R., Dajani, A.S. and Wannamaker, L.W.
Ryan, M.P., Rea, M.C., Hill, C. and Ross, R.P. (1976) Bacteriocins of Gram-positive bacteria.
(1996) An application in cheddar cheese Bacteriological Reviews 40, 722756.
manufacture for a strain of Lactococcus lactis Thomas, T.D. and Crow, V.L. (1983) Mechanism of
producing a novel broad-spectrum bacteriocin, D()-lactic acid formation in Cheddar cheese.
lacticin 3147. Applied and Environmental New Zealand Journal of Dairy Science and
Microbiology 62, 612619. Technology 18, 131141.
Ryan, M.P., Flynn, J., Hill, C., Ross, R.P. and Twomey, D.P., Wheelock, A.I., Flynn, J., Meaney,
Meaney, W.J. (1999a) The natural food grade W.J., Hill, C. and Ross, R.P. (2000) Protection
inhibitor, lacticin 3147, reduced the incidence of against Staphylococcus aureus mastitis in dairy
mastitis after experimental challenge with cows using a bismuth-based teat seal containing
Streptococcus dysgalactiae in nonlactating the bacteriocin, lacticin 3147. Journal of Dairy
dairy cows. Journal of Dairy Science 82, 2625 Science 83, 19811988.
2631. van den Hooven, H.W., Lagerwerf, F.M., Heerma,
Ryan, M.P., Jack, R.W., Josten, M., Sahl, H.G., W., Haverkamp, J., Piard, J.C., Hilbers, C.W.,
Jung, G., Ross, R.P. and Hill, C. (1999b) Siezen, R.J., Kuipers, O.P. and Rollema, H.S.
Extensive post-translational modification, (1996) The structure of the lantibiotic lacticin
including serine to D-alanine conversion, in the 481 produced by Lactococcus lactis: location of
two-component lantibiotic, lacticin 3147. Journal the thioether bridges. FEBS Letters 391, 317
of Biological Chemistry 274, 3754437550. 322.
Lantibiotic-related Research and Application 39
Wescombe, P.A. and Tagg, J.R. (2003) Purification function. Annual Review of Microbiology 61,
and characterization of streptin, a type A1 477501.
lantibiotic produced by Streptococcus pyogenes. Xie, L. and van der Donk, W.A. (2004) Post-
Applied and Environmental Microbiology 69, translational modifications during lantibiotic
27372747. biosynthesis. Current Opinion in Chemical
Wiedemann, I., Breukink, E., van Kraaij, C., Kuipers, Biology 8, 498507.
O.P., Bierbaum, G., de Kruijff, B. and Sahl, H.G. Ye, S.Y., Koponen, O., Qiao, M., Immonen, T. and
(2001) Specific binding of nisin to the Saris, P.E. (1995) NisP is related to nisin
peptidoglycan precursor lipid II combines pore precursor processing and possibly to immunity
formation and inhibition of cell wall biosynthesis in Lactococcus lactis. Journal of Tongji Medical
for potent antibiotic activity. Journal of Biological University 15, 193197.
Chemistry 276, 17721779. Yonezawa, H. and Kuramitsu, H.K. (2005) Genetic
Wiedemann, I., Bottiger, T., Bonelli, R.R., Wiese, analysis of a unique bacteriocin, Smb, produced
A., Hagge, S.O., Gutsmann, T., Seydel, U., by Streptococcus mutans GS5. Antimicrobial
Deegan, L., Hill, C., Ross, P. and Sahl, H.G. Agents and Chemotherapy 49, 541548.
(2006) The mode of action of the lantibiotic Yuan, J., Zhang, Z.Z., Chen, X.Z., Yang, W. and
lacticin 3147 a complex mechanism involving Huan, L.D. (2004) Site-directed mutagenesis of
specific interaction of two peptides and the cell the hinge region of nisinZ and properties of
wall precursor lipid II. Molecular Microbiology nisinZ mutants. Applied Microbiology and
61, 285296. Biotechnology 64, 806815.
Willey, J.M. and van der Donk, W.A. (2007)
Lantibiotics: peptides of diverse structure and
3 Antimicrobial Peptides in Plants
Abstract
This chapter provides an overview of plant antimicrobial peptides. It mainly focuses on one particular
class of plant defence peptides, namely the cyclotides, which have been discovered over the last decade
in plants from the Rubiaceae, Violaceae and Cucurbitaceae families. Cyclotides have a head-to-tail cyclized
peptide backbone and a cystine knot motif formed from their six conserved cysteine residues, which
makes them exceptionally stable. This chapter describes their isolation and characterization, structure
and biosynthesis, and applications. The structural stability of cyclotides makes them excellent scaolds
for the engineering of novel therapeutic proteins. Advances in methods for the production of cyclotides
and their potential clinical applications are also described.
non-specific manner, without a preference and plant growth and development are
for a particular type of lipid. In vitro, these linked with LTPs (Yeats and Rose, 2008).
peptides have been shown to transfer lipids Membrane targeting of LTPs from plants has
from one membrane to another. In an early been shown to inhibit some bacterial (Molina
example, transfer of radioactively labelled et al., 1993) and fungal (Terras et al., 1992)
phosphatidylcholine from artificial vesicles plant pathogens in vitro. Terras et al. (1992)
to chloroplasts from spinach was shown to showed that increasing the ionic strength of
be mediated by an LTP isolated from spinach the assay medium abrogates antifungal
leaves (Miquel et al., 1988). Similarly, electron activity, indicating a charge-dependent mech-
paramagnetic resonance and fluorescence anism of action, most likely involving
experiments have been used to show that an interactions with the charged head groups of
LTP from maize seeds was able to shuttle lipids in the fungal membrane.
lipids containing spin labels from artificial The first crystal structure of a plant LTP
vesicles to membranes from human was for an example from Zea mays in
erythrocytes and fluorescently labelled lipids complex with palmitate (Shin et al., 1995).
from artificial vesicles to bovine chroman The solution structures of LTPs from Triticum
granules, respectively (Geldwerth et al., aestivum (wheat) (Gincel et al., 1994), Hordeum
1991). In that study, negatively charged vulgare (barley) (Heinemann et al., 1996) and
lipids with phosphatidylcholine, phospha- Z. mays were early examples solved by
tidylethanolamine, phosphatidylserine and nuclear magnetic resonance (NMR) and are
phosphatic acid head groups were found to shown in Fig. 3.1A (Gomar et al., 1996). The
be transferred to the target membrane at structures of LTP from barley in complex
similar rates; thus, a direct charge interaction with palmitate-coenzyme A (Lerche et al.,
between positively charged LTP and 1997) and palmitate (Lerche and Poulsen,
negatively charged lipid head groups 1998) were also solved by NMR. The global
appears to be important. This lipid-transfer fold of LTPs reveals a bundle of four helices
activity has been implicated in plant cell that cage lipid chains in the hydrophobic
development (Nieuwland et al., 2005), but core formed between them (Fig. 3.1B and Fig.
generally a variety of bioactivities, including 3.1C). Only minor changes in the C-terminal
defence against pathogens, cuticle synthesis region of LTPs have been reported when
Fig. 3.1. Structural features of lipid-transfer proteins (LTPs). (A) The apo (lipid-free) form of LTP from Zea
mays comprises four -helices (Protein Data Bank (PDB) access code: 1afh). (B) Palmitate (black, stick
representation, carboxyl group on top) is deeply buried in the cage formed by the four helices of LTP from
barley (PDB: 1be2). (C) Palmitate-coenzyme A (CoA) conjugate binds to barley LTP in a similar manner;
the lipid (black) is buried inside the peptide, whereas CoA is located on the surface of the peptide (PDB:
1jtb). Note that these are two complexes solved for the same LTP; for sake of clarity, C is rotated by 180
in the view shown.
AMPs in Plants 43
binding to lipids (Lerche et al., 1997; Lerche The first two thionins to be discovered
and Poulsen, 1998). were identified (Balls et al., 1942a,b) and
In general, plant AMPs are of much isolated (Fisher et al., 1968) from T. aestivum
interest, not only for the control of plant (wheat) germ. Their names, - and
diseases (Montesinos, 2007), but also due to -purothionins, reflect that they are wheat
their ability to attack human pathogens, peptides (Greek: puro = wheat) and that they
including Candida and Aspergillus species have a high sulfur content (Greek: thio =
(Thevissen et al., 2007). These properties, sulfur). Mixtures of - and -purothionins,
together with the growing problem of resist- as well as purified peptides, were tested
ance to conventional antibiotics (Hancock against a range of phytopathogenic bacteria.
and Sahl, 2006), make plant AMPs interesting Activity was shown against Pseudomonas
as novel human therapeutic leads. However, solanacearum, Xanthomonas phaseoli, Corynebac-
as far as LTPs are concerned, such applica- terium michiganense and others, but not
tions need to be evaluated carefully because against Pseudomonas savastanoi (Fernandez de
some plant LTPs have been identified as Caleya et al., 1972). Tests with purified
allergens in food (Zuidmeer and van Ree, purothionins showed that -purothionin was
2007). For example, studies have reported more active against X. phaseoli than
that allergy to hazelnut (Flinterman et al., -purothionin, which in turn was more active
2008) and wheat (Inomata, 2009) may be against P. solanacearum (Fernandez de Caleya
associated with these peptides. et al., 1972). These complementary activities
of - and -purothionins exemplify how
the diversity of AMPs in a single plant
3.1.2 Thionins confers resistance to a diverse spectrum of
pathogens.
Thionins comprise 4547 residues. They are The antimicrobial activity of thionins is
divided into five classes depending on the
thought to be enabled via an interaction
number of disulfide bonds, number of basic
with the head groups of lipids in membranes
residues and overall charge. Type I thionins
and has been studied in detail. Hughes et al.
have four disulfide bonds and are highly
(2000) showed that the presence of
basic, with a charge of +10. Type II thionins
negatively charged phosphatidylserine in
also have four disulfide bonds, but have a
artificial lipid mixtures resulted in the
reduced basic character with a charge of +7.
formation of membrane pores acting as ion
Type III and IV thionins both have three
channels. However, NMR and infrared
disulfide bonds. Type III thionins have a
charge of +7, whereas type IV thionins are spectroscopic evidence exists that in the
neutral. Type V thionins appear to be absence of negatively charged lipids,
truncated variants of other thionins thionins are also capable of binding both
(Bohlmann and Apel, 1991; Stec, 2006). In neutral lipids and those with positive
general, it appears that antimicrobial activity charges (Richard et al., 2002, 2005). This
is correlated with the overall charge: the broad binding ability may explain their
higher the positive charge, the higher the broad antibacterial spectrum.
activity (Stec, 2006). Overall, thionins are a widely studied
Thionins adopt an overall shape similar class of plant defence peptides. They
to the letter L (Stec, 2006). The structure potentially have applications as drug leads,
consists of a -strand followed by two reflecting their presence as active compounds
-helices (helix-turn-helix motif) and a in traditional medicines from plants used
second -strand completing a double- since prehistoric times (Stec, 2006). Their
stranded -sheet. Several structures have activities against phytopathogenic microbes
been found to accommodate lipid moieties in have also led to their incorporation in
the groove formed by the two -helices and transgenic plants to increase or confer
two -sheets (Fig. 3.2) (Stec et al., 1995; resistance to plant pests (Carmona et al.,
Debreczeni et al., 2003). 1993; Chan et al., 2005).
44 Q. Kaas et al.
Fig. 3.3. Structure of Rs-AFP1 (Raphanus sativus antifungal protein 1; Protein Data Bank (PDB) access
code: 1ayj) and active residues of Psd1 (Pisum sativum defensin 1; PDB: 1jkz). (A) Rs-AFP2 adopts the
typical fold of plant defensins. The N- and C-termini are in close proximity due to a disulfide bond (ball
and stick representation) from C3 to C51. (B) Backbone trace of Psd1. Residues of loop 1 (A7N17) and
turn 3 (H36W38) and C35 are highlighted by stick representation. (C) Residues that experience
chemical shift perturbation in the presence of phosphatidylcholine vesicles are shaded. (D) In the
presence of 10% monohexosylceramide in the phosphatidylcholine vesicles, the distribution of residues
affected by chemical shift perturbations changes significantly. Loop 1 is affected from G12 to N17, as is
W38 in turn 3 and the neighbouring K39 (N17, L30 and W38 are not labelled for clarity).
showed much higher activity to a set of fungi They also have some activity against Gram-
in vitro. The mode of action is most likely positive bacteria, although to a lesser extent.
disruption of the fungal cell membrane; it Their amino acid sequence bears some
was shown using fluorescently labelled resemblance to other chitin-binding peptides
peptide and confocal microscopy, in but they lack the C-terminal domain, which
conjunction with electron microscopy, that includes two cysteine residues (Broekaert et
Pn-AMP1 accumulates in the septa of fungal al., 1997). Both peptides show marked anity
hyphae (Koo et al., 1998). to chitin at neutral pH. In other recent
Antifungal activity has also been evidence demonstrating the importance of
observed for two small peptides, Ac-AMP1 chitin binding, mutations in the chitin-
and Ac-AMP2, from Amaranthus caudatus binding protein Cy-AMP1 (Cycas revoluta,
(pendant amaranth) (Broekaert et al., 1992). cycad) that decrease anity for chitin also
AMPs in Plants 47
decrease antimicrobial activity against fungi non-natural amino acids in complex with
but not Gram-positive and Gram-negative chitotriose, that was solved in solution using
bacteria (Yokoyama et al., 2009). This indi- NMR (Chavez et al., 2005). In the synthetic
cates that binding to chitin is essential for the peptide, phenylalanine in position 18 and
antimicrobial activity of chitin-binding tyrosine in position 20 were both substitu-
peptides. However, the detailed mechanism ted with 4-fluorophenylalanine. The complex
of membrane disruption is still unknown. reveals the interaction of the aromatic side
Figure 3.4 shows the structure of a chain of residues 18 and 20 with the rings of
mutant of Ac-AMP2, incorporating two the central N-acetyl glucosamine residue and
Fig. 3.4. Chitin and chitin-binding peptides. (A) 1,4-linked N-acetylglucosamine (top) is the building
block of chitin. Chitotriose (bottom) is a defined oligomer of N-acetylglucosamine. (B) An overlay of the
solution structures of hevein (grey, Protein Data Bank (PDB) access code: 1hev) (Andersen et al., 1993)
and Ac-AMP2 (Amaranthus caudatus; dark grey, PDB: 1mmc) (Martins et al., 1996) reveals their
structural similarity. The C-terminus of hevein is highlighted in light grey to clarify the extension of hevein
relative to Ac-AMP2. The C-termini are labelled. (C) Solution structure of Ac-AMP2. (D) Solution structure
of a synthetic Ac-AMP2 analogue (in the same orientation as in C in complex with chitotriose (dark grey)).
The synthetic peptide incorporates two non-natural para-fluorophenylalanine (Pff) residues in positions
18 and 20 (arrows) to stabilize the complex with the carbohydrate through aromatic interactions. The
rings of the side chains of residues 18 and 20 are aligned with the plane of the carbohydrate rings (PDB:
1znt).
48 Q. Kaas et al.
the residue on the non-reducing end, binding lectins have been shown to be toxic
respectively, a common recognition motif in to insects (Hegedus et al., 2009).
carbohydrate binding (Jimenez-Barbero et al.,
2006).
In general, the broad importance of
chitin-binding proteins arises from the 3.1.5 Miscellaneous AMPs from plants
ubiquitous importance of their targets.
Complex carbohydrates are essential com- In addition to the major classes of plant AMPs
ponents of cell surfaces and glycoproteins in noted above, there are several peptides that
many organisms, including pathogens and do not fall into large families, but none the
humans (Gabius et al., 2004). Lectins with less have important activities or novel
high specificity for certain carbohydrate structures. For example, a series of AMPs that
motifs, including N-acetylglucosamine and are expressed as a single precursor peptide
its oligomers, are valuable research tools and and processed into their final form occur in
have potential as therapeutics (Gabius et al., Impatiens balsamina (rose balsam) (Tailor et al.,
2004; Andr et al., 2009). For example, lectins, 1997). These relatively short peptides,
including chitin-binding lectins, have been comprising approximately 20 residues, are
investigated for their ability to inhibit human highly homologous and are stabilized by two
immunodeficiency virus (HIV) (Balzarini, disulfide bonds to form well-defined
2006) and cancer cells (Liu et al., 2010). These structures (Patel et al., 1998). As can be seen in
studies have mainly focused on proteins, but Fig. 3.5, the Ib-AMPs are expressed as
the results may encourage further research precursors that contain six highly homol-
into chitin-binding peptides to exploit their ogous repeats. Ib-AMP1 is present in three
therapeutic and pharmaceutical advantages copies (1a, 1b and 1c), whereas the other
over proteins (da Rocha Pitta and Galdino, peptides appear only as single copies. The
2010). Chitin-binding ability is linked to plant precursor protein comprises a signal peptide
protection against herbivore insects. The and seven propeptide regions of approxi-
insect gut is lined with the peritrophic mately 28 residues located before each
membrane, which contains chitin, and chitin- mature peptide region.
Fig. 3.5. Organization of Ib-AMP precursor (from Impatiens balsamina) and mature AMPs. (A) The
precursor contains six repeats encoding four individual peptides. Ib-AMP1 is present in three identical
copies (ac). The mature peptide regions are preceded by an acidic propeptide region that is
proteolytically cleaved during the maturation process. (B) The sequence alignment of the mature Ib-AMPs
reveals the high homology and identity of the three copies of Ib-AMP1. The disulfide connectivity is
indicated. * indicates identical residues and : indicates highly similar residues.
AMPs in Plants 49
Fig. 3.6. Structural features of MiAMP1 (Macadamia integrifolia AMP1; Protein Data Bank access code:
1c01). (A) Three-dimensional structure. The disulfide bonds are shown in a ball and stick representation
with N- and C-termini indicated. The Greek key motifs are in light grey (motif 1) and dark grey (motif 2).
The representations are rotated by 180 for clarity. (B) Schematic showing the two -sheets in MiAMP1
and the -strand swap between Greek key motif 1 (white filling) and motif 2 (grey filling). (C) An
alignment of Greek key motif 1 (residues 138) and motif 2 (residues 3976) reveals a weak sequence
identity, but high similarity in the arrangement of the -strands (arrows). Similarity: *, identical; :, highly
similar; ., similar. The symmetrical disulfide bonds within the motifs are indicated by solid lines on the top
and bottom, respectively, and the disulfide bond tethering the two motifs together is indicated as a
dashed line between the sequences.
AMPs in Plants 51
Fig. 3.7. Cyclopeptide alkaloids from plants. The arrows indicate the stereochemistry at position 3, which
differs in scutianine M (right) compared with condaline A (left) and scutianine E (middle). The
stereocentre occurs as a result of a cross-link to the -carbon of phenylalanine or valine, respectively.
Condaline A and scutianine M both have an isoleucine residue (grey highlight), whereas scutianine E
features the unusual amino acid -phenylserine at this position. The dashed outline highlights N-methyl
phenylalanine. The breadth of the activity spectrum and activity decreases from left to right (Morel et al.,
2005).
in at least two plant families, the Violaceae tides, but some individual cyclotides have
(violet) and Rubiaceae (coee) families been discovered in multiple plants. For
(Gruber et al., 2008). Most plants express instance kalata S, also known as varv A
non-overlapping suites of dierent cyclo- (Gransson et al., 1999), has been isolated
from six dierent Violaceae species (Viola
odorata, Viola tricolor, Viola arvensis, Viola
baoshanensis, Viola yedoensis and Viola biflora)
and one Rubiaceae species (O. anis). Thirty-
four cyclotides have been isolated from the
Rubiaceae family and 142 cyclotides from the
Violaceae family. Two cyclic proteins, MCoTI-I
and MCoTI-II, having the same structural
topology but with unrelated sequences to
other cyclotides, are found in the
Cucurbitaceae family (Momordica cochin-
chinensis) (Hernandez et al., 2000). Because of
their plant origin and cyclic cystine knot
motif we classify them as cyclotides, but they
are also referred to as cyclic knottins (Heitz
et al., 2001).
CyBase is a database established to
Fig. 3.8. The three-dimensional structure and
sequence of the prototypical cyclotide kalata B1. catalogue the sequences and structures of
Cyclotides are characterized by a cyclic cystine naturally occurring and engineered cyclic
knot motif, which includes a head-to-tail cyclic proteins, including cyclotides (Wang et al.,
backbone and knotted arrangement of three 2008a). It currently provides information on
cystines. The cystine side chains are represented 158 dierent sequences of wild-type cyclo-
in a ball and stick format in the structure and the tides. Figure 3.9 shows a screenshot of a web
disulfide connectivities are shown by black thick page from this database.
lines above the sequence. The half-cystines are An inspection of all the sequences
numbered using Roman numerals. Kalata B1 has a
currently in CyBase shows that cyclotides
small -sheet, represented by arrows on the
range from 27 to 37 amino acids in size. They
structure.
52 Q. Kaas et al.
Fig. 3.9. Alignment of cyclotide sequences carried out with CyBase. This database incorporates specific
tools for the study of cyclic proteins, including the alignment of cyclotide sequences, as shown here. The
cysteine residues of the cyclotide cystine knot motif are aligned and gaps are inserted into the middle of
the inter-cysteine regions. The vertical menu on the left provides access to search pages, lists and tools
of cyclic protein and nucleic acid sequences.
invariably contain six cysteines forming the its conformation is relatively conserved (Fig.
three disulfide bonds that constitute their 3.10) despite a high level of sequence
characteristic cystine knot motif, as shown in variability (Fig. 3.11). Loop 6 accommodates
an overlay of structures in Fig. 3.10 (Craik et the longest loop sequence seen so far in
al., 1999). The inter-cysteine regions are cyclotides (i.e. ten amino acids). By contrast,
referred to as loops and have dierent ranges loops 1 and 4 are short and conserved in
of amino acid lengths and sequence length, with three and one amino acids,
variability (Fig. 3.11). The loops with the respectively. Some positions seem to be
greatest sequence diversity are loops 3, 5 and crucial for cyclotide folding, including the
6. Loop 3 is particularly interesting because cysteines and a glutamate in the second
AMPs in Plants 53
Fig. 3.10. Overlay of cyclotide structures. Cystine side chains are represented in dark grey and the
cyclotide backbones are in light grey, except for the backbone of kalata B1, which is in black. The cystine
knot motif is highly conserved and the cystine side chain positions align well. The conformations of loops
1, 2, 3 and 4 are highly conserved, whereas loops 5 and 6 are more variable. The structures shown are:
circulin A (Protein Data Bank access code: 1bh4), circulin B (2eri), cycloviolacin O1 (1nbj), cycloviolacin
O2 (2kcg), cycloviolacin O14 (2gj0), kalata B1 (1nb1), kalata B2 (1pt4), kalata B7 (2jwm), kalata B8
(2b38), palicourein (1r1f), tricyclon A (1yp8), varv peptide F (1k7g), vhl-1 (1za8) and vhr 1 (1vb8).
position of loop 1, which forms hydrogen family (Simonsen et al., 2005; Burman et al.,
bonds with the backbone amides of the first 2010), but their occurrence in Rubiaceae
two positions in loop 5 (Rosengren et al., species is more sparse, with <10% of
2003; Gransson et al., 2009). The penultimate examined species of this family expressing
position of loop 5 is also important as it is cyclotides (Gruber et al., 2008). The Violaceae
often occupied by a cis-proline, which confers and Rubiaceae belong to the monophyletic
a conceptual twist in loop 5, leading to the groups rosids and asterids, respectively
analogy of a Mbius strip (Craik et al., 1999). which are both part of the eudicot class. The
Depending on the presence or absence of this rosids and asterids are thought to have
cis-proline, cyclotides have been divided into diverged 100150 million years ago (Yang et
Mbius and bracelet structural subfamilies al., 1999). To explain the gaps in cyclotide
(Craik et al., 1999). The impact of sequence expression in the Rubiaceae and also the
variations on cyclotide activities is discussed occurrence of cyclotides in both the Violaceae
in Section 3.2.4. and Rubiaceae, it has been hypothesized that
Methods that detect cyclotide peptide the current cyclotide distribution results
expression in plants (see Section 3.2.2) and from convergent evolution in dierent plant
the isolation of transcripts using molecular families (Gruber et al., 2008). This hypothesis
biology techniques have facilitated an is supported by the fact that the enzymes
increase in knowledge on cyclotide diversity that appear to be responsible for the
(Simonsen et al., 2005; Gruber et al., 2008). cyclization process are ubiquitous in plants
Screening has been carried out in many (discussed in Section 3.2.3). In support of the
tissues from hundreds of plant species convergent evolution hypothesis, cyclotide-
(Simonsen et al., 2005; Gruber et al., 2008) and like sequences have been detected in the
it is clear that cyclotides are present in all Poaceae family (grass), which includes
parts of plants. Cyclotides appear to be important cereal crop species such as wheat,
expressed in all species from the Violaceae maize and rice (Mulvenna et al., 2006b).
54 Q. Kaas et al.
Fig. 3.11. Sequence variability of cyclotide loops. Each loop is identified on the three-dimensional
structure at the centre of the figure. The number of different sequences is indicated. For loops 2, 3, 5 and
6, a bar graph represents the number of loops with a given length. For a given loop length, the loops
having identical sequences are counted independently. Therefore, the total number of sequences
represented in the graph is 158, which is the total number of cyclotide sequences in CyBase. Loops 1
and 4 do not vary in length, and their length and number of different sequences are provided as text.
pro-region and one or several repeats, each an aspartate at the position usually occupied
comprising an N-terminal repeat, a cyclotide by the conserved asparagine. AEP has
domain and a C-terminal region (CTR) (Kaas reduced catalytic activity at an aspartate
and Craik, 2010). The structure of the O. residue (Mntz et al., 2002) and, consequently,
anis kalata B2 precursor is shown in Fig. other enzymes might be involved in cyclotide
3.12. backbone cyclization. Interestingly, during a
An asparagine endopeptidase (AEP) has small-scale expressed sequence tag project, a
been shown to be involved in the backbone transcript coding for an asparaginase was
cyclization of cyclotides (Saska et al., 2007; isolated from O. anis (Qin et al., 2010).
Gillon et al., 2008). This enzyme cleaves the Asparaginases catalyse the hydrolysis of
peptide bond after a conserved asparagine asparagine into aspartate. Therefore, the
that occupies the last position of the cyclotide unusual aspartate could have been formed
domain. It has been postulated that before by post-translational modification of the
cleavage occurs, the first positions of the CTR conserved asparagine after cyclization (Qin
could bind in pockets on the surface of AEP et al., 2010).
(Gillon et al., 2008). After cleavage and the A protein disulfide isomerase (PDI) from
release of the CTR, the first positions of the O. anis has been shown to interact with a
cyclotide domain, which have similar linear kalata B1 precursor protein and
sequences to the first positions of the CTR, improve its oxidative folding (Gruber et al.,
occupy the pockets. The N- and C-termini of 2007b). In the absence of the PDI, a stable
the peptide would then be in close proximity intermediate in the folding pathway of kalata
and could be ligated by AEP, therefore B1 in vitro does not lead directly to the native
cyclizing the protein (Gillon et al., 2008). product and requires disulfide shuing for
Twenty-three of the 158 cyclotide eventual formation of the cystine knot (Daly
sequences currently recorded in CyBase have et al., 2003). Thus, PDI activity in vivo might
Fig. 3.12. Oldenlandia affinis kalata B2 precursor. The precursor comprises an endoplasmic reticulum
(ER) signal sequence (dark grey background), a pro-region (white background) and three N-terminal
repeats (NTRs, light grey background), three kalata B2 domains (black background) and three C-terminal
regions (CTRs, diagonal stripes). An NTR, a domain and a CTR consecutive in the sequence form a
repeat unit. At the bottom of the figure, the three repeats in the kalata B2 precursor are aligned and the
positions presenting sequence variability are on a grey background. Whereas the NTR is highly
conserved between repeats, the CTR is more variable. The three first positions of the CTR (underlined in
the last repeat) have been identified as important for the enzymatic cyclization process and are
homologous to the first three amino acids of the mature domain (also underlined). The last position of the
domain is usually an asparagine but sometimes an aspartate, as is the case for kalata B2. This last
position and the second position of the CTR are crucial for enzymatic cyclization to occur and are marked
with a black background.
56 Q. Kaas et al.
be important to prevent a significant number Helicoverpa are among the most polyphagous
of cyclotides being trapped into intermediates and cosmopolitan pests.
with non-native disulfide bonds. Moreover, Cyclotides also have potent activity
similarities between hydrophobic-solvent- against Haemonchus contortus and Tricho-
assisted folding and folding using O. anis strongylus colubriformis, two economically
PDI suggest that the chaperone activity of important gastrointestinal nematode para-
PDI might also improve the folding of sites of livestock. This shows that the
cyclotides (Gruber et al., 2007b). Cyclotides pesticidal properties of cyclotides are not
have a hydrophobic patch on their surface specific to Helicoverpa (Colgrave et al., 2008).
that might interact with a hydrophobic Cyclotides also have molluscicidal activity
domain of the PDI. The cyclotide hydro- against Pomacea canaliculata, a serious pest of
phobic patch has also been shown to be rice in South-east Asia, with a comparable
important for the binding of cyclotides to potency to a commercial pesticide (Plan et al.,
dodecylphosphocholine micelles, used as 2008). P. canaliculata causes billions of dollars
model membranes (Shenkarev et al., 2006, worth of damage on rice plantations each
2008; Wang et al., 2009). year (Sin, 2003) and there may be a role for
cyclotides as a novel class of molluscicidal
agents.
Cyclotides seem to act by a mechanism
3.2.4 Biological activities of cyclotides
that aects cell membrane integrity. Upon
ingestion of kalata B1, disruption of the gut
Cyclotides first came to notice for their
membrane of caterpillars has been observed
uterotonic activity employed in a native
by light scanning microscopy and trans-
medicine application (Gran, 1970), but they
mission electron microscopy (Barbeta et al.,
have a range of other bioactivities including
2008). The integrity of a hydrophobic patch
haemolytic (Daly et al., 1999), cardiotoxic
on the surface of the kalata B1 structure was
(Gran, 1973b), antitumour (Herrmann et al.,
found to be important not only for
2008), antifungal (Tam et al., 1999), anthel-
insecticidal activity, but also for membrane-
mintic (Colgrave et al., 2009), anti-HIV
binding anity, as revealed by alanine-
(Gustafson et al., 1994, 2004; Daly et al., 2006;
scanning mutagenesis (Simonsen et al., 2008;
Wang et al., 2008b) and, reportedly, anti-
Huang et al., 2009). This suggests that the
bacterial activities (Tam et al., 1999), as well
membrane-binding and insecticidal activities
as inhibition of trypsin (Hernandez et al.,
of cyclotides are correlated.
2000) and neurotensin (Witherup et al., 1994).
In this section, we focus on their purported
antimicrobial properties. First, however, we Anti-HIV activity
give a brief overview of their pesticidal
Initially two cyclotides, circulin A and
activities, because we believe that this host-
circulin B, were discovered in the course of
defence activity is the primary biological
screening for anti-HIV natural products in a
function of cyclotides.
drug discovery programme at the US
National Cancer Institute (Gustafson et al.,
1994). In the anti-HIV assay, plant extracts
Pesticidal activity
were added to cultured T cells and
The natural function of cyclotides appears to subsequently exposed to infectious virus.
be to defend their host plants from insect The number of surviving cells was quantified
pests (Jennings et al., 2001, 2005; Gruber et al., after 6 days of incubation and compared
2007a; Craik, 2009; Daly et al., 2009). Kalata with uninfected cells and untreated cells
B1 is a potent inhibitor of the growth and (Gustafson et al., 2004). With this protocol,
development of Helicoverpa species (Jennings compounds that inhibit early steps of the
et al., 2001), moths whose larvae feed on a HIV infection could be detected. In the
wide array of plants, including a range of primary anti-HIV screen, extracts from
agricultural plants (maize and cotton). Chassalia parvifolia were found to be active,
AMPs in Plants 57
and circulin A and circulin B were identified insights in the antiviral mode of action of
as the bioactive compounds (Gustafson et al., cyclotides.
2004). Following this study, several other The natural cyclotides tested so far for
native cyclotides were reported to have anti- their anti-HIV activity have a low in vitro
HIV activity (Daly et al., 2004, 2006; therapeutic index (i.e. the ratio of their
Gustafson et al., 2004; Chen et al., 2005; therapeutic eects to toxic eects) (Gustafson
Ireland et al., 2008; Wang et al., 2008b). et al., 1994), which has limited their pro-
The anti-HIV activity of cyclotides is gression to the clinic as candidates for anti-
interesting, but the mechanism of action is HIV therapeutics. Nevertheless, other natural
still unclear. It has been proposed that cyclotides have been shown to possess higher
cyclotides could prevent HIV from inter- therapeutic indices (i.e. 44 for cycloviolacin
acting or fusing with host cells (Henriques Y5) (Wang et al., 2008b), thus reviving the
and Craik, 2010). For ecient viral infection, possibility of cyclotide-based anti-HIV drugs.
viruses must deliver their genomes into the The possibility of decreasing the toxic eects
interior of target cells. Before virus entry into of cyclotides by a single mutation (Simonsen
the cell, the HIV infection process is initiated et al., 2008; Huang et al., 2009), together with
by HIV receptor recognition at the surface of a more complete understanding of the
host cells, followed by fusion of the viral and mechanism of action, might accelerate the
host cell membranes (Cooley and Lewin, process.
2003). The cytoprotective eect of cyclotides
has been reported to be associated with a
Antibacterial and antifungal activity
decreased level of infectious virions
(Gustafson et al., 1994), whereas no eect on Cyclotides have distinct hydrophobic and
HIV reverse transcriptase activity was hydrophilic patches (Fig. 3.13), which
detected (Gustafson et al., 2004). Such resemble the amphipathic nature of AMPs.
observations support the hypothesis that These properties led Tam et al. (1999) to
cyclotides exert their eect before the entry of hypothesize that cyclotides might exhibit
the virus into the cell, inhibiting membrane antimicrobial activity. Four cyclotides were
targeting and/or membrane fusion. tested against selected bacteria and fungi,
Cyclotide bioactivities seem to correlate and selective antimicrobial activity was
with membrane-binding properties, as reported, as summarized in Table 3.2.
supported by biophysical studies with model Although low micromolar minimal inhibitory
membranes (Shenkarev et al., 2006, 2008; concentrations (MICs) were reported for
Huang et al., 2009; Wang et al., 2009) and by some microbes (e.g. the MIC against
cell membrane disruption observed in the Staphylococcus aureus was 0.26 M in the
insecticidal studies referred to above (Barbeta absence of salt), the antimicrobial activity
et al., 2008). As a hydrophobic patch has been was ablated in the presence of physiological
found to be crucial for the interactions with salt concentrations. Salt-dependent activity is
membranes (Huang et al., 2009), it is reason- often seen for AMPs, as high salt levels can
able to propose that cyclotides modulate aect electrostatic interactions with bacterial
their protective eect by a membrane- membranes (Bals et al., 1998; Wei et al., 2007).
binding mechanism inhibiting HIV entry into Currently, is not possible to determine if an
the cell (Henriques and Craik, 2010). increase in antimicrobial activity correlates
Specifically, anti-HIV eciency shows a with increased electrostatic attractions
correlation with the extent of a hydrophobic between cyclotides and bacterial membranes,
patch on the surface of cyclotides (Ireland et as the eect of salt concentration on cyclotide
al., 2008). Nevertheless, at this stage it is membrane anity has not been reported.
premature to hypothesize that cyclotides Nevertheless, a lack of activity at physio-
exert their eects by interacting with either logical conditions brings into question the
the HIV membrane or the host cell antimicrobial value of the cyclotides tested,
membrane, or with both. Investigation of probably accounting for the fact that since
direct virucidal activity would give more the study in 1999, no follow-up has been
58 Q. Kaas et al.
Fig. 3.13. Surface characteristics and charges of wild-type cyclotides with known structure. The surfaces
of all known wild-type cyclotides are represented by two views that differ by a rotation of 180 along the
vertical axis. Structures located below each other have similar orientations (alignment of the cystine knot).
The surfaces are darkened according to the properties of the amino acid under the surface: hydrophobic
(white), hydrophilic but not charged (grey) and charged (black). The charge of the amino acids is shown
on the surface. The number of positive and negative charges is given in columns 4 and 5, respectively,
and the total charge of the cyclotide is provided in the last column. Protein Database numbers are shown
between the structures.
AMPs in Plants 59
Organism Salt No salt Salt No salt Salt No salt Salt No salt Salt No salt Salt No salt Salt No salt
Gram-negative
Escherichia coli >500 >500 >500 0.41 >500 1.55 >500c >500 9 9 9 9
Pseudomonas
>500 >500 48.0 25.5 50.2 13.5 >500 >500
aeruginosa
Proteus vulgaris >500 54.6 >500 6.80 >500 13.2 >500 >500
Klebsiella oxytoca >500 >500 15.6 8.20 13.2 5.80 >500 54.8
Q. Kaas et al.
Haemophilus
influenzae
Gram-positive
Staphylococcus
>500 0.19 >500 13.5 >500 39.0 >500 0.26
aureus
Micrococcus luteus >500 >500 >500 >500 >500 48.0 >500 40.4
Fungi
Candida kefyr >500 18.6 >500 29.0 48.0 14.0 >500 21.4
Candida tropicalis >500 19.4 >500 >500 >500 56.5 >500 >500
Candida albicans >500 >500 >500 >500 >500 >500 >500 >500
a Minimum inhibitory concentrations (MICs) were determined after testing seven concentrations of peptide in a radial diffusion assay with underlay gel containing 1% agarose and
10 mM phosphate buffer without salt (no salt) or with 100 mM NaCl (salt); 5 l of each concentration was incubated at 37C for 3 h +1624 h. The bacteria clear zones were
measured and MICs were determined from the doseresponse curves (Tam et al., 1999).
b The antibiotic effect on Escherichia coli was tested after addition of 10 l of 5 mg ml1 peptide stock solution to agar plates; peptides were incubated at 37C for 3 days. Antibacterial
effects on Staphylococcus aureus and Haemophilus influenzae were evaluated by adding peptide directly to the growth medium. Peptide concentrations of 2, 4, 8 or 16 mg l1 were
incorporated and plates were inoculated with either S. aureus or H. influenzae and incubated at 37C for 1 day (Gran et al., 2008).
c Grey shading highlights contradictory results between the two independent studies.
AMPs in Plants 61
published apart from one recent report (Gran (Giangaspero et al., 2001). Although
et al., 2008). The results obtained by the only cyclotides have distinct hydrophobic and
two reports on the antibacterial activities of hydrophilic patches, most are not highly
cyclotides are compared in Table 3.2. positively charged; the total charge is close to
Kalata B1 is the only cyclotide that is zero and most of the currently known
common to both studies and contradictory cyclotides have a charge between 1 and +2
results were obtained. Tam et al. (1999) (see Figs 3.13 and 3.14). In addition, kalata B1
reported that kalata B1 is inactive against does not have a preference for negatively
Escherichia coli and active against Staph. charged membranes at physiological ionic
aureus, whereas Gran et al. (2008) reported strength (Huang et al., 2009). Overall, cyclo-
the opposite. In these two studies, activity tides have a poor profile as antibacterial
was assessed using dierent experimental peptides.
set-ups. Tam et al. incubated the bacterial Some cyclotides have a global charge of
strains with dierent concentrations of kalata +3 (Fig. 3.14), but they have not yet been
B1 and determined the MIC in a radial tested for their antimicrobial properties. The
diusion assay, and concluded that kalata B1 limited studies on the antibacterial activity of
is inactive against E. coli (MIC >500 M in the cyclotides make it dicult to judge the
presence or absence of salt). On the other potential applications of native cyclotides as
hand, Gran et al. concluded that kalata B1 antimicrobial drugs; however, many more
was active against E. coli based on the eect cyclotides wait to be discovered and new
of 10 l of 5 mg ml1 kalata B1 (approximately examples might reveal dierent char-
1700 M) added to the agar plate. The very acteristics, including higher charge and
high concentration used in the Gran et al. potentially stronger antimicrobial activities.
study might explain the dierence of activity The cyclotides so far reported have
observed for E. coli between the two studies. revealed the remarkable plasticity of the
Regarding the results obtained against cyclotide framework. Their tolerance for
Staph. aureus, Tam et al. concluded that kalata substitution suggests that cyclotides can be
B1 was active against Staph. aureus as the used as a scaold and engineered to include
MIC was found to be 0.26 M in conditions a foreign sequence with a desired activity
without salt, but inactive in conditions with (Craik et al., 2006a). As already mentioned,
salt (MIC >500 M). In the study by Gran et
al., a lack of activity against Staph. aureus was
concluded based on incorporation of kalata
B1 into the bacterial growth medium,
whereby 16 mg ml1 of kalata B1 (approxi-
mately 5.5 M) did not aect bacterial
growth. The reasons for the dierences in the
two studies are not fully understood.
Classic antibacterial peptides selectively
target negatively charged bacterial mem-
branes over neutral mammalian cells and kill
microbes through a membrane disruption
mechanism (Yeaman and Yount, 2003). An
amphipathic structure, with well-defined
and distinct hydrophobic and positively
charged patches, has been identified as a
determinant for bacterial membrane target-
ing by antibacterial peptides (Dathe et al., Fig. 3.14. Total charge of cyclotides based on 158
1997). The most frequent global charge cyclotide sequences in CyBase. Arginines and
identified in classical antibacterial peptides is lysines are positively charged, while aspartates
+5 or +6, emphasizing the importance of the and glutamates are negatively charged. Histidines
net positive charge for antimicrobial activity are taken as neutral.
62 Q. Kaas et al.
the bioactivities of cyclotides seem to broadly and thus optimization is sometimes required
correlate with membrane-binding anity. to find the most appropriate combination of
Whereas the disruption of the hydrophobic grafted sequences and sites (Gunasekera et
patch in kalata B1 leads to loss of function al., 2008). Studies investigating the tolerance
and a decrease in membrane-binding proper- of the cyclotide framework to substitution
ties (Huang et al., 2009, 2010), the insertion of and grafting have been recently reviewed
positive charges in selected locations (Henriques and Craik, 2010).
increases bioactivity and membrane leakage The next step towards the development
properties (Huang et al., 2010). These of cyclotides as pharmaceuticals will be to
properties suggest that cyclotides can be discover or design examples that have high
modulated so that toxic properties can be potencies and selectivities for proven
eliminated and antimicrobial activity therapeutic targets. Assuming that such
improved. In this sense, cyclotide frame- results are forthcoming in the near future, it
works with their circular structure and will then be necessary to achieve high-yield
high resistance to thermal, enzymatic and production for scale-up testing. Unfortu-
chemical degradation are a potential new nately, the plant-based production of
class of antibiotics that could be designed to engineered cyclotides is problematic as
have both high stability and membrane- transgenic plants have so far only produced
disrupting properties. low yields (Saska et al., 2007; Gillon et al.,
2008). The most probable explanation for the
low yields is that the transgenic plants lack
3.2.5 Cyclotide engineering and mass optimized enzymes required for the correct
production processing of cyclotides. At least three
alternative cyclotide production strategies
Despite being rapidly degraded by proteases have been attempted, including solid-phase
in vivo and having generally low stability, synthetic chemistry, production in bacteria
peptides have generated much interest in and plant cell culture.
drug design because they bind to their Solid-phase peptide synthesis is a
molecular targets with high anity and classical and straightforward method to
specificity. The cyclotide cyclic cystine knot produce peptides, but it has a high cost that
motif confers high resistance to dierent is incompatible with mass production. A
kinds of denaturants and can potentially biomimetic strategy based on chemoenzym-
overcome the stability and degradation atic cyclization by an immobilized protease
problems of peptides (Craik et al., 2006a, has been developed to complement this
2009; Henriques and Craik, 2010). The fact method (Thongyoo et al., 2007), and has
that a range of loop sizes is tolerated by the shown improved yields over the usual native
cyclic cystine knot framework suggests that chemical ligation/cyclization reaction. More
cyclotides are potentially amenable to recently, Austin et al. (2009) developed an
substitution by a range of foreign epitopes in ingenious strategy to biosynthesize a
protein engineering and drug design studies cyclotide-based library in E. coli cells. E. coli
(Craik et al., 2006b). Indeed, cyclotides may lacks the required enzymes to cyclize
be thought of as a natural combinatorial proteins, but an alternative approach based
library that is expressed on an ultra-stable on a modified protein-splicing unit, or intein,
structural scaold (Craik et al., 2001). As was put into practice and directly employed
explained previously, loops 2, 3, 5 and 6 are inside living E. coli cells (Fig. 3.15). This
the most naturally variable and are the method generally produced good yields of
preferential sites for grafting biologically cyclic proteins.
active epitopes. Because there is a degree of Plant cell-culture technology is a
cooperatively between sequences in the promising strategy because cyclotides can be
dierent loops of the cyclotide scaold (Daly directly cultivated in cells from cyclotide-
et al., 2006; Gunasekera et al., 2009), not all producing plants (Drnenburg, 2009). This
grafted peptides can be successfully folded should overcome the limitations of cyclotide
AMPs in Plants 63
Fig. 3.15. The cyclization strategy used by Austin et al. (2009) to produce cyclotides in Escherichia coli.
An intein was used as a protein-splicing unit and the last step of cyclization was performed by an
intramolecular native chemical ligation reaction. A cellulose-binding domain (CBD) was fused at the
C-terminal end of the intein region. Figure adapted from Austin et al. (2009). MCoTI-I, Momordica
cochinchinensis trypsin inhibitor I.
production in model plants such as tobacco. laboratory are focused primarily on one class
Figure 3.16 illustrates cyclotide production of these molecules, the cyclotides, the
using this approach. principal natural function of which is
thought to be as insecticidal agents. In
addition to this presumed native function,
3.3 Concluding Remarks and however, cyclotides have been reported to
Perspectives have a variety of antimicrobial properties.
Most extensively studied have been their
As is clear from this chapter, plants produce antiviral properties, with a large number of
a variety of structurally diverse peptides that cyclotides showing significant activity
they use for defence purposes against against HIV. Although they have promising
microbes and other pests. Studies in our potencies, their therapeutic index is not at
64 Q. Kaas et al.
Fig. 3.16. Oldenlandia affinis cell culture growth and production kinetics of the batch cultivation process.
Kalata B1 accumulation is maximized after 7 days, corresponding to the beginning of the stationary
phase. DW, dry weight. Figure adapted from Drnenburg (2009).
present suciently high to justify their One of the major unsolved problems in
clinical use as antiviral agents in humans. cyclotide research is the mechanistic basis of
Nevertheless, with a large number of their action. They have an impressive array
cyclotides predicted to be discovered over of biological activities and it would be
the coming years, it is possible that natural simplistic to imagine that all of these
examples with better therapeutic indices may activities can be accounted for by a single
be found. Furthermore, with several routes mechanism. However, if there is a common
to their synthesis available, the design of theme, then membrane binding appears to
synthetic analogues with improved potency be a central player. Although it is equivocally
and reduced toxicity is a promising area for established that cyclotides bind to
future investigation. membranes and some aspects of their
Cyclotides have been widely reported in binding have been delineated, the way in
the review literature to have antibacterial which they form pores in membranes
activity. However, as we have indicated here, remains a puzzle. Another mystery is how
a careful examination of the literature shows plants themselves are unaected by the
that only two original research papers have membrane-disrupting properties of cyclo-
reported this activity and to an extent they tides. Cyclotides are probably stored inside
present conflicting results. Thus, there is a membrane-containing organelles within
question about the significance and relevance plants, yet they do not harm the producing
of the reported antibacterial activity of plants. Thus, the systematic study of the
cyclotides. This is an area that needs further interactions of cyclotides with a range of
investigation and, in particular, pathogens dierent plant membranes is an important
relevant to plants should be examined. The area of investigation.
studies so far have focused on human Finally, it will be of interest to determine
pathogenic bacteria, rather than on bacteria why individual plants produce so many
that might be typically encountered by cyclotides in some cases, up to 100
plants. cyclotides per plant. The variation from one
AMPs in Plants 65
cyclotide to another within a producing plant (UniProt). Nucleic Acids Research 33,
is often not particularly large and raises the D154-D159.
question of why a plant would devote a Balls, A.K., Hale, W.S. and Harris, T.H. (1942a) A
significant amount of its energy resources to crystalline protein obtained from a lipoprotein of
wheat flour. Cereal Chemistry 19, 279288.
producing a suite of similar peptides. Per-
Balls, A.K., Hale, W.S. and Harris, T.H. (1942b)
haps some of the questions raised here are Further observations on a crystalline wheat
related, and the pore-forming ability of protein. Cereal Chemistry 19, 840844.
cyclotides might involve a mix-and-match Bals, R., Wang, X., Zasloff, M. and Wilson, J.M.
interaction between dierent cyclotides. At (1998) The peptide antibiotic LL-37/hCAP-18 is
this stage this is speculation, but it is an expressed in epithelia of the human lung where
interesting area for future studies. The it has broad antimicrobial activity at the airway
studies of membrane binding of cyclotides surface, Proceedings of the National Academy
reported so far show that purified individual of Sciences of the USA 95, 95419546.
cyclotides can certainly disrupt membranes, Balzarini, J. (2006) Inhibition of HIV entry by
carbohydrate-binding proteins. Antiviral
but we do not yet know how mixtures of
Research 71, 237247.
cyclotides, as occurs naturally in plants,
Barbeta, B.L., Marshall, A.T., Gillon, A.D., Craik,
might aect membranes. D.J. and Anderson, M.A. (2008) Plant cyclotides
disrupt epithelial cells in the midgut of
lepidopteran larvae. Proceedings of the National
Academy of Sciences of the USA 105, 1221
References 1225.
Bohlmann, H. and Apel, K. (1991) Thionins. Annual
Aerts, A.M., Francois, I.E., Meert, E.M., Li, Q.T., Review of Plant Physiology and Plant Molecular
Cammue, B.P. and Thevissen, K. (2007) The Biology 42, 227240.
antifungal activity of RsAFP2, a plant defensin Broekaert, W.F., Marien, W., Terras, F.R., De Bolle,
from Raphanus sativus, involves the induction M.F., Proost, P., Van Damme, J., Dillen, L.,
of reactive oxygen species in Candida albicans. Claeys, M., Rees, S.B., Vanderleyden, J. and
Journal of Molecular Microbiology and Cammue, B.P. (1992) Antimicrobial peptides
Biotechnology 13, 243247. from Amaranthus caudatus seeds with
Almeida, M.S., Cabral, K.M., Kurtenbach, E., sequence homology to the cysteine/glycine-rich
Almeida, F.C. and Valente, A.P. (2002) Solution domain of chitin-binding proteins. Biochemistry
structure of Pisum sativum defensin 1 by high 31, 43084314.
resolution NMR: plant defensins, identical Broekaert, W.F., Cammue, B.P.A., De Bolle, M.F.C.,
backbone with different mechanisms of action. Thevissen, K., De Samblanx, G.W. and Osborn,
Journal of Molecular Biology 315, 749757. R.W. (1997) Antimicrobial peptides from plants.
Andersen, N.H., Cao, B., Rodriguez-Romero, A. Critical Reviews in Plant Sciences 16, 297
and Arreguin, B. (1993) Hevein: NMR assign- 323.
ment and assessment of solution-state folding Burman, R., Gruber, C.W., Rizzardi, K., Herrmann,
for the agglutinin-toxin motif. Biochemistry 32, A., Craik, D.J., Gupta, M.P. and Gransson, U.
14071422. (2010) Cyclotide proteins and precursors from
Andr, S., Kozr, T., Kojima, S., Unverzagt, C. and the genus Gloeospermum: filling a blank spot in
Gabius, H.-J. (2009) From structural to functional the cyclotide map of Violaceae. Phytochemistry
glycomics: core substitutions as molecular 71, 1320.
switches for shape and lectin affinity of Carmona, M.J., Molina, A., Fernandez, J.A., Lopez-
N-glycans. Biological Chemistry 390, 557565. Fando, J.J. and Garcia-Olmedo, F. (1993)
Austin, J., Wang, W., Puttamadappa, S., Shekhtman, Expression of the alpha-thionin gene from
A. and Camarero, J.A. (2009) Biosynthesis and barley in tobacco confers enhanced resistance
biological screening of a genetically encoded to bacterial pathogens. The Plant Journal 3,
library based on the cyclotide MCoTI-I. 457462.
Chembiochem 10, 26632670. Carvalho, A.D. and Gomes, V.M. (2009) Plant
Bairoch, A., Apweiler, R., Wu, C.H., Barker, W.C., defensins prospects for the biological functions
Boeckmann, B., Ferro, S., Gasteiger, E., Huang, and biotechnological properties. Peptides 30,
H., Lopez, R., Magrane, M., Martin, M.J., Natale, 10071020.
D.A., ODonovan, C., Redaschi, N. and Yeh, L.S. Chan, Y.L., Prasad, V., Sanjaya, Chen, K.H., Liu,
(2005) The Universal Protein Resource P.C., Chan, M.T. and Cheng, C.P. (2005)
66 Q. Kaas et al.
Transgenic tomato plants expressing an Craik, D.J., Daly, N.L. and Waine, C. (2001) The
Arabidopsis thionin (Thi2.1) driven by fruit- cystine knot motif in toxins and implications for
inactive promoter battle against phytopathogenic drug design. Toxicon: Official Journal of the
attack. Planta 221, 386393. International Society on Toxinology 39, 4360.
Chavez, M.I., Andreu, C., Vidal, P., Aboitiz, N., Craik, D.J., Cemazar, M. and Daly, N.L. (2006a)
Freire, F., Groves, P., Asensio, J.L., Asensio, G., The cyclotides and related macrocyclic peptides
Muraki, M., Canada, F.J. and Jimenez-Barbero, as scaffolds in drug design. Current Opinion in
J. (2005) On the importance of carbohydrate- Drug Discovery and Development 9, 251260.
aromatic interactions for the molecular Craik, D.J., Cemazar, M., Wang, C.K.L. and Daly,
recognition of oligosaccharides by proteins: N.L. (2006b) The cyclotide family of circular
NMR studies of the structure and binding affinity miniproteins: natures combinatorial peptide
of AcAMP2-like peptides with non-natural template. Biopolymers 84, 250266.
naphthyl and fluoroaromatic residues. Chemistry Craik, D.J., Mylne, J.S. and Daly, N.L. (2009)
11, 70607074. Cyclotides: macrocyclic peptides with
Chen, B., Colgrave, M.L., Daly, N.L., Rosengren, applications in drug design and agriculture.
K.J., Gustafson, K.R. and Craik, D.J. (2005) Cellular and Molecular Life Sciences 67, 916.
Isolation and characterization of novel cyclotides da Rocha Pitta, M.G. and Galdino, S.L. (2010)
from Viola hederaceae: solution structure and Development of novel therapeutic drugs in
anti-HIV activity of vhl-1, a leaf-specific humans from plant antimicrobial peptides.
expressed cyclotide. Journal of Biological Current Protein and Peptide Science 11, 236
Chemistry 280, 2239522405. 247.
Colgrave, M.L. and Craik, D.J. (2004) Thermal, Daly, N.L., Love, S., Alewood, P.F. and Craik, D.J.
chemical, and enzymatic stability of the cyclotide (1999) Chemical synthesis and folding pathways
kalata B1: the importance of the cyclic cystine of large cyclic polypeptides: studies of the
knot. Biochemistry 43, 59655975. cystine knot polypeptide kalata B1. Biochemistry
Colgrave, M.L., Jones, A. and Craik, D.J. (2005) 38, 1060610614.
Peptide quantification by matrix-assisted laser Daly, N.L., Clark, R.J. and Craik, D.J. (2003)
desorption ionisation time-of-flight mass Disulfide folding pathways of cystine knot
spectrometry: investigations of the cyclotide proteins. Tying the knot within the circular
kalata B1 in biological fluids. Journal of backbone of the cyclotides. Journal of Biological
Chromatography A 1091, 187193. Chemistry 278, 63146322.
Colgrave, M.L., Kotze, A.C., Huang, Y.H., OGrady, Daly, N.L., Gustafson, K.R. and Craik, D.J. (2004)
J., Simonsen, S.M. and Craik, D.J. (2008) The role of the cyclic peptide backbone in the
Cyclotides: natural, circular plant peptides that anti-HIV activity of the cyclotide kalata B1. FEBS
possess significant activity against Letters 574, 6972.
gastrointestinal nematode parasites of sheep. Daly, N.L., Clark, R.J., Plan, M.R. and Craik, D.J.
Biochemistry 47, 55815589. (2006) Kalata B8, a novel antiviral circular
Colgrave, M.L., Kotze, A.C., Kopp, S., McCarthy, protein, exhibits conformational flexibility in the
J.S., Coleman, G.T. and Craik, D.J. (2009) cystine knot motif. Biochemical Journal 393,
Anthelmintic activity of cyclotides: in vitro studies 619626.
with canine and human hookworms. Acta Daly, N.L., Rosengren, K.J. and Craik, D.J. (2009)
Tropica 109, 163166. Discovery, structure and biological activities of
Colilla, F.J., Rocher, A. and Mendez, E. (1990) cyclotides. Advanced Drug Delivery Reviews
Gamma-purothionins: amino acid sequence of 61, 918930.
two polypeptides of a new family of thionins Dathe, M., Wieprecht, T., Nikolenko, H., Handel, L.,
from wheat endosperm. FEBS Letters 270, Maloy, W.L., MacDonald, D.L., Beyermann, M.
191194. and Bienert, M. (1997) Hydrophobicity,
Cooley, L.A. and Lewin, S.R. (2003) HIV-1 cell entry hydrophobic moment and angle subtended by
and advances in viral entry inhibitor therapy, charged residues modulate antibacterial and
Journal of Clinical Virology 26, 121132. haemolytic activity of amphipathic helical
Craik, D.J. (2009) Circling the enemy: cyclic proteins peptides. FEBS Letters 403, 208212.
in plant defence. Trends in Plant Science 14, de Medeiros, L.N., Angeli, R., Sarzedas, C.G.,
328335. Barreto-Bergter, E., Valente, A.P., Kurtenbach,
Craik, D.J., Daly, N.L., Bond, T. and Waine, C. E. and Almeida, F.C.L. (2010) Backbone
(1999) Plant cyclotides: a unique family of cyclic dynamics of the antifungal Psd1 pea defensin
and knotted proteins that defines the cyclic and its correlation with membrane interaction by
cystine knot structural motif. Journal of Molecular NMR spectroscopy. Biochimica et Biophysica
Biology 294, 13271336. Acta 1798, 105113.
AMPs in Plants 67
Debreczeni, J.E., Girmann, B., Zeeck, A., Kratzner, Gransson, U., Luijendijk, T., Johansson, S., Bohlin,
R. and Sheldrick, G.M. (2003) Structure of L. and Claeson, P. (1999) Seven novel macro-
viscotoxin A3: disulfide location from weak SAD cyclic polypeptides from Viola arvensis. Journal
data. Acta Crystallographica. Section D: of Natural Products 62, 283286.
Biological Crystallography 59, 21252132. Gransson, U., Herrmann, A., Burman, R.,
Drnenburg, H. (2009) Progress in kalata peptide Haugaard-Jnsson, L.M. and Rosengren, K.J.
production via plant cell bioprocessing. (2009) The conserved Glu in the cyclotide
Biotechnology Journal 4, 632645. cycloviolacin O2 has a key structural role.
Fant, F., Vranken, W., Broekaert, W. and Borremans, Chembiochem 10, 23542360.
F. (1998) Determination of the three-dimensional Gran, L. (1970) An oxytocic principle found in
solution structure of Raphanus sativus antifungal Oldenlandia affinis DC. Meddelelser fra Norsk
protein 1 by 1H NMR. Journal of Molecular Farmaceutisk Selskap 12, 173180.
Biology 279, 257270. Gran, L. (1973a) Isolation of oxytocic peptides from
Fernandez de Caleya, R., Gonzalez-Pascual, B., Oldenlandia affinis by solvent extraction of tetra-
Garcia-Olmedo, F. and Carbonero, P. (1972) phenylborate complexes and chromatography
Susceptibility of phytopathogenic bacteria to on sephadex LH-20. Lloydia 36, 207208.
wheat purothionins in vitro. Applied Microbiology Gran, L. (1973b) On the effect of a polypeptide
23, 9981000. isolated from Kalata-Kalata (Oldenlandia affinis
Fisher, N., Redman, D.G. and Elton, G.A.H. (1968) DC) on the oestrogen dominated uterus. Acta
Fractionation and characterization of purothionin. Pharmacologica et Toxicologica 33, 400408.
Cereal Chemistry 45, 4857. Gran, L., Sandberg, F. and Sletten, K. (2000)
Flinterman, A.E., Akkerdaas, J.H., Knulst, A.C., van Oldenlandia affinis (R&S) DC. A plant containing
Ree, R. and Pasmans, S.G. (2008) Hazelnut uteroactive peptides used in African traditional
allergy: from pollen-associated mild allergy to medicine. Journal of Ethnopharmacology 70,
severe anaphylactic reactions. Current Opinion 197203.
in Allergy and Clinical Immunology 8, 261265. Gran, L., Sletten, K. and Skjeldal, L. (2008) Cyclic
Gabius, H.-J., Siebert, H.-C., Andr, S., Jimnez- peptides from Oldenlandia affinis DC. Molecular
Barbero, J. and Rdiger, H. (2004) Chemical and biological properties. Chemistry &
biology of the sugar code. Chembiochem 5, Biodiversity 5, 20142022.
740764. Gruber, C.W., Cemazar, M., Anderson, M.A. and
Geldwerth, D., de Kermel, A., Zachowski, A., Craik, D.J. (2007a) Insecticidal plant cyclotides
Guerbette, F., Kader, J.C., Henry, J.P. and and related cystine knot toxins. Toxicon 49,
Devaux, P.F. (1991) Use of spin-labeled and 561575.
fluorescent lipids to study the activity of the Gruber, C.W., Cemazar, M., Clark, R.J., Horibe, T.,
phospholipid transfer protein from maize Renda, R.F., Anderson, M.A. and Craik, D.J.
seedlings. Biochimica et Biophysica Acta 1082, (2007b) A novel plant protein-disulfide
255264. isomerase involved in the oxidative folding of
Giangaspero, A., Sandri, L. and Tossi, A. (2001) cystine knot defence proteins. Journal of
Amphipathic alpha helical antimicrobial peptides. Biological Chemistry 282, 2043520446.
European Journal of Biochemistry 268, 5589 Gruber, C.W., Elliott, A.G., Ireland, D.C., Delprete,
5600. P.G., Dessein, S., Gransson, U., Trabi, M.,
Gillon, A.D., Saska, I., Jennings, C.V., Guarino, Wang, C.K., Kinghorn, A.B., Robbrecht, E. and
R.F., Craik, D.J. and Anderson, M.A. (2008) Craik, D.J. (2008) Distribution and evolution of
Biosynthesis of circular proteins in plants. The circular miniproteins in flowering plants. The
Plant Journal 53, 505515. Plant Cell 20, 24712483.
Gincel, E., Simorre, J.P., Caille, A., Marion, D., Ptak, Gunasekera, S., Foley, F.M., Clark, R.J., Sando, L.,
M. and Vovelle, F. (1994) Three-dimensional Fabri, L.J., Craik, D.J. and Daly, N.L. (2008)
structure in solution of a wheat lipid-transfer Engineering stabilized vascular endothelial
protein from multidimensional 1H-NMR data. A growth factor-A antagonists: synthesis, structural
new folding for lipid carriers. European Journal characterization, and bioactivity of grafted
of Biochemistry 226, 413422. analogues of cyclotides. Journal of Medicinal
Gomar, J., Petit, M.C., Sodano, P., Sy, D., Marion, Chemistry 51, 76977704.
D., Kader, J.C., Vovelle, F. and Ptak, M. (1996) Gunasekera, S., Daly, N., Clark, R. and Craik, D.J.
Solution structure and lipid binding of a (2009) Dissecting the oxidative folding of circular
nonspecific lipid transfer protein extracted from cystine knot miniproteins. Antioxidants & Redox
maize seeds. Protein Science 5, 565477. Signaling 11, 971980.
68 Q. Kaas et al.
Gustafson, K.R., Sowder, R.C. II, Henderson, L.E., cyclotide kalata B1 for the optimisation of
Parsons, I.C., Kashman, Y., Cardellina, J.H. II, nematocidal activity. Journal of Biological
McMahon, J.B., Buckheit, R.W. Jr, Pannell, L.K. Chemistry 285,1079710805.
and Boyd, M.R. (1994) Circulins A and B: novel Hughes, P., Dennis, E., Whitecross, M., Llewellyn,
HIV-inhibitory macrocyclic peptides from the D. and Gage, P. (2000) The cytotoxic plant
tropical tree Chassalia parvifolia. Journal of the protein, -purothionin, forms ion channels in
American Chemical Society 116, 93379338. lipid membranes. Journal of Biological Chemistry
Gustafson, K.R., McKee, T.C. and Bokesch, H.R. 275, 823827.
(2004) Anti-HIV cyclotides. Current Protein & Hutchinson, E.G. and Thornton, J.M. (1993) The
Peptide Science 5, 331340. Greek key motif: extraction, classification and
Hammami, R., Ben Hamida, J., Vergoten, G. and analysis. Protein Engineering 6, 233245.
Fliss, I. (2009) PhytAMP: a database dedicated Inomata, N. (2009) Wheat allergy. Current Opinion
to antimicrobial plant peptides. Nucleic Acids in Allergy and Clinical Immunology 9, 238243.
Research 37, D963D968. Ireland, D.C., Wang, C.K.L., Wilson, J.A., Gustafson,
Hancock, R.E. and Sahl, H.G. (2006) Antimicrobial K.R. and Craik, D.J. (2008) Cyclotides as natural
and host-defense peptides as new anti-infective anti-HIV agents. Biopolymers 90, 5160.
therapeutic strategies. Nature Biotechnology Jennings, C., West, J., Waine, C., Craik, D. and
24, 15511557. Anderson, M. (2001) Biosynthesis and
Hegedus, D., Erlandson, M., Gillott, C. and Toprak, insecticidal properties of plant cyclotides: the
U. (2009) New insights into peritrophic matrix cyclic knotted proteins from Oldenlandia affinis.
synthesis, architecture, and function. Annual Proceedings of the National Academy of
Review of Entomology 54, 285302. Sciences of the USA 98, 1061410619.
Heinemann, B., Andersen, K.V., Nielsen, P.R., Jennings, C.V., Rosengren, K.J., Daly, N.L., Plan,
Bech, L.M. and Poulsen, F.M. (1996) Structure M., Stevens, J., Scanlon, M.J., Waine, C.,
in solution of a four-helix lipid binding protein. Norman, D.G., Anderson, M.A. and Craik, D.J.
Protein Science 5, 1323. (2005) Isolation, solution structure, and
Heitz, A., Hernandez, J.F., Gagnon, J., Hong, T.T., insecticidal activity of kalata B2, a circular
Pham, T.T., Nguyen, T.M., Le-Nguyen, D. and protein with a twist: do Mbius strips exist in
Chiche, L. (2001) Solution structure of the nature? Biochemistry 44, 851860.
squash trypsin inhibitor MCoTI-II. A new family Jimenez-Barbero, J., Canada, F.J., Asensio, J.L.,
for cyclic knottins. Biochemistry 40, 79737983. Aboitiz, N., Vidal, P., Canales, A., Groves, P.,
Henriques, S.T. and Craik, D.J. (2010) Cyclotides Gabius, H.J. and Siebert, H.C. (2006) Hevein
as templates in drug design. Drug Discovery domains: an attractive model to study
Today 15, 5764. carbohydrateprotein interactions, at atomic
Hernandez, J.F., Gagnon, J., Chiche, L., Nguyen, resolution. Advances in Carbohydrate Chemistry
T.M., Andrieu, J.P., Heitz, A., Trinh Hong, T., and Biochemistry 60, 303354.
Pham, T.T. and Le Nguyen, D. (2000) Squash Joulli, M.M. and Richard, D.J. (2004) Cyclopeptide
trypsin inhibitors from Momordica alkaloids: chemistry and biology. Chemical
cochinchinensis exhibit an atypical macrocyclic Communications (Cambridge, England) 18,
structure. Biochemistry 39, 57225730. 20112015.
Herrmann, A., Burman, R., Mylne, J.S., Karlsson, Kaas, Q. and Craik, D.J. (2010) Analysis and
G., Gullbo, J., Craik, D.J., Clark, R.J. and classification of circular proteins in CyBase.
Gransson, U. (2008) The alpine violet, Viola Biopolymers Peptide Science 94, 584591.
biflora, is a rich source of cyclotides with potent Kessler, A. and Baldwin, I.T. (2002) Plant responses
cytotoxicity. Phytochemistry 69, 939952. to insect herbivory: the emerging molecular
Howe, G.A. and Jander, G. (2008) Plant immunity analysis. Annual Review of Plant Biology 53,
to insect herbivores. Annual Review of Plant 299328.
Biology 59, 4166. Koo, J.C., Lee, S.Y., Chun, H.J., Cheong, Y.H., Choi,
Huang, Y.H., Colgrave, M.L., Daly, N.L., Keleshian, J.S., Kawabata, S., Miyagi, M., Tsunasawa, S.,
A., Martinac, B. and Craik, D.J. (2009) The Ha, K.S., Bae, D.W., Han, C.D., Lee, B.L. and
biological activity of the prototypic cyclotide Cho, M.J. (1998) Two hevein homologs isolated
kalata B1 is modulated by the formation of from the seed of Pharbitis nil L. exhibit potent
multimeric pores. Journal of Biological Chemistry antifungal activity. Biochimica et Biophysica
284, 2069920707. Acta 1382, 8090.
Huang, Y.H., Colgrave, M.L., Clark, R.J., Kotze, Latge, J.P. (2007) The cell wall: a carbohydrate
A.C. and Craik, D.J. (2010) Lysine-scanning armour for the fungal cell. Molecular
mutagenesis reveals an amendable face of the Biotechnology 66, 279290.
AMPs in Plants 69
Lay, F.T. and Anderson, M.A. (2005) Defensins Molina, A., Segura, A. and Garcia-Olmedo, F.
components of the innate immune system in (1993) Lipid transfer proteins (nsLTPs) from
plants. Current Protein & Peptide Science 6, barley and maize leaves are potent inhibitors of
85101. bacterial and fungal plant pathogens. FEBS
Lerche, M.H. and Poulsen, F.M. (1998) Solution Letters 316, 119122.
structure of barley lipid transfer protein Montesinos, E. (2007) Antimicrobial peptides and
complexed with palmitate. Two different binding plant disease control. FEMS Microbiology
modes of palmitate in the homologous maize Letters 270, 111.
and barley nonspecific lipid transfer proteins. Morel, A.F., Maldaner, G., Ilha, V., Missau, F., Silva,
Protein Science 7, 24902498. U.F. and Dalcol, II (2005) Cyclopeptide alkaloids
Lerche, M.H., Kragelund, B.B., Bech, L.M. and from Scutia buxifolia Reiss and their antimicrobial
Poulsen, F.M. (1997) Barley lipid-transfer protein activity. Phytochemistry 66, 25712576.
complexed with palmitoyl CoA: the structure Mulvenna, J.P., Wang, C. and Craik, D.J. (2006a)
reveals a hydrophobic binding site that can CyBase: a database of cyclic protein sequence
expand to fit both large and small lipid-like and structure. Nucleic Acids Research 34,
ligands. Structure 5, 291306. D192D194.
Liu, B., Bian, H.-J. and Bao, J.-K. (2010) Plant Mulvenna, J.P., Mylne, J.S., Bharathi, R., Burton,
lectins: potential antineoplastic drugs from R.A., Shirley, N.J., Fincher, G.B., Anderson,
bench to clinic. Cancer Letters 287, 112. M.A. and Craik, D.J. (2006b) Discovery of
Lobo, D.S., Pereira, I.B., Fragel-Madeira, L., cyclotide-like protein sequences in graminaceous
Medeiros, L.N., Cabral, L.M., Faria, J., Bellio, crop plants: ancestral precursors of circular
M., Campos, R.C., Linden, R. and Kurtenbach, proteins? The Plant Cell 18, 21342144.
E. (2007) Antifungal Pisum sativum defensin 1 Mntz, K., Blattner, F.R. and Shutov, A.D. (2002)
interacts with Neurospora crassa cyclin F Legumains a family of asparagine-specific
related to the cell cycle. Biochemistry 46, 987
cysteine endopeptidases involved in
996.
propolypeptide processing and protein
Marcus, J.P., Goulter, K.C., Green, J.L., Harrison,
breakdown in plants. Journal of Plant Physiology
S.J. and Manners, J.M. (1997) Purification,
159, 12811293.
characterisation and cDNA cloning of an
Nieuwland, J., Feron, R., Huisman, B.A., Fasolino,
antimicrobial peptide from Macadamia
A., Hilbers, C.W., Derksen, J. and Mariani, C.
integrifolia. European Journal of Biochemistry
(2005) Lipid transfer proteins enhance cell wall
244, 743749.
extension in tobacco. The Plant Cell 17, 2009
Martins, J.C., Maes, D., Loris, R., Pepermans, H.A.,
2019.
Wyns, L., Willem, R. and Verheyden, P. (1996)
1H NMR study of the solution structure of OKeefe, B.R. (2001) Biologically active proteins
from natural product extracts. Journal of Natural
Ac-AMP2, a sugar binding antimicrobial protein
isolated from Amaranthus caudatus. Journal of Products 64, 13731381.
Molecular Biology 258, 322333. Parijs, J., Broekaert, W.F., Goldstein, I.J. and
McManus, A.M., Nielsen, K.J., Marcus, J.P., Peumans, W.J. (1991) Hevein: an antifungal
Harrison, S.J., Green, J.L., Manners, J.M. and protein from rubber-tree (Hevea brasiliensis)
Craik, D.J. (1999) MiAMP1, a novel protein from latex. Planta 183, 258264.
Macadamia integrifolia adopts a Greek key Patel, S.U., Osborn, R., Rees, S. and Thornton,
beta-barrel fold unique amongst plant J.M. (1998) Structural studies of Impatiens
antimicrobial proteins. Journal of Molecular balsamina antimicrobial protein (Ib-AMP1).
Biology 293, 629638. Biochemistry 37, 983990.
Mendez, E., Moreno, A., Colilla, F., Pelaez, F., Pelegrini, P.B. and Franco, O.L. (2005) Plant
Limas, G.G., Mendez, R., Soriano, F., Salinas, -thionins: novel insights on the mechanism of
M. and de Haro, C. (1990) Primary structure and action of a multi-functional class of defence
inhibition of protein synthesis in eukaryotic cell- proteins. The International Journal of
free system of a novel thionin, -hordothionin, Biochemistry & Cell Biology 37, 22392253.
from barley endosperm. European Journal of Plan, M.R.R., Saska, I., Cagauan, A.G. and Craik,
Biochemistry 194, 533539. D.J. (2008) Backbone cyclised peptides from
Miquel, M., Block, M.A., Joyard, J., Dorne, A.-J., plants show molluscicidal activity against the
Dubacq, J.-P., Kader, J.-C. and Douce, R. (1988) rice pest Pomacea canaliculata (golden apple
Protein-mediated transfer of phosphatidylcholine snail). Journal of Agricultural and Food
from liposomes to spinach chloroplast envelope Chemistry 56, 52375241.
membranes. Biochimica et Biophysica Acta 937, Qin, Q., McCallum, E.J., Kaas, Q., Suda, J., Saska,
219228. I., Craik, D.J. and Mylne, J.S. (2010) Identification
70 Q. Kaas et al.
of candidates for cyclotide biosynthesis and Simonsen, S.M., Sando, L., Rosengren, K.J., Wang,
cyclisation by expressed sequence tag analysis C.K., Colgrave, M.L., Daly, N.L. and Craik, D.J.
of Oldenlandia affinis. BMC Genomics 11, 111. (2008) Alanine scanning mutagenesis of the
Richard, J.A., Kelly, I., Marion, D., Pezolet, M. and prototypic cyclotide reveals a cluster of residues
Auger, M. (2002) Interaction between essential for bioactivity. Journal of Biological
-purothionin and dimyristoylphosphatidyl- Chemistry 283, 98059813.
glycerol: a 31P-NMR and infrared spectroscopic Sin, T.S. (2003) Damage potential of the golden
study. Biophysical Journal 83, 20742083. apple snail Pomacea canaliculata (Lamarck) in
Richard, J.A., Kelly, I., Marion, D., Auger, M. and irrigated rice and its control by cultural
Pezolet, M. (2005) Structure of -purothionin in approaches. International Journal of Pest
membranes: a two-dimensional infrared Management 49, 49.
correlation spectroscopy study. Biochemistry Stec, B. (2006) Plant thionins the structural per-
44, 5261. spective. Cellular and Molecular Life Sciences
Rosengren, K.J., Daly, N.L., Plan, M.R., Waine, C. 63, 13701385.
and Craik, D.J. (2003) Twists, knots, and rings in Stec, B., Rao, U. and Teeter, M.M. (1995)
proteins. Structural definition of the cyclotide
Refinement of purothionins reveals solute
framework. Journal of Biological Chemistry 278,
particles important for lattice formation and
86068616.
toxicity. Part 2: structure of -purothionin at 1.7
Ryan, C.A. (2000) The systemin signaling pathway:
resolution. Acta Crystallographica. Section D:
differential activation of plant defensive genes.
Biochimica et Biophysica Acta 1477, 112121. Biological Crystallography 51, 914924.
Ryan, C.A., Huffaker, A. and Yamaguchi, Y. (2007) Tailor, R.H., Acland, D.P., Attenborough, S.,
New insights into innate immunity in Arabidopsis. Cammue, B.P., Evans, I.J., Osborn, R.W., Ray,
Cellular Microbiology 9, 19021908. J.A., Rees, S.B. and Broekaert, W.F. (1997) A
Saether, O., Craik, D.J., Campbell, I.D., Sletten, K., novel family of small cysteine-rich antimicrobial
Juul, J. and Norman, D.G. (1995) Elucidation of peptides from seed of Impatiens balsamina is
the primary and three-dimensional structure of derived from a single precursor protein. Journal
the uterotonic polypeptide kalata B1. of Biological Chemistry 272, 2448024487.
Biochemistry 34, 41474158. Tam, J.P., Lu, Y.A., Yang, J.L. and Chiu, K.W. (1999)
Saska, I., Gillon, A.D., Hatsugai, N., Dietzgen, R.G., An unusual structural motif of antimicrobial
Hara-Nishimura, I., Anderson, M.A. and Craik, peptides containing end-to-end macrocycle and
D.J. (2007) An asparaginyl endopeptidase cystine-knot disulfides. Proceedings of the
mediates in vivo protein backbone cyclization. National Academy of Sciences of the USA 96,
Journal of Biological Chemistry 282, 29721 89138918.
29728. Tan, N.H. and Zhou, J. (2006) Plant cyclopeptides.
Shenkarev, Z.O., Nadezhdin, K.D., Sobol, V.A., Chemical Reviews 106, 840895.
Sobol, A.G., Skjeldal, L. and Arseniev, A.S. Terras, F.R., Goderis, I.J., Van Leuven, F.,
(2006) Conformation and mode of membrane Vanderleyden, J., Cammue, B.P. and Broekaert,
interaction in cyclotides. Spatial structure of W.F. (1992) In vitro antifungal activity of a radish
kalata B1 bound to a dodecylphosphocholine (Raphanus sativus L.) seed protein homologous
micelle. European Journal of Biochemistry 273, to nonspecific lipid transfer proteins. Plant
26582672.
Physiology 100, 10551058.
Shenkarev, Z.O., Nadezhdin, K.D., Lyukmanova,
Terras, F.R., Eggermont, K., Kovaleva, V., Raikhel,
E.N., Sobol, V.A., Skjeldal, L. and Arseniev, A.S.
N.V., Osborn, R.W., Kester, A., Rees, S.B.,
(2008) Divalent cation coordination and mode
Torrekens, S., Van Leuven, F., Vanderleyden, J.
of membrane interaction in cyclotides: NMR
et al. (1995) Small cysteine-rich antifungal
spatial structure of ternary complex Kalata B7/
Mn2+/DPC micelle. Journal of Inorganic proteins from radish: their role in host defence.
Biochemistry 102, 12461256. The Plant Cell 7, 573588.
Shin, D.H., Lee, J.Y., Hwang, K.Y., Kim, K.K. and Thevissen, K., Cammue, B.P., Lemaire, K.,
Suh, S.W. (1995) High-resolution crystal Winderickx, J., Dickson, R.C., Lester, R.L.,
structure of the non-specific lipid-transfer protein Ferket, K.K., Van Even, F., Parret, A.H. and
from maize seedlings. Structure 3, 189199. Broekaert, W.F. (2000a) A gene encoding a
Simonsen, S.M., Sando, L., Ireland, D.C., Colgrave, sphingolipid biosynthesis enzyme determines
M.L., Bharathi, R., Gransson, U. and Craik, the sensitivity of Saccharomyces cerevisiae to
D.J. (2005) A continent of plant defence peptide an antifungal plant defensin from dahlia (Dahlia
diversity: cyclotides in Australian Hybanthus merckii). Proceedings of the National Academy
(Violaceae). The Plant Cell 17, 31763189. of Sciences of the USA 97, 95319536.
AMPs in Plants 71
Thevissen, K., Osborn, R.W., Acland, D.P. and (2008b) Anti-HIV cyclotides from the Chinese
Broekaert, W.F. (2000b) Specific binding sites medicinal herb Viola yedoensis. Journal of
for an antifungal plant defensin from dahlia Natural Products 71, 4752.
(Dahlia merckii) on fungal cells are required for Wang, C.K., Colgrave, M.L., Ireland, D.C., Kaas, Q.
antifungal activity. Molecular PlantMicrobe and Craik, D.J. (2009) Despite a conserved
Interactions 13, 5461. cystine knot motif, different cyclotides have
Thevissen, K., Francois, I.E., Takemoto, J.Y., Ferket, different membrane binding modes. Biophysical
K.K., Meert, E.M. and Cammue, B.P. (2003) Journal 97, 14711481.
DmAMP1, an antifungal plant defensin from Wang, Z. and Wang, G. (2004) APD: the
dahlia (Dahlia merckii), interacts with Antimicrobial Peptide Database. Nucleic Acids
sphingolipids from Saccharomyces cerevisiae. Research 32, D590D592.
FEMS Microbiology Letters 226, 169173. Wei, G.X., Campagna, A.N. and Bobek, L.A. (2007)
Thevissen, K., Warnecke, D.C., Francois, I.E., Factors affecting antimicrobial activity of MUC7
Leipelt, M., Heinz, E., Ott, C., Zahringer, U., 12-mer, a human salivary mucin-derived
Thomma, B.P., Ferket, K.K. and Cammue, B.P. peptide. Annals of Clinical Microbiology and
(2004) Defensins from insects and plants Antimicrobials 6, 14.
interact with fungal glucosylceramides. Journal Witherup, K.M., Bogusky, M.J., Anderson, P.S.,
of Biological Chemistry 279, 39003905. Ramjit, H., Ransom, R.W., Wood, T. and
Thevissen, K., Kristensen, H.-H., Thomma, B.P.H.J., Sardana, M. (1994) Cyclopsychotride A, a
Cammue, B.P.A. and Franois, I.E.J.A. (2007) biologically active, 31-residue cyclic peptide
Therapeutic potential of antifungal plant and isolated from Psychotria longipes. Journal of
insect defensins. Drug Discovery Today 12, Natural Products 57, 16191625.
966971. Yang, Y.W., Lai, K.N., Tai, P.Y. and Li, W.H. (1999)
Thomma, B.P., Cammue, B.P. and Thevissen, K. Rates of nucleotide substitution in angiosperm
(2002) Plant defensins. Planta 216, 193202. mitochondrial DNA sequences and dates of
Thomma, B.P., Cammue, B.P. and Thevissen, K. divergence between Brassica and other
(2003) Mode of action of plant defensins angiosperm lineages. Journal of Molecular
suggests therapeutic potential. Current Drug Evolution 48, 597604.
Targets. Infectious Disorders 3, 18. Yeaman, M.R. and Yount, N.Y. (2003) Mechanisms
Thongyoo, P., Jaulent, A.M., Tate, E.W. and of antimicrobial peptide action and resistance.
Leatherbarrow, R.J. (2007) Immobilized Pharmacological Reviews 55, 2755.
protease-assisted synthesis of engineered Yeats, T.H. and Rose, J.K. (2008) The biochemistry
cysteine-knot microproteins. Chembiochem 8, and biology of extracellular plant lipid-transfer
11071109. proteins (LTPs). Protein Science 17, 191198.
Trabi, M. and Craik, D.J. (2004) Tissue-specific Yokoyama, S., Iida, Y., Kawasaki, Y., Minami, Y.,
expression of head-to-tail cyclized miniproteins Watanabe, K. and Yagi, F. (2009) The chitin-
in Violaceae and structure determination of the binding capability of Cy-AMP1 from cycad is
root cyclotide Viola hederacea root cyclotide1. essential to antifungal activity. Journal of Peptide
The Plant Cell 16, 22042216. Science 15, 492497.
Trabi, M., Mylne, J.S., Sando, L. and Craik, D.J. Yu, H., Zhang, L., Li, L., Zheng, C., Guo, L., Li, W.,
(2009) Circular proteins from Melicytus Sun, P. and Qin, L. (2010) Recent developments
(Violaceae) refine the conserved protein and and future prospects of antimicrobial metabolites
gene architecture of cyclotides. Organic & produced by endophytes. Microbiological
Biomolecular Chemistry 7, 23782388. Research 165, 437449.
UniProt Consortium (2010) The Universal Protein Zhang, J., Liao, B., Craik, D.J., Li, J.-T., Hu, M. and
Resource (UniProt) in 2010. Nucleic Acids Shu, W.-S. (2009) Identification of two suites of
Research 38, D142D148. cyclotide precursor genes from metallophyte
Wang, C.K.L., Kaas, Q., Chiche, L. and Craik, D.J. Viola baoshanensis: cDNA sequence variation,
(2008a) CyBase: a database of cyclic protein alternative RNA splicing and potential cyclotide
sequences and structures, with applications in diversity. Gene 431, 2332.
protein discovery and engineering. Nucleic Zuidmeer, L. and van Ree, R. (2007) Lipid transfer
Acids Research 36, D206D210. protein allergy: primary food allergy or pollen/
Wang, C.K.L., Colgrave, M.L., Gustafson, K.R., food syndrome in some cases. Current Opinion
Ireland, D.C., Gransson, U. and Craik, D.J. in Allergy and Clinical Immunology 7, 269273.
4 Database-aided Prediction and Design
of Novel Antimicrobial Peptides
Guangshun Wang
Abstract
The isolation and characterization of potent antimicrobial peptides from natural sources has opened a
new avenue to the development of future antimicrobials. To further expand the peptide space, new
antimicrobial peptides can be predicted or designed based on natural peptide templates. By using the
knowledge derived from mature peptides, propeptides, or both (i.e. precursor proteins), a variety of
peptide prediction tools have been developed. Furthermore, bacteriocins are predicted under the
genomic context by considering not only the peptide itself, but also the accompanying genes for its
expression, processing and export. This chapter also discusses database-aided peptide design based on
database screening, sequence shuing, grammar-based design and de novo design. Finally, major
strategies for improving peptide selectivity and stability are highlighted.
Table 4.1. Amino acid properties useful for AMP prediction and design.
Residue Gotf
AAa AAb mass BacP (%)c PlaP (%)d AniP (%)e (kcal mol1)f PVg AASIh
I Ile 113.16 6.25 4.29 6.72 2.45 0.198i 1.97
V Val 99.13 6.52 4.7 5.97 1.66 0.200 2.37
L Leu 113.16 5.8 3.31 10.43 2.31 0.246 1.74
F Phe 147.18 2.66 2.82 4.5 2.43 0.246 1.53
C Cys 103.14 5.00 17.96 5.28 2.09 0.165 1.73
M Met 131.20 1.61 0.51 1.18 1.67 0.265 2.50
A Ala 71.08 11.35 3.9 8.86 0.42 0.307 1.89
W Trp 186.21 3.64 1.61 1.27 3.06 0.172 2.00
Y Tyr 163.18 3.49 3.64 1.99 1.31 0.185 2.01
G Gly 57.05 15.04 11.29 11.89 0.0 0.265 2.67
P Pro 97.12 2.92 5.81 4.94 0.98 0.327 0.22
T Thr 101.11 5.57 7.43 3.39 0.35 0.242 2.18
S Ser 87.08 7.18 7.64 5.15 0.05 0.281 2.14
Q Gln 128.13 2.13 1.91 2.29 0.30 0.248 3.05
N Asn 114.10 5.88 5.18 3.13 0.82 0.240 2.33
E Glu 129.12 1.86 3.39 2.12 0.87 0.449 3.14
D Asp 115.09 2.03 2.34 2.23 1.05 0.479 3.13
H His 137.14 1.99 1.21 1.97 0.18 0.202 3.00
K Lys 128.17 6.77 6.15 10.86 1.35 0.111 2.28
R Arg 156.19 2.05 5.27 5.84 1.37 0.106 1.91
a Amino acid in the single-letter code.
b Amino acid in the three-letter code.
c,d,e Amino acid percentage of AMPs from bacteria (BacP), plants (PlaP) and animals (AniP), respectively (Wang et al.,
2009). Slight variations in these values are anticipated with increases in the number of AMPs in the database (Wang
et al., 2009).
f Hydrophobic parameter, as represented by the transfer of free energy of an amino acid side chain from the octanol
phase to water (Fauchere and Pliska, 1983). An application of this was illustrated in Wang et al. (2005).
g Bactericidal propensity value (PV) (Torrent et al., 2009).
h Amino acid selectivity index (AASI) (Juretic et al., 2009).
i Some outstanding values are in bold.
analysis revealed a distinct peak in the power similar to the human brain. The artificial
spectrum of peptide sequences. This finding neural network (ANN) is a powerful
forms the basis for identifying potential machine-learning method that is insensitive
AMPs by scanning the protein sequences to noise and correlated inputs. The ANN can
derived from the genomic data. As a test, this be trained using carefully registered known
Fourier transformation filter found three AMP data and perform predictions based on
positive hits from 10,000 randomly generated the extracted rules. The support vector
sequences of 16 residues each. The machine (SVM) approach includes a set of
antimicrobial activities of those peptides related supervised learning methods used
have not yet been evaluated experimentally. for classification and regression. In
conjugation with peptide terminal sequence
analysis, the authors reported an accuracy of
Machine-learning methods
88% for ANN and 92% for SVM. A recent
Also based on the positive AMP dataset of update of the APD (Wang et al., 2009) led to
the APD (Wang and Wang, 2004), Lata et al. the improvement of this prediction program.
(2007) applied machine-learning algorithms By including new peptide entries and source
to AMP classification and prediction. Neural information annotated in the APD, the
networks handle information in a way overall prediction accuracy reached 98.95%
Prediction and Design of Novel AMPs 75
(Lata et al., 2010). We made some test runs of discovery. While the amino acid sequences
the program (www.imtech.res.in/raghava/ for the mature AMP region are highly
antibp2/submit.html) using the newly variable, the peptide sequences prior to the
registered AMPs, which are not likely to be mature peptide portion (propeptide) are
part of the training set. When SVM and full remarkably similar. This observation laid
sequence composition were selected, the the basis for identifying novel cathelicidins
program gave no prediction for AMPs with (Fig. 4.1B). In analogy to swine PR-39, a
<15 residues. Among 17 AMPs with >15 human version sequence was initially pre-
residues, 12 were correctly predicted as dicted as FALL-39 (Agerberth et al., 1995).
antibacterial (i.e. 71% accuracy). Subsequently, Srensen et al. (2001, 2003)
isolated LL-37 from human neutrophils and
ALL-38 from the reproductive system. In
AMP sequence motifs
this case, the experimentally elucidated
Based on a manually compiled list of protease cleavage sites on the precursor
disulfide-containing AMPs such as defensins protein diered slightly from those
from plants and animals, Yount and Yeaman predicted. This example illustrates that the
(2004) identified a well-conserved GXC precursor sequence of each AMP is also
sequence motif. This motif is common in useful for AMP prediction. Indeed, the
defensins, ranging from plants to bacteria, precursors of ranid AMPs also possess a
fungi and animals. In the three-dimensional common and highly conserved pro-region,
structures of peptides, this sequence motif usually acidic and terminated at a typical
forms a -core. Using this signature model, processing signal lysine/arginine. This find-
the authors were able to identify previously ing has been utilized as a basis for
unidentified AMPs such as brazzein and identifying AMPs on a large scale in
charybdotoxin that exerted direct antimicro- amphibians (Li et al., 2007).
bial activity against bacteria and Candida
albicans. This motif does not apply to the
cysteine-bridged Rana box at the C-termini
of some amphibian AMPs (Conlon et al., 4.1.3 Prediction based on both
2004). propeptides and mature peptides
Structural motif-based prediction has
also been performed at the gene level. Based In the AMPer prediction model, Fjell et al.
on the conserved six-cysteine -defensin (2007) considered both propeptide sequences
structural motif, Schutte et al. (2002) were and mature peptides (Fig. 4.1C). Data from
able to identify 28 new human and 43 new the Antimicrobial Sequences Database
mouse -defensin genes in five syntenic (AMSDb) (Tossi and Sandri, 2002) were
chromosomal regions by combining the validated and expanded as a training dataset.
HMMER and BLAST analysis tools. HMMER The basic idea was to classify the peptides in
(http://hmmer.janelia.org/) is a bioinformatics the database into multiple groups (or
tool based on the hidden Markov models. clusters) and then calculate the property
These newly discovered genes were missed profiles as a basis for subsequent prediction.
in previous annotations of human and mouse Fjell et al. (2007) identified 146 clusters for
genomes. Future studies will elucidate when, mature peptides and 40 clusters for pro-
where and why particular defensins are peptides (186 clusters in total). The prediction
expressed or silenced. was accomplished by matching the profiles
of the unknowns with those of the known
186 clusters. The mutual overlap between the
4.1.2 Prediction based on highly matched pairs should be >90%. In this
conserved propeptide sequences manner, they identified an additional 229
mature peptides from the 230,133 peptides
The sequence alignment of cathelicidin collected in the Swiss-Prot database. Most of
precursor proteins has led to an interesting these peptides could be associated with
76 G. Wang
known activity data in the literature. This 4.2 Database-aided Peptide Design
tool achieved a high prediction accuracy of and Improvement
99%.
4.2.1 Database screening for HIV-
inhibitory antimicrobial peptides
4.1.4 Prediction based on processing
Natural AMPs show inhibitory activities
enzymes
against human immunodeficiency virus
(HIV). Examples are insect melittin and
It has been illustrated that sequence
cecropin, amphibian AMPs, mammalian
similarities among polypeptide modification
cathelicidins, defensins and plant cyclotides
enzymes such as LanM are useful in
(Wachinger et al., 1998; VanCompernolle et
identifying novel lantibiotics. Because this
al., 2005; Jenssen et al., 2006; Lehrer, 2007;
approach does not utilize polypeptide
Ireland et al., 2008). Because eective HIV
information of either AMPs or their pre-
vaccines are not yet available, there is a need
cursors, it can be regarded as a fourth
to develop topical microbicides that prevent
method (Fig. 4.1D). Both haloduracin and
the sexual transmission of HIV (Turpin, 2002;
lichenicidin were identified in the genomes
Buckheit et al., 2010). Currently, <5% of the
using this approach (McClerren et al., 2006;
AMPs in the APD are known to be HIV
Begley et al., 2009).
inhibitory. We have hypothesized that a large
number of AMPs in our database (Wang and
Wang, 2004), with a variety of sequences,
4.1.5 Genomic context-based bacteriocin contain useful templates for developing
prediction novel agents that may prevent HIV trans-
mission. To test this hypothesis, we recently
A possible reason for missing AMPs during conducted a database-based peptide screen
gene annotations is the small size of their against HIV type 1 (HIV-1). In total, 30
open reading frames (ORFs). De Jong et al. natural peptides were selected by considering
(2006) developed BAGEL (http://bioinform peptide length, charge, cysteine content,
atics.biol.rug.nl/websoftware/bagel) to detect toxicity to mammalian cells and uniqueness
AMPs in a bacterial genome. This program of the candidate sequences (Wang et al.,
incorporates a few published ORF prediction 2010). The well-accepted and highly
tools, including Glimmer/RBSfinder, Zcurve standardized cytopathic eects inhibition
and GeneMark, for the detection of small assay in CEM-SS cells was utilized to
ORFs for AMPs. In addition, the program evaluate the ecacy and toxicity of candidate
also considers the existence of gene clusters peptides. The therapeutic index (TI) is
that encode proteins for processing, modi- defined as the ratio of TC50 to EC50, where
fication, transport, regulation and/or EC50 is the concentration required for 50%
immunity of bacteriocins. Finally, the inhibition of viral replication and TC50 is the
program compares the candidate with those concentration required for a 50% reduction
in the knowledge-based bacteriocin database in cell viability. We found that 11 peptides
generated by the authors. Because this displayed an EC50 of <10 M. These include
approach is of an integrated nature and an uperin 7.1 mutant, temporin-PTa, clavanin
diers from all the other methods discussed B, ponericin L2, spinigerin, piscidin 3,
above, we shall name it as genomic context- brevinin-2-related, maculatin 1.3, ascaphin-8,
based AMP prediction (Fig. 4.1E). Using melectin and temporin-LTc. Of these peptides,
BAGEL, the authors found one additional frog temporin-PTa, temporin-LTc, insect
possible bacteriocin from the genomic ponericin L2 and spinigerin had a TI of >10.
sequences of both Streptococcus pneumoniae For longer peptides such as human
TIGR4 and R6. This online tool has also been cathelicidin LL-37 and bovine BMAP-27, we
used to identify putative bacteriocin genes also identified the major HIV-inhibitory
(Majchrzykiewicz et al., 2010). regions (Wang et al., 2008). In the case of
Prediction and Design of Novel AMPs 77
LL-37, FK-13 was identified as the smallest that the hybrid peptide became less
HIV-inhibitory region; KR-12, which diers antimicrobial after sequence reversal. In
from FK-13 by just one N-terminal phenyl- another case, sequence reversal did not cause
alanine (Table 4.2), turned out to be inactive. a selective loss in haemolytic activity
Among the series of peptides tested, the (Subbalakshmi et al., 2001). We found that the
LL-37-derived GI-20 displayed one of LL-37-derived core AMP (FK-13) was active
the highest TIs. Likewise, BMAP-18, the against both Escherichia coli and HIV. After
N-terminal 18 residues of BMAP-27, was sequence reversal, retro-FK13 retained its
found to have a TI of >20 (Table 4.2). We are bactericidal activity against E. coli (Li et al.,
now developing anti-HIV microbicides based 2006b), but lost its HIV-inhibitory activity
on these peptide templates. (Table 4.2). These examples indicate that the
We found excellent overlap between the sequence order of AMPs determines the
minimal antibacterial region (Li et al., 2006a) activity spectrum. Sequence reversal, however,
and the smallest anti-HIV region of human is only a special case of sequence shuing or
LL-37 identified experimentally (Wang et al., rearrangement (Fig. 4.2B), since the latter can
2008). Torrent et al. (2009) developed a produce numerous new sequences (i.e. a
theoretical method to spot active regions in miniature combinatorial library).
antimicrobial proteins. A bactericidal pro- To evaluate the eect of sequence
pensity index value (PV) was calculated for shuing on anti-HIV-1 activity, we created
each amino acid (Table 4.1). The PVs for some new peptides based on an aurein 1.2
residues arginine, lysine, cysteine, trypto- analogue, where phenylalanine 13 of aurein
phan, tyrosine and isoleucine are 0.2 and 1.2 was mutated to tryptophan 13 (sequence:
are favoured in AMPs. A potentially active GLFDIIKKIAESW). The peptides were
region should have a low PV value on generated by rearranging the 13 amino acid
average. This method achieved a prediction residues of peptide 1 and by modelling
accuracy of 85%. known helical AMPs. These peptides were
subjected to HIV-1 inhibition evaluation as
described above. Among the eight peptides
4.2.2 Sequence shuffling is a primitive synthesized, two displayed reduced TIs,
combinatorial approach three showed little variation and two showed
improved TIs. Hence, sequence shuing
Sequence reversal influences the activity of provides an approach for generating better
AMPs (Fig. 4.2B). Merrifield et al. (1995) found HIV-inhibitory peptides (Wang et al., 2010).
Table 4.2. Select HIV-1-inhibitory peptides identified from the APD. Adapted from Wang et al.
(2008, 2010).
Name Peptide sequence EC50 (M)a TC50 (M)a TIa
AZTb 0.009 >0.5 >55.6
KR-12 KRIVQRIKDFLR-NH2 >63.5 >63.5
FK-13 FKRIVQRIKDFLR-NH2 3.4 10.4 3.1
Retro-FK13 RLFDKIRQVIRKF-NH2 >58.1 33.7 <0.58
GF-17 GFKRIVQRIKDFLRNLV-NH2 0.98 8.9 9.1
GI-20 GIKEFKRIVQRIKDFLRNLV-NH2 1.08 22.7 21
BMAP-18 GRFKRFRKKFKKLFKKIS 0.35 8.45 24.1
GLK-19 GLKKLLGKLLKKLGKLLLK >47.5 25.1 <0.53
GLR-19 GLRRLLGRLLRRLGRLLLR 4.4 25.7 5.8
DRS S9 GLRSKIWLWVLLMIWQESNKFKKM 31.6 >32.9 >1.04
DRS S9r3 GLRSRIWLWVLLMIWQESNRFKRM 1.25 >32.1 >25.7
a EC50, the concentration required for 50% inhibition of viral replication; TC50, the concentration required for a 50%
reduction in cell viability; TI, therapeutic index.
b Azidothymidine (AZT; also known as zidovudine) is a Food and Drug Association-approved anti-HIV drug that is used
to prevent HIV transmission from infected pregnant women to their children.
78 G. Wang
peptides and five for -sheet proteins (Villain peptides in the APD contain the LGK
et al., 2000). A prototype helix design only sequence. When highly used motifs are
involved lysine and leucine, which represent chosen, the likelihood of the peptide being
positively charged and hydrophobic com- antimicrobial increases. By following the
ponents, respectively. Such peptides are amphipathic pattern, the number of peptides
referred to as LK peptides. Among a series of can be further reduced. We found that
peptides constructed, 14- or 15-residue GLK-19, a 19-residue peptide consisting of
peptides in the amphipathic helix pattern only glycine, leucine and lysine (Table 4.2),
were found to be most active against bacteria was more active against E. coli K12 than
(Blondelle and Houghten, 1992). Shorter human LL-37 (Wang et al., 2009). The
peptides are inactive and longer peptides inclusion of a low glycine content may
tend to be haemolytic. Wang et al. (2009) improve peptide selectivity, as peptides
found that a 12-residue LK peptide was consisting only of leucine and lysine are
inactive. Kang et al. (2008) were able to obtain known to be cytotoxic to human cells
a highly active LK-peptide with merely 11 (Braunstein et al., 2004). Recently, Jureti et al.
residues only after including a tryptophan (2009) arrived at an amino acid selectivity
residue, an excellent membrane anchor index based on TIs and amino acid
(discussed in Chapter 9). Monroc et al. occurrences in peptides (Table 4.1).
(2006a) revealed that the linear form of de Adepantin-1 showed the highest TI. The seven
novo-designed peptides of 410 residues glycine residues in adepantin-1 (sequence
displayed no antimicrobial activity. However, GIGKHVGKALKGLKGLLKGLGES) could be
cyclization enhanced the hydrophobicity of important for peptide selectivity. Interestingly,
the peptides and rendered them antibacterial. this peptide is also rich in glycine, leucine and
These results agree with the fact that the lysine residues (17 out of 23).
shortest helical peptides collected in the APD In both the grammar-based (Fig. 4.2C)
are of 1012 residues. and frequently occurring amino acid-based
Wang et al. (2009) found that glycine, (Fig. 4.2D) approaches, amphipathic segrega-
leucine and lysine are the three most tion is along the peptide backbone (Fig.
frequently occurring residues of AMPs from 4.3A). Based on the APD, Duval et al. (2009)
animals (AniP in Table 4.1), including frogs. designed a two-segment amphipathic struc-
This sheds light on the biological significance ture (Fig. 4.3B). The peptide was found to be
of the earlier choice of leucine and lysine in active against both Gram-positive and Gram-
peptide design, above. Using these three negative bacteria, indicating the feasibility of
residues, we designed a GLK peptide in three an alternative peptide design as previously
steps (Fig. 4.2D). In brief, these three residues demonstrated by Glukhov et al. (2008). It is of
can form dierent sequence motifs such as outstanding interest to note that dermaseptin
GLK and LGK, which can in turn be S9 (DRS S9 in Table 4.2), a natural AMP from
combined into multiple peptides. The number the South American hylid frog (Lequin et al.,
of AMPs containing a specific sequence motif 2006), possesses the amphipathic structure
is searchable in the APD. For example, 64 depicted in Fig. 4.3B.
(A) (B)
Fig. 4.3. Amphipathic structures can be designed in (A) the classic way or (B) the two-segment mode,
where + and L represent hydrophilic and hydrophobic moieties of the design, respectively. The
amphipathic nature would be blurred if an end-on view is displayed for model B.
80 G. Wang
Fig. 4.4. (A) Average contents of a group of amino acids in AMPs with different activities. Hydrophobic
residues include isoleucine, valine, leucine, phenylalanine, alanine, methionine, cysteine and tryptophan;
positive residues are histidine, lysine and arginine; negative residues are glutamic acid and aspartic acid;
polar residues comprise serine, threonine, asparagine, glutamine and tyrosine; and the GP group contains
glycine and proline residues. (B) Average content of lysine (K) and arginine (R) in the five groups of AMPs.
AB, antibacterial; AC, anticancer; AF, antifungal; AV, antiviral; MC, toxic to mammalian cells.
Peptide hydrophobicity can also be (Table 4.2). When d-amino acids were
scaled down by partial incorporation of introduced at residues 20, 24 and 28 of
d-amino acids (Sharon et al., 1999; Chen et GF-17 (numbered as in LL-37), we obtained
al., 2005). By combining d-amino acid a peptide that retained toxicity to E. coli
incorporation with disulfide-bond linked K12, but displayed no toxic eect to human
cyclization, Oren and Shai (2000) found cells. The structural basis for peptide
that the engineered LK peptide (i.e. selectivity improvement by d-amino acid
leucine- and lysine-based peptide) adopted incorporation is described in Chapter 9. Li
a helical structure with improved cell et al. (2006a) proposed that a decrease in
selectivity. Li et al. (2006a) identified a hydrophobicity is a unified approach to
major antimicrobial region corresponding improving cell selectivity of membrane-
to residues 1732 of human LL-37 (GF-17) targeting AMPs.
82 G. Wang
Dathe, M., Nikolenko, H., Klose, J. and Bienert, M. amphipathic interface. Journal of Peptide
(2004) Cyclization increases the antimicrobial Science 15, 583588.
activity and selectivity of arginine- and trypto- Lata, S., Sharma, B.K. and Raghava, G.P.S. (2007)
phan-containing hexapeptides. Biochemistry Analysis and prediction of antibacterial peptides.
43, 91409150. BMC Bioinformatics 8, 263.
De Jong, A., van Hijum, S.A., Bijlsma, J.J., Kok, J. Lata, S., Mishra, N.K. and Raghava, G.P.S. (2010)
and Kuipers, O.P. (2006) BAGEL: a web-based AntiBP2: improved version of antibacterial
bacteriocin genome mining tool. Nucleic Acids peptide prediction. BMC Bioinformatics 11
Research 34 (Web server issue), W273W279. (Suppl. 1), S19.
Duval, E., Zatylny, C., Laurencin, M., Baudy-Floch, Lee, K.H., Lee, H.Y., Slutsky, M.M., Anderson, J.T.
M. and Henry, J. (2009) KKKKPLFGLFFGLF: a and Marsh, E.N. (2004) Fluorous effect in
cationic peptide designed to exert antibacterial proteins: de novo design and characterization
activity. Peptides 30, 16081612. of a four--helix bundle protein containing
Ehrlich, A., Heyne, H.-U., Winter, R., Beyermann, hexafluoroleucine. Biochemistry 43, 16277
M., Carpino, L. A. and Bienert, M. (1996) Rapid 16284.
cyclization on all-L-pentapeptides by means of Lehrer, R.I. (2007) Multispecific myeloid defensins.
1-hydroxy-7-azabenzotriazole-derived uronium Current Opinion in Hematology 14, 1621.
and phosphonium reagents. Journal of Organic Lequin, O., Ladram, A., Chabbert, L., Bruston, F.,
Chemistry 61, 88318838. Convert, O., Vanhoye, D., Chassaing, G.,
Fauchere, J.-L. and Pliska, V. (1983) Hydrophobic Nicolas, P. and Amiche, M. (2006) Dermaseptin
parameters of amino acid side chains from the S9, an -helical antimicrobial peptide with a
partioning of N-acetyl-amino acid amides. hydrophobic core and cationic termini. Bio-
European Journal of Medicinal Chemistry 18, chemistry 45, 468480.
369375. Li, J., Xu, X., Xu, C., Zhou, W., Zhang, K., Yu, H.,
Zhang, Y., Zheng, Y., Rees, H.H., Lai, R., Yang,
Fjell, C.D., Hancock, R.E. and Cherkasov, A. (2007)
D. and Wu, J. (2007) Anti-infection peptidomics
AMPer: a database and an automated discovery
of amphibian skin. Molecular & Cellular
tool for antimicrobial peptides. Bioinformatics
Proteomics 6, 882894.
23, 11481155.
Li, X., Li, Y., Han, H., Miller, D.W. and Wang, G.
Glukhov, E., Burrows, L.L. and Deber, C.M. (2008)
(2006a) Solution structures of human LL-37
Membrane interactions of designed cationic
fragments and NMR-based identification of a
antimicrobial peptides: the two thresholds.
minimal membrane-targeting antimicrobial and
Biopolymers 89, 360371.
anticancer region. Journal of the American
Gottler, L.M., Lee, H.Y., Shelburne, C.E.,
Chemical Society 128, 57765785.
Ramamoorthy, A. and Marsh, E.N. (2008) Using
Li, X., Li, Y., Peterkofsky, A. and Wang, G. (2006b)
fluorous amino acids to modulate the biological
NMR studies of aurein 1.2 analogs. Biochimica
activity of an antimicrobial peptide. Chembio- et Biophysica Acta 1758, 12031214.
chem 9, 370373. Loose, C., Jensen, K., Rigoutsos, I. and
Hong, S.Y., Oh, J.E. and Lee, K.H. (1999) Effect of Stephanopoulos, G. (2006) A linguistic model
D-amino acid substitution on the stability, the
for the rational design of antimicrobial peptides.
secondary structure, and the activity of Nature 443, 867869.
membrane-active peptide. Biochemical Majchrzykiewicz, J.A., Lubelski, J., Moll, G.N.,
Pharmacology 58, 177580. Kuipers, A., Bijlsma, J.J., Kuipers, O.P. and Rink,
Ireland, D.C., Wang, C.K., Wilson, J.A., Gustafson, R. (2010) Production of a class II two-component
K.R. and Craik, D.J. (2008) Cyclotides as natural lantibiotic of Streptococcus pneumoniae using
anti-HIV agents. Biopolymers 90, 5160. the class I nisin synthetic machinery and leader
Jenssen, H., Hamill, P. and Hancock, R.E. (2006) sequence. Antimicrobial Agents and Chemo-
Peptide antimicrobial agents. Clinical Micro- therapy 54, 14981505.
biology Reviews 19, 491511. McClerren, A.L., Cooper, L.E., Quan, C., Thomas,
Juretic, D., Vukicevic, D., Ilic, N., Antcheva, N. and P.M., Kelleher, N.L. and van der Donk, W.A.
Tossi, A. (2009) Computational design of highly (2006) Discovery and in vitro biosynthesis of
selective antimicrobial peptides. Journal of haloduracin, a two-component lantibiotic. Pro-
Chemical Information and Modeling 49, 2873 ceedings of the National Academy of Sciences
2882. of the USA 103, 1724317248.
Kang, S.J., Won, H.S., Choi, W.S. and Lee, B.J. Meng, H. and Kumar, K. (2007) Antimicrobial
(2008) De novo generation of antimicrobial LK activity and protease stability of peptides
peptides with a single tryptophan at the critical containing fluorinated amino acids. Journal of
Prediction and Design of Novel AMPs 85
the American Chemical Society 129, Sharon, M., Oren, Z., Shai, Y. and Anglister, J.
1561515622. (1999) 2D-NMR and ATR-FTIR study of the
Merrifield, R.B., Juvvadi, P., Andreu, D., Ubach, J., structure of a cell-selective diastereomer of
Boman, A. and Boman, H.G. (1995) Retro and melittin and its orientation in phospholipids.
retroenantio analogs of cecropinmelittin Biochemistry 38, 1530515316.
hybrids. Proceedings of the National Academy Skerlavaj, B., Gennaro, R., Bagella, L., Merluzzi, L.,
of Sciences of the USA 92, 34493453. Risso, A. and Zanetti, M. (1996) Biological
Monroc, S., Badosa, E., Feliu, L., Planas, M., characterization of two novel cathelicidin-
Montesinos, E. and Bardaj, E. (2006a) De novo derived peptides and identification of structural
designed cyclic cationic peptides as inhibitors of requirements for their antimicrobial and cell lytic
plant pathogenic bacteria. Peptides 27, 2567 activities. Journal of Biological Chemistry 271,
2574. 2837528381.
Monroc, S., Badosa, E., Besalu, E., Planas, M., Song, Y.M., Park, Y., Lim, S.S., Yang, S.T., Woo,
Bardaj, E., Montesinos, E. and Feliu, L. (2006b) E.R., Park, I.S., Lee, J.S., Kim, J.I., Hahm, K.S.,
Improvement of cyclic decapeptides against Kim, Y. and Shin, S.Y. (2005) Cell selectivity and
plant pathogenic bacteria using a combinatorial mechanism of action of antimicrobial model
chemistry approach. Peptides 27, 25752584. peptides containing peptoid residues. Bio-
Mowery, B.P., Lindner, A.H., Weisblum, B., Stahl, chemistry 44, 1209412106.
S.S. and Gellman, S.H. (2009) Structureactivity Srensen, O.E., Follin, P., Johnsen, A.J., Calafat,
relationship among random nylon-3 copolymers J., Tjabringa, G.S., Hiemstra, P.S. and
that mimic antibacterial host-defense peptides. Borregaard, N. (2001) Human cathelicidin,
Journal of the American Chemical Society 131, hCAP-18, is processed to the antimicrobial
97359745. peptide LL-37 by extracellular cleavage with
Muller, K., Faeh, C. and Diederich, F. (2007) proteinase 3. Blood 97, 39513959.
Fluorine in pharmaceuticals: looking beyond Srensen, O.E., Gram, L., Johnsen, A.J.,
intuition. Science 317, 18811886. Andersson, E., Bangsboll, S., Tjabringa, G.S.,
Nagarajan, V., Kaushik, N., Murali, B., Zhang, C., Hiemstra, P.S., Malm, J., Egesten, A. and
Lakhera, S., Elasri, M.O. and Deng, Y. (2006) A Borregaard, N. (2003) Processing of seminal
Fourier transformation based method to mine plasma hCAP-18 to ALL-38 by gastricsin: a
peptide space for antimicrobial activity. BMC novel mechanism of generating antimicrobial
Bioinformatics 7 (Suppl. 2), S2. peptides in vagina. Journal of Biological
Niemz, A. and Tirrell, D.A. (2001) Self-association Chemistry 278, 2854028546.
and membrane-binding behavior of mellitins Subbalakshmi, C., Nagaraj, R. and Sitaram, N.
containing trifluorpleucine. Journal of the (2001) Biological activities of retro and diastereo
American Chemical Society 123, 74077413. analogs of a 13-residue peptide with anti-
Oren, Z. and Shai, Y. (2000) Cyclization of a cyto- microbial and hemolytic activities. Journal of
lytic amphipathic -helical peptide and its Peptide Research 57, 5967.
diastereomer: effect on structure, interaction Tanabe, H., Ayabe, T., Maemoto, A., Ishikawa, C.,
with model membranes, and biological function. Inaba, Y., Sato, R., Moriichi, K., Okamoto, K.,
Biochemistry 39, 61036114. Watari, J., Kono, T., Ashida, T. and Kohgo, Y.
Pouny, Y. and Shai, Y. (1992) Interaction of D-amino (2007) Denatured human -defensin attenuates
acid incorporated analogues of pardaxin with the bactericidal activity and the stability against
membranes. Biochemistry 31, 94829490. enzymatic digestion. Biochemical and
Rajabi, M., de Leeuw, E., Pazgier, M., Li, J., Biophysical Research Communications 358,
Lubkowski, J. and Lu, W. (2008) The conserved 349355.
salt bridge in human -defensin 5 is required for Torrent, M., Nogus, V.M. and Boix E. (2009) A
its precursor processing and proteolytic stability. theoretical approach to spot active regions in
Journal of Biological Chemistry 283, antimicrobial proteins. BMC Bioinformatics 10,
2150921518 373.
Schutte, B.C., Mitros, J.P., Bartlett, J.A., Walters, Tossi, A. and Sandri, L. (2002) Molecular diversity
J.D., Jia, H.P., Welsh, M.J., Casavant, T.L. and in gene-coded, cationic antimicrobial poly-
McCray, P.B. Jr (2002) Discovery of five peptides. Current Pharmaceutical Design 8,
conserved -defensin gene clusters using 743761.
computational search strategy. Proceedings of Tossi, A., Sandri, L. and Giangaspero, A. (2000)
the National Academy of Sciences of the USA Amphipathic, alpha-helical antimicrobial pep-
99, 21292133. tides. Biopolymers 55, 430.
86 G. Wang
Turpin, J.A. (2002) Considerations and development peptide KR-12 in lipid micelles. Journal of
of topical microbicides to inhibit the sexual Biological Chemistry 283, 3263732643.
transmission of HIV. Expert Opinion on Wang, G., Li, Y. and Li, X. (2005) Correlation of three-
Investigational Drugs 11, 10771097. dimensional structures with the antibacterial
Unger, T., Oren, Z. and Shai, Y. (2001) The effect of activity of a group of peptides designed based on
cyclization of magainin 2 and melittin analogues a non-toxic bacterial membrane anchor. Journal
on structure, function, and model membrane of Biological Chemistry 280, 58035811.
interactions: implication to their mode of action. Wang, G., Watson, K.M. and Buckheit, R.W. Jr
Biochemistry 40, 63886397. (2008) Anti-human immunodeficiency virus type
VanCompernolle, S.E., Taylor, R.J., Oswald-Richter, 1 activities of antimicrobial peptides derived from
K., Jiang, J., Youree, B.E., Bowie, J.H., Tyler, human and bovine cathelicidins. Antimicrobial
M.J., Conlon, J.M., Wade, D., Aiken, C., Agents and Chemotherapy 52, 34383440.
Dermody, T.S., KewalRamani, V.N., Rollins- Wang, G., Li, X. and Wang, Z. (2009) APD2: the
Smith, L.A. and Unutmaz, D. (2005) Antimicrobial updated antimicrobial peptide database and its
peptides from amphibian skin potently inhibit application in peptide design. Nucleic Acids
human immunodeficiency virus infection and Research 37 (Database issue), D933D937.
transfer of virus from dendritic cells to T cells. Wang, G., Watson, K.M., Peterkofsky, A. and
Journal of Virology 79, 1159811606. Buckheit, R.W. Jr (2010) Identification of novel
Villain, M., Jackson, P.L., Manion, M.K., Dong, W.J., human immunodeficiency virus type 1 inhibitory
Su, Z., Fassina, G., Johnson, T.M., Sakai, T.T., peptides based on the antimicrobial peptide
Krishna, N.R. and Blalock, J.E. (2000) De novo database. Antimicrobial Agents and Chemo-
design of peptides targeted to the EF hands of therapy 54, 13431346.
calmodulin. Journal of Biological Chemistry 275, Wang, Z. and Wang, G. (2004) APD: the
26762685. antimicrobial peptide database. Nucleic Acids
Wachinger, M., Klenschmidt, A., Winder, D., von Research 32 (Database issue), D590D592.
Pechmann, N., Ludvigsen, A., Neumann, M., Yount, N.Y. and Yeaman, M.R. (2004) Multi-
Holle, R., Salmons, B., Erfle, V. and Brack- dimensional signatures in antimicrobial peptides.
Werner, R. (1998) Antimicrobial peptides melittin Proceedings of the National Academy of
and cecropin inhibit replication of human Sciences of the USA 101, 73637368.
immunodeficiency virus 1 by suppressing viral Zhu, W.L., Hahm, K.S. and Shin, S.Y. (2007)
gene expression. Journal of General Virology Cathelicidin-derived Trp/Pro-rich antimicrobial
79, 731740 peptides with lysine peptoid residue (Nlys):
Wang, G. (2008) Structures of human host defense therapeutic index and plausible mode of action.
cathelicidin LL-37 and its smallest antimicrobial Journal of Peptide Science 13, 529535.
5
Discovery of Novel Antimicrobial
Peptides Using Combinatorial Chemistry
and High-throughput Screening
William C. Wimley
Abstract
The field of antimicrobial peptide (AMP) research has now spanned three decades, in which hundreds of
AMPs have been discovered, designed or engineered. Yet despite a vast literature, obvious structure
function relationships are rare and this has created a bottleneck in the discovery of novel AMPs. Instead
of rigorous structurefunction principles, AMP activity may be best addressed using the physical
chemistry concept of interfacial activity, a concept that does not help one predict or engineer AMP
activity. In this chapter, I address one method of circumventing this engineering bottleneck: combinatorial
chemistry and high-throughput screening. Combinatorial methods are first discussed from the
perspective of library synthesis techniques, for both indexed and non-indexed methods. This is followed
by a discussion of available high-throughput screening techniques. Finally, I address the accomplishments
to date generated using combinatorial chemistry and high-throughput screening and future directions
the field may take. Combinatorial chemistry and high-throughput screening are powerful and eective
tools for discovering novel AMPs. The future of this field holds great promise.
5.1 The Interfacial Activity Model of 2005; Mowery et al., 2007; Rausch et al., 2007).
AMP Activity Instead it depends on interfacial activity,
which has been referred to as the ability of a
Antimicrobial peptides (AMPs) exert their molecule to bind to a membrane, partition
biological activity by first acting on microbial into the membranewater interface and to
membranes (Steiner et al., 1981; White et al., alter the packing and organization of the
1995), sometimes followed by additional lipids (Rathinakumar and Wimley, 2008;
eects on the microbial cell. Yet, despite Rathinakumar et al., 2009). Interfacial activity
decades of intense study, compelling (Fig. 5.1) is derived from the appropriate
structurefunction relationships for AMPs are balance of interactions between and among
rarely found and evidence for stable, well- peptides, water and membrane lipids. These
defined transmembrane pores (Fig. 5.1) is interactions depend more on the amino acid
rarely observed. Recent literature suggests composition of a peptide and on its physical
that physical impact on membranes, such as chemical properties than on its exact sequence
lipid domain formation (Chapter 7), is not or secondary/tertiary structure (Rathinakumar
dependent on specific amino acid sequences and Wimley, 2008; Rathinakumar et al., 2009;
or on specific three-dimensional peptide Chapter 7). In support of this idea, Hilpert
structures (Hilpert et al., 2005, 2006; Jin et al., et al. (2006) have shown that a high
Barrel-stave pore
Toroidal pore
Interfacial activity
Fig. 5.1. Some schematic models of antimicrobial peptide activity. The literature contains numerous
mechanistic models to explain the action of antimicrobial peptides on lipid bilayers. The barrel-stave and
toroidal pore models, while commonly referenced, do not explain the experimentally observed actions of
most antimicrobial peptides. The interfacial activity model (Rathinakumar and Wimley, 2008) can explain
the actions of most AMPs. Most importantly, the interfacial activity model can be used as a basis for
designing compositionally varied combinatorial peptide libraries.
percentage of random versions of a potent charge, hydrophobicity; see Chapter 1). This
AMP retain good activity and some even would allow the design of rational libraries
have improved activity. from which the most active sequences can
Although AMPs are known to adopt a readily be determined by high-throughput
variety of three-dimensional structures in screening. In this chapter, I explore the
lipid bilayers, the rational design of AMPs design, selection and screening of com-
based on structure-specific ideas is rare binatorial peptide libraries toward the
(Chapter 9). However, design based on the ecient discovery of novel AMPs.
principle of interfacial activity is not yet
possible because the physical chemical basis
of interfacial activity has not been 5.2 Combinatorial Chemistry Methods
parameterized. In light of these considera-
tions, the use of combinatorial chemistry and 5.2.1 Overview of library synthesis
high-throughput screening is an especially
attractive approach to discovering novel Almost as soon as the Merrifield method of
AMPs. To design a library, one can use the solid-phase peptide synthesis became widely
principle of interfacial activity by requiring known, combinatorial peptide synthesis was
libraries to contain peptides that adhere to envisioned as a natural extension of the
common attributes of known AMPs (e.g. chemistry. The power of combinatorial
peptide length, amino acid composition, peptide synthesis is derived, in part, from
Discovery of Novel AMPs 89
the fact that the chemistry required to make of peptide synthesis for deconvolution, and
a library is no more complex than to perform risks the possibility that peptides in the
a non-combinatorial synthesis. No novel mixture will act synergistically or
reaction protocols, equipment or protection antagonistically, it is simple and eective, as
strategies are needed. Combinatorial peptide shown by Houghton and colleagues, and can
chemistry is thus directly accessible to successfully lead to the discovery of novel
researchers with a moderate chemistry potent AMPs.
knowledge. In this section, I describe some of Perhaps an even more powerful
the more commonly used synthetic approach to non-indexed combinatorial
approaches to combinatorial peptide library peptide libraries is the one-bead:one-
synthesis. sequence method, also known as split and
recombine libraries. In a one-bead:one-
sequence library, a researcher takes advan-
5.2.2 Non-indexed methods tage of the discrete nature of solid-phase
synthesis resins, which are typically in the
In any high-throughput screen, one must be form of polymer microbeads, often made
able to select for active compounds and then from polystyrene and sometimes with
identify the active molecule or molecules grafted polyethylene glycol. A synthesis
exactly. Combinatorial peptide libraries can slurry of resin beads can be combined into a
be separated into two broad categories: (i) single vessel when common chemistry is
indexed libraries, discussed in detail below, performed; it can be split into separate
which use spatial or chemical indexing that vessels for the addition of separate amino
allows active sequences to be known during acids when a combinatorial site is reached.
the course of the screen; and (ii) non-indexed Recombination of the resin beads into one
libraries, in which indirect analytical vessel allows for randomization before the
methods, such as Edman sequencing, mass next split. Each bead thus has a unique
spectrometry or iterative deconvolution, history of amino acid additions, and contains
must be used to identify active sequences only peptides of a single sequence. Spatial
after they have been selected in the screen. separation of the beads is required for
Here, I discuss some commonly used screening. Typical one-bead:one-sequence
non-indexed combinatorial library methods. libraries have 50,0001,000,000 beads g1 of
Houghten and colleagues were some of resin or 0.22.5 nmol of peptide per bead.
the earliest users of combinatorial chemistry Table 5.1 shows the statistics for some
and high-throughput screening to identify commonly used library beads. One-bead:one-
novel AMPs (Blondelle et al., 1996). They sequence libraries are spatially segregated
used several simple, partially indexed library but are usually not indexed. Screening is
design methods in which the peptides performed on individual beads and the
screened initially were random mixtures of positive sequences are identified post facto
peptides containing fixed residues at some using chemical sequencing, mass spec-
positions and random mixtures of amino trometry or deconvolution.
acids at others. The relative abundance of AMP discovery requires that peptides
each amino acid in the varied position was tested are free in solution, a fact that places
controlled by adjusting amino acid concen- limitations on the types of combinatorial
trations according to their relative reaction libraries that can be used. For example,
rates. Identification of active peptides was classical phage display or selectide-type
accomplished by positional scanning or peptide libraries (Lam et al., 2003), designed
iterative deconvolution. When necessary, to identify peptide sequences that bind to
mixtures with activity were further particular targets, must be modified to allow
subdivided by iterative deconvolution based peptides to be released. For one-bead:one-
on rounds of additional synthesis and testing sequence libraries, an orthogonal approach
until single active sequences were obtained. to synthesis and release must be employed.
While this approach requires a large amount Typically, researchers use a photolabile or
90 W.C. Wimley
(A) (B)
Combinatorial High-throughput
library synthesis screening
Number of Number of
peptides peptides
Fig. 5.2. Combinatorial library synthesis and high-throughput screening. Library design and the high-
throughput screening must be balanced. (A) Library size increases very rapidly with its complexity. The
larger the library, the greater the amount of sequence space that can be explored. (B) High-throughput
screening must be able to identify a small number of highly active library members in a reasonable
amount of time and effort. Screens with higher throughput and better discrimination allow a researcher to
design more complex libraries.
Fig. 5.3. Broth dilution assay as a high-throughput screen. In this example of a 96-well screen, each well
contains a 2.2 M concentration of one member of a combinatorial peptide library, rich growth medium
and an inoculum of 103 Escherichia coli bacteria. After overnight incubation the wells are either opaque,
indicating stationary-phase growth, or they are transparent, indicating no growth (i.e. the bacteria have
been sterilized by the peptide). This is a very stringent high-throughput screen, which requires no
sophisticated equipment to perform. Wells C11H11 have no added peptides and wells C12H12 have
no added bacteria.
(A) (B)
1 cm 1 cm
(C)
1 cm
Fig. 5.4. Agar diffusion assay for high-throughput screening of one-bead:one-sequence libraries. (A,B)
For controls, the bovine AMP indolicidin was linked to synthesized resin microbeads using a photolabile
linker. Indolicidin beads were placed on a thin layer of nutrient agar seeded with Escherichia coli bacteria.
(A) The photolabile linker is cleaved with UV light and the released peptides create a large clearance
zone where no bacteria grow. (B) The linker is not cleaved and no clearance zone is created. (C) Beads
from a combinatorial peptide library described elsewhere (Rathinakumar and Wimley, 2008) are scattered
on to an agar diffusion plate. This is the same library screened by broth dilution in Fig. 5.3. Some beads
(black arrows) create large zones of clearance, while others (white arrows) are less active.
94 W.C. Wimley
cationic residues and, as a result, contain Tables 5.2 and 5.3 give information on these
many antimicrobial members. experiments.
Broad-spectrum antimicrobial activity, Biological assays have been used
defined as activity against multiple classes of successfully to select AMPs from combina-
microbes, is desirable, but is rarer and more torial libraries. However, the single-species
dicult to select than species-specific nature of most experiments and the diculty
activity. Selecting directly for broad-spectrum in performing multispecies experiments with
activity using biological screens can be high throughput are problems that have not
achieved if a single library member is yet been overcome. The next section de-
screened in parallel against microbes from scribes lipid-vesicle-based high-throughput
multiple classes. This approach requires a screens, which are simpler to perform and
complex experiment that reduces the have been shown to select specifically for
throughput of a screen. However, it has the peptides with broad-spectrum activity and
advantage over other screens that broad- low toxicity.
spectrum peptides are identified directly and
unambiguously. To test the eectiveness of
this approach, I have performed parallel 5.3.3 Non-biological assays
multispecies screening using a sample of a
one-bead:one-sequence library described in Countless studies in the literature show that
the literature (Rathinakumar and Wimley, AMPs interact with and perturb lipid bilayer
2008; Rathinakumar et al., 2009). We screened membranes in vitro as well as in vivo. A
in parallel for activity against Escherichia coli typical in vitro experiment is performed
(a Gram-negative bacterium), Staphylococcus using unilamellar lipid vesicles with an
aureus (a Gram-positive bacterium) and entrapped marker for assaying membrane
Cryptococcus neoformans (a fungus). We found permeability (Rausch and Wimley, 2001).
species-specific antimicrobial activity to be Experimental lipid compositions vary widely
surprisingly common, while overlapping (i.e. between laboratories, but mixtures of anionic
broad-spectrum) activity was quite rare. and zwitterionic lipids are often used to
95
sequence alone is inactive and is used for a control peptide.
Abbreviations: HEK293, human embryonic kidney cell line 293; PMSD, perfringolysin membrane-spanning domain; ND, no data; RBC, red blood cell.
96 W.C. Wimley
mimic microbial membranes, while phos- chelator dipicolinic acid (DPA) added to the
phatidylcholine and/or phosphatidylcholine/ external solution. The Tb3+DPA complex,
cholesterol are used to mimic mammalian which forms only when the membranes have
membranes. Because cationic/hydrophobic been permeabilized, is highly fluorescent and
AMPs with good interfacial activity are can be quantitated. Assays are performed in
expected to perturb any lipid bilayer the 96-well plate format and fluorescence is
membrane to which they bind well enough, used to rate permeabilization. Figure 5.5
the exact anionic lipid content is probably shows high-throughput screening using this
not a crucial factor in vesicle-based char- method. By adjusting the concentration of
acterization of AMPs. lipid vesicles added to each well, the
We have developed a vesicle-based high- stringency of the assay can be adjusted to
throughput screen to select for potent vesicle- suit the library being studied. Figure 5.6
permeabilizing peptides from combinatorial shows permeabilization data for a combina-
libraries (Rausch and Wimley, 2001; Rausch torial library screened by this method.
et al., 2005; Rathinakumar and Wimley, When performing high-throughput
2008; Rathinakumar et al., 2009). The high- screens for AMPs, it is important to also select
throughput screen utilizes large unilamellar against peptides that have poor solubility. In
vesicles with the lanthanide metal terbium the vesicle-based screen described above, we
(III) (Tb3+) trapped inside and the aromatic also used a premixing step in which library
Fig. 5.5. A vesicle-based high-throughput screen for bilayer-permeabilizing peptides. Large unilamellar
vesicles containing entrapped terbium (III) (Tb3+) and external dipicolinic acid (DPA) are added to each
well, which also contains about 10 M of one peptide from a combinatorial library. Permeabilization of the
vesicles results in Tb3+DPA complex formation, which is highly fluorescent. These plates were
photographed under UV light. (A) A screen with a lower lipid concentration gives high sensitivity, but lower
stringency. (B) A screen conducted with a higher lipid concentration used for lower sensitivity and higher
stringency screening. In practice, stringency is altered to provide the desired number of positive peptides
(Rausch et al., 2005).
Discovery of Novel AMPs 97
a whole animal infection model. ACS Chemical Academy of Sciences of the USA 102,
Biology 4, 527533. 1051110515.
Rathinakumar, R. and Wimley, W.C. (2008) Rausch, J.M., Marks, J.R., Rathinakumar, R. and
Biomolecular engineering by combinatorial Wimley, W.C. (2007) -Sheet pore-forming
design and high-throughput screening: small, peptides selected from a rational combinatorial
soluble peptides that permeabilize membranes. library: mechanism of pore formation in lipid
Journal of the American Chemical Society 130, vesicles and activity in biological membranes.
98499858. Biochemistry 46, 1212412139.
Rathinakumar, R., Walkenhorst, W.F. and Wimley, Steiner, H., Hultmark, D., Engstrom, A., Bennich, H.
W.C. (2009) Broad-spectrum antimicrobial and Boman, H.G. (1981) Sequence and
peptides by rational combinatorial design and specificity of two antibacterial proteins involved
high-throughput screening: the importance of in insect immunity. Nature 292, 246248.
interfacial activity. Journal of the American White, S.H., Wimley, W.C. and Selsted, M.E. (1995)
Chemical Society 131, 76097617. Structure, function, and membrane integration
Rausch, J.M. and Wimley, W.C. (2001) A high- of defensins. Current Opinion in Structural
throughput screen for identifying transmembrane Biology 5, 521527.
pore-forming peptides. Analytical Biochemistry Wiegand, I., Hilpert, K. and Hancock, R.E. (2008)
293, 258263. Agar and broth dilution methods to determine
Rausch, J.M., Marks, J.R. and Wimley, W.C. (2005) the minimal inhibitory concentration (MIC) of
Rational combinatorial design of pore-forming antimicrobial substances. Nature Protocols 3,
-sheet peptides. Proceedings of the National 163175.
6 Chemical Mimics with Systemic
Efficacy
Amram Mor
Abstract
Host-derived cationic antimicrobial peptides (AMPs) and their synthetic variants are widely regarded as
a potential source of future therapeutic agents against a broad range of pathogens. This is due to a
remarkable set of advantageous properties, ranging from molecular simplicity that readily allows for
time- and cost-ecient establishment of structureactivity relationships, to an essentially non-specific
multitargeted mode of action, which significantly limits the capacity of pathogens to develop ecient
resistance mechanisms. Nevertheless, dicult challenges need to be overcome as we move towards their
eventual use in therapeutics, including improved bioavailability, toxicity and production costs. To
address these issues, an increasingly rich variety of strategies for improving AMP properties are currently
available for designing chemical mimics that reproduce the most critical biophysical characteristics of
AMPs. In this chapter, I review the main strategies for the de novo design of AMP mimics that have
generated systemically active lead compounds and showed promise for therapeutic development.
Conceptually, the known designs can be distinguished on the basis of backbone rigidity. Thus, after a
brief discussion of recent advances on representative approaches used to generate relatively sti
antimicrobial structures, this chapter focuses on the acyl-lysyl approach, which allows the design of
structurally bendable molecules that none the less can selectively target a variety of microbial cells.
as well as sometimes fatal illnesses that may one or more two-component sensor/regulator
not be cured by conventional antibiotics. All systems, concern was voiced that the general
the evidence indicates that, in contrast to use of AMPs might provoke the evolution of
common antibiotics, the antibacterial action resistance that will compromise our natural
of these peptides follows a multiple-hit defences against infection. In that sense,
scheme with several potential targets, AMP mimics that retain antibacterial activity
including the cytoplasmic membrane, cell but lack the ability to activate PhoQ or its
division and cell-wall and macromolecule orthologues would represent preferable thera-
synthesis (Shai, 2002; Zaslo, 2002; Brogden, peutics.
2005), making it dicult for the pathogen to A rich range of strategies have been put
acquire resistance. forward in the attempt to alleviate one or
The therapeutic potential of AMPs more of these shortcomings. These have
became evident very early after their included the use of peptides composed of
discovery. As therapeutic agents, AMPs d-amino acids (Bessalle et al., 1990; Wade et
present various a priori advantages over al., 1990; Shai and Oren, 1996), combinatorial
antibiotics, including molecular simplicity, libraries (Eichler and Houghten, 1995;
broad-spectrum activity (Tossi et al., 2000; Blondelle et al., 1996; Blondelle and Lohner,
Radzishevsky et al., 2005; Jenssen et al., 2006), 2000) and a wealth of sequence templates or
potentially low levels of induced resistance minimalistic approaches (Tossi et al., 1997,
(Perron et al., 2006) and concomitant broad 2000; Epand et al., 2003; Jing et al., 2003;
anti-inflammatory activities (McIntur et al., Dartois et al., 2005; Deslouches et al., 2005).
2005; Guarna et al., 2006; Easton et al., 2009). These modification strategies have yielded
Nevertheless, as potential drug candidates, several peptide versions with systemic
HDPs can present a variety of shortcomings antibacterial activities; to name a few
that need to be addressed, particularly examples, a 24-mer peptide termed WLBU2,
towards development as systemic therapeutic which is based solely on cationic (arginine)
agents. Namely, antimicrobial activity of and hydrophobic (valine and tryptophan)
many HDPs is significantly reduced in the residues (Deslouches et al., 2005), and cyclic
presence of salts and divalent cations d,l-hexapeptides composed of lysine and
(Goldman et al., 1997; Bals et al., 1998; Minahk tryptophan only (Dartois et al., 2005). Most
and Morero, 2003) and is susceptible to pH recently, a dimeric proline-rich peptide
changes (Lee et al., 1997; Rydlo et al., 2006) (A3-APO) was designed to attack both the
and plasma components (Oh et al., 1999; bacterial membrane and the Entero-
Rozek et al., 2003; Radzishevsky et al., 2005). bacteriaceae-specific domain of the heat-
HDPs can also suer from poor pharmaco- shock protein DnaK in order to reduce
kinetic properties and systemic toxicity, as toxicity (Szabo et al., 2010). Although
well as high production costs (Bradshaw, A3-APO was bactericidal to Escherichia coli at
2003; Yeaman and Yount, 2003; Gordon et al., a relatively high concentration (30 g ml1) in
2005; Marr et al., 2006). vitro, the designer peptide was eective
Another concern for therapeutic uses of against systemic E. coli infections in dierent
AMPs pertains to the emergence of extreme mouse models after multiple dosing of 10 mg
resistance phenomena to the host defence kg1, whereas the maximal tolerated dose
system. In addition to their ability to develop (MTD) was >20 mg kg1.
resistance mechanisms such as eux pumps, Another set of strategies has opted for
secreted proteases and alterations of the cell the de novo design of peptide-like constructs
surface, bacteria may also sense AMPs via a that mimic HDP structure or function
variety of two-component sensor/regulator (representative structures are shown in Fig.
systems (e.g. PhoPQ, PmrAB). These regulate 6.1). While diering with respect to their
specific gene expression leading to greater prime philosophies, these strategies share a
stability of the outer membrane and adapting common rationale, which is based on the idea
bacteria for survival (Gunn, 2008; Otto, 2009). that reproduction of the critical biophysical
As some AMPs have been found to activate characteristics in unnatural oligomers should
102 A. Mor
Fig. 6.1. Chemical formulae of representative building blocks used for the de novo design of AMP
mimics. The -peptide sequence (centre) can be replaced with -peptide (top), peptoid (bottom),
arylamide (upper left), phenylene ethynylene (lower left), aminoacyl-lysyl (upper right) or lysyl-aminoacyl-
lysyl (lower right) subunits.
Chemical Mimics with Systemic Efficacy 103
Unfortunately, few in vivo data exist in An OAK library including >150 sequences
the literature to reflect the possible composed of either (aminoacyl-lysyl) or
therapeutic potential of these amphiphiles. (lysyl-aminoacyl-lysyl) subunits has been
While, the MTD of mPE in mice was found produced and characterized (Radzishevsky et
to be 10 mg kg1 (Tew et al., 2006), suggesting al., 2007a,b, 2008; Livne et al., 2009). Analysis
a potential therapeutic window, to my of the data obtained from this library enabled
knowledge no ecacy studies have been pertinent conclusions to be drawn as to the
published since. In contrast, short arylamide properties required for the potency and
foldamers were recently reported to exhibit selectivity of OAKs, as detailed in the
systemic ecacy in vivo (Choi et al., 2009). following paragraphs.
The most potent variants reduced staphylo-
coccal load to various extents upon the
systemic administration of 110 mg kg1, 6.3.2 Structureactivity relationships
while MTD was found at 20 mg kg1.
Collectively, these studies seem to imply Figure 6.2 depicts a plot of the charge (Q)
that rigidifying the conformation of the and hydrophobicity (H) of the OAKs tested.
peptidomimetic oligomers may lead to It also highlights relationships that exist
improved antibacterial activity and selectivity, between HQ values and selective sequences
and consequently suggest potential adverse sequences that combine low haemolysis
eects of molecular flexibility in these (LC50 100 M, as assessed against human
respects. erythrocytes) with high antibacterial activity
(MIC 3.1 M, as assessed against
representatives of Gram-negative and Gram-
6.3 Acyl-lysyl Oligomers positive bacteria, E. coli and Staphylococcus
aureus, respectively). Table 6.1 shows the
6.3.1 Design biophysical properties of the most active
sequences (highlighted in Fig. 6.2).
Oligomers of acylated lysines (OAKs) were One of the main trends that emerged
originally designed to test the necessity for a from these data was that active sequences
stable fold in AMPs (Radzishevsky et al., presented a bell-shape behaviour with an
2007a,b). To promote the lack of a stable optimal window of HQ values per tested
secondary structure, acyl bridges were variable (Radzishevsky et al., 2008; Livne et
intercalated between cationic amino acid al., 2009). As shown in Fig. 6.2, selective
residues. The lack of hydrogen-bonding sequences also displayed a specific (relatively
opportunities in such constructs is expected narrow) window of HQ values for each
to limit the formation of stable folds because activity tested. Both the relative positions and
of the rotational freedom of the carbon atoms slopes are indicative of distinct HQ
within short-acyl chains (the particular case requirements for selective antibacterial
of long acyls will be discussed further activities. Thus, for E. coli, hydrophobicity
below). On the other hand, the OAK requirements were quite strict (i.e. H = 4550),
molecule is provided with a chance to reveal while charge levels were distributed over a
antimicrobial activity as it is composed of large band of values (Q = 69). These values
cationic and hydrophobic residues; both were strikingly dierent for S. aureus, both in
properties have long been suspected to tolerating a wider range of hydrophobicity
represent essential requirements for values (H = 4050) and in requiring lower
antimicrobial activity. This method therefore charge values (Q = 35). As detailed further
enables a fold-independent systematic tool below, selective antiplasmodial activity seems
for a gradual control of molecular hydro- to require a dierent yet distinct window of
phobicity (i.e. by changing acyl chain HQ values. This trend may therefore provide
lengths) and charge (by changing the nature, a useful guideline in the search for new and
position or number of cationic residues). improved OAK sequences.
Chemical Mimics with Systemic Efficacy 105
60
50
Hydrophobicity
40
30
20
0 1 2 3 4 5 6 7 8 9 10 11 12
Charge
Fig. 6.2. A charge (Q) and hydrophobicity (H) map of representative oligomers of an acylated lysine
(OAK) library. Plotted are the HQ values of each of >150 OAK sequences. Details are found elsewhere
(Radzishevsky et al., 2008; Livne et al., 2009). The distributions of the most selective OAKs (listed in
Table 6.1), defined as sequences that combine low haemolysis (median lethal concentration 100 M)
with high antibacterial activity (minimum inhibitory concentration 3.1 M), are highlighted. The light grey
and dark grey arrows represent the active window against Staphylococcus aureus and Escherichia coli,
respectively.
Table 6.1. List of the most selective oligomers of acylated lysines (OAKs; highlighted in Fig. 6.2).
OAK designation a OAK sequencea Qb Hb
Active against Gram-positive bacteria
*C12(7)K-12c C12(7)K-KC12Ka 3 50
C8212 C8-KC12K-KC12Ka 4 45
NC12212 NC12-KC12K-KC12Ka 5 38.5
Active against Gram-negative bacteria
C12K-58 C12K-C8K-C8K-C8K-C8K-C8Ka 6 49.7
C12K-68 C12K-C8K-C8K-C8K-C8K-C8K-C8Ka 7 50
C12K-78 C12K-C8K-C8K-C8K-C8K-C8K-C8K-C8Ka 8 47.5
C12K-88 C12K-C8K-C8K-C8K-C8K-C8K-C8K-C8K-C8Ka 9 48.5
*C12K-310 C12K-KC10K-KC10K-KC10Ka 7 44.9
C12K-410 C12K-KC10K-KC10K-KC10K-KC10Ka 9 44.7
a For N-terminal acyls: C8, octanoyl; C10, decanoyl; C12, dodecanoyl; NC12, aminododecanoyl; C12(7), dodecenoyl (all
intercalating residues are aminoacyl derivatives); a, amide.
b Q, charge; H, estimated hydrophobicity (percentage of acetonitrile eluent) as determined by reversed-phase high-
performance liquid chromatography using a C18 column.
c Asterisks indicate OAK sequences that displayed systemic antibacterial efficacy using the mouse thigh-infection
model.
106 A. Mor
self-assembly in aqueous media appear to be Cell death was apparently caused by the
responsible for poor antibacterial per- OAKs ability to interfere with DNA
formance and potential toxicity, as reflected functions in a similar manner to that of
by haemolysis. This issue was initially C12K-78 (Rotem et al., 2008), as evidenced by
addressed by attempting to manipulate a both the direct interaction with DNA under
highly aggregated short sequence (C16K-12) live conditions and the resulting inhibition of
that exhibited both antibacterial and biosynthesis.
haemolytic properties. Substitution of the There is a growing list of conventional
N-terminal acyl with its unsaturated AMPs such as PR-39 (Boman et al., 1993),
counterpart (C16(7)K-12) significantly altered tPMP-1 and HNP-1 (Xiong et al., 1999),
the OAKs supramolecular organization, even buforin II (Park et al., 2000) and pyrrhocoricin
though the critical aggregation concentration (Kragol et al., 2001) that are proposed to exert
(CAC) did not change (Sarig et al., 2008). antibacterial activity through interactions
Thus, although antibacterial performance did with intracellular targets (Nicolas, 2009).
improve, haemolytic activity remained high Combined with the OAK findings, the
enough to compromise its potential use in collective data show that small dierences
systemic therapy. Extending this study, the (such as a single charge, backbone length or
N-terminal acyl was replaced with the less even a single double bond) are required for
hydrophobic shorter acyl, dodecanoyl (Sarig an antibacterial compound to switch from
et al., 2010). As a result, the C12(7)K-12 CAC one mechanism to another. Further studies
became significantly higher than that of are, however, needed to validate this
C12K-12 (CACs of approximately 50 and 10 hypothesis, including investigation into the
M, respectively). Reversed-phase high- mechanism of action of a compound against
performance liquid chromatography analysis a large panel of bacterial species in order to
of these compounds confirmed that the acyl determine the possible generalization of this
substitution was accompanied by reduced phenomenon. The vast majority of mech-
hydrophobicity, as reflected by the elution anistic insights published in the literature are
time (50% and 54% acetonitrile eluent, based on investigations that have used a
respectively). As anticipated, disassembly single strain of a single bacterial species,
was responsible for reducing toxicity to which prohibits conclusions on whether the
mammalian cells as assessed with erythro- compound always uses a particular
cytes and primary fibroblasts. Yet C12(7)K-12 mechanism, or whether it uses distinct
displayed potent growth inhibitory activity mechanisms on distinct strains or species. It
against Gram-positive bacteria (MIC 2.55 g is also possible that an antibacterial com-
ml1), while Gram-negative bacteria were pound can use multiple or combined
generally less susceptible. Furthermore, the mechanisms. This is often touted in the
introduction of the double bond also literature, but has yet to be convincingly
drastically aected the antibacterial mech- demonstrated experimentally. Stretching this
anism of action. Thus, unlike C12K-12, which idea further, the data support the notion that
displayed classically rapid membrane simultaneous treatment of infections with a
disruptive bactericidal activity, the cocktail of compounds acting by distinct
unsaturated analogue C12(7)K-12 exhibited mechanisms may represent a more ecient
a strict bacteriostatic mode of action (Sarig et treatment approach. Nature seems to use this
al., 2010). In addition, recent unpublished strategy via the concomitant production and
data indicate that the more hydrophobic delivery of multiple closely related HDPs
unsaturated analogue C16(7)K-12 reverted (Levy et al., 1994; Mor et al., 1994).
back to the bactericidal mode of action Various preliminary in vivo investi-
(causing 99% death within 2 h) as assessed gations performed so far with four OAK
against multiple strains of dierent bacterial sequences have revealed a fair ability of
species. Interestingly, however, C16(7)K-12 OAK-based compounds to aect bacterial
did not disrupt the bacterial membrane(s). viability in mice and moreover highlighted
Chemical Mimics with Systemic Efficacy 109
shown these abilities in other cell types unclear. None the less, these studies suggest
(Rotem et al., 2008; Held-Kuznetsov et al., that ecient antimalarial therapeutic drugs
2009). can be engineered based on the physico-
The most recent attempts to generate chemical attributes imbedded in the mol-
improved antiplasmodial OAKs have re- ecular formulae of AMPs.
vealed additional octanoyl-based OAKs that
inhibit parasite growth at sub-micromolar
concentrations (mean IC50 0.3 0.1 M) 6.4 Concluding Remarks and
(unpublished data). Both the ring and Perspectives
trophozoite stages of the parasite develop-
mental cycle were sensitive to the most potent The fact that most HDPs and their synthetic
OAK, which moreover exhibited no mimics essentially act by non-specific
haemolytic activity at least up to 150 M mechanisms has long created serious doubts
(<0.4% haemolysis). Interestingly, the most as to their ability to exert sucient selectivity
potent non-haemolytic OAKs all localized to be considered ideal candidates for drug
near the S. aureus HQ window (Fig. 6.2), but development, particularly for systemic
contained distinct sequences (i.e. did not therapies. In that respect, an increasing
display antibacterial activity). Preliminary in number of recent studies have provided a
vivo data from acute toxicity, pharmacokinetic variety of counterarguments that significantly
and ecacy studies provide evidence for the contribute to dissipating this doubt.
potential of OAK sequences generated during Through distinct approaches for design-
this study to be considered suitable ing chemical mimics of HDPs, at least three
candidates for development as systemic major achievements have been realized:
antimalarials. Namely, upon daily intra- simplifying the pharmacophore composition,
peritoneal administration (4 25 mg kg1 increasing its robustness and miniaturizing
day1) to Plasmodium vinckei-infected mice, its size. These achievements should signifi-
the OAK displayed high blood concentrations cantly enhance the eciency of future
(approximately 100 M) that were sustained structureactivity relationship studies in
for at least several hours. It also demonstrated better defining the active principles, which
the ability to eliminate parasitaemia, albeit will in turn drive the discovery of new and
not without chronic toxicity. Nevertheless, the improved compounds.
encouraging results obtained in this study Beyond their capacity to aect microbial
regarding the selectivity and pharmacokinetic viability directly, various AMPs have been
properties of some compounds justify further proposed to significantly modulate host
investigation, with the aim of delivering immune responses. Another unmet challenge
antimalarial analogues with high safety. to be addressed in the future is therefore the
Although antiparasitic properties have eect of these chemical mimics on the host
not yet been investigated as thoroughly as immune system and its contribution to the
antibacterial activities, an increasing amount observed in vivo ecacies.
of experimental evidence supports the view
that the antiparasitic activity of AMPs also
emanates from interactions with multiple
targets. Remarkably, at least a few peptides Acknowledgement
exhibit in vitro high potency and selectivity
factors of several orders of magnitude, and This research was supported by the Israel
appear to be endowed with the formidable Science Foundation (grant 283/08).
ability to cross a number of membrane
systems before reaching their target(s) within
the intracellular parasite. While dierences References
in membrane composition are likely to
contribute to this selectivity, the molecular Adessi, C. and Soto, C. (2002) Converting a peptide
basis for these observations remains largely into a drug: strategies to improve stability and
Chemical Mimics with Systemic Efficacy 111
bioavailability. Current Medicinal Chemistry 9, Boman, H.G., Wade, D., Boman, I.A., Wahlin, B.
963978. and Merrifield, R.B. (1989) Antibacterial and
Andreu, D. and Rivas, L. (1998) Animal antimicrobial antimalarial properties of peptides that are
peptides: an overview. Biopolymers 47, cecropinmelittin hybrids. FEBS Letters 259,
415433. 103106.
Armand, P., Kirshenbaum, K., Goldsmith, R.A., Boman, H.G., Agerberth, B. and Boman, A. (1993)
Farr-Jones, S., Barron, A.E., Truong, K.T.V., Dill, Mechanisms of action on Escherichia coli of
K.A., Mierke, D.F., Cohen, F.E., Zuckermann, cecropin P1 and PR-39, two antibacterial
R.N. and Bradley, E.K. (1998) NMR determination peptides from pig intestine. Infection and
of the major solution conformation of a peptoid immunity 61, 29782984.
pentamer with chiral side chains. Proceedings Bradshaw, J.P. (2003) Cationic antimicrobial
of the National Academy of Sciences of the peptides issues for potential clinical use.
USA 95, 43094314. Biodrugs 17, 233240.
Arnt, L. and Tew, G.N. (2002) New Bremner, J.B., Keller, P.A., Pyne, S.G., Boyle, T.P.,
poly(phenyleneethynylene)s with cationic, Brkic, Z., David, D.M., Garas, A., Morgan, J.,
facially amphiphilic structures. Journal of the Robertson, M., Somphol, K., Miller, M.H., Howe,
American Chemical Society 124, 76647665. A.S., Ambrose, P., Bhavnani, S., Fritsche, T.R.,
Arnt, L. and Tew, G.N. (2003) Cationic facially Biedenbach, D.J., Jones, R.N., Buckheit, R.W.
amphiphilic poly(phenylene ethynylene)s Jr, Watson, K.M., Baylis, D., Coates, J.A.,
studied at the airwater interface. Langmuir 19, Deadman, J., Jeevarajah, D., McCracken, A.
24042408. and Rhodes, D.I. (2010) Binaphthyl-based
Avrahami, D., Oren, Z. and Shai, Y. (2001) Effect of dicationic peptoids with therapeutic potential.
multiple aliphatic amino acids substitutions on Angewandte Chemie (International ed. in
the structure, function, and mode of action of English) 49, 537540.
diastereomeric membrane active peptides. Brogden, K.A. (2005) Antimicrobial peptides: pore
Biochemistry 40, 1259112603. formers or metabolic inhibitors in bacteria?
Bals, R., Wang, X., Wu, Z., Freeman, T., Bafna, V., Nature Reviews Microbiology 3, 238250.
Zasloff, M. and Wilson, J.M. (1998) Human beta- Bruce-Chwatt, L.J. (1982) Chemoprophylaxis of
defensin 2 is a salt-sensitive peptide antibiotic malaria in Africa: the spent magic bullet. British
expressed in human lung. Journal of Clinical Medical Journal 285, 674676.
Investigation 102, 874880. Bulet, P., Stocklin, R. and Menin, L. (2004) Anti-
Beckloff, N., Laube, D., Castro, T., Furgang, D., microbial peptides: from invertebrates to
Park, S., Perlin, D., Clements, D., Tang, H., vertebrates. Immunological Reviews 198, 169
Scott, R.W., Tew, G.N. and Diamond, G. (2007) 184.
Activity of an antimicrobial peptide mimetic Chen, X., Tang, H., Even, M.A., Wang, J., Tew, G.N.
against planktonic and biofilm cultures of oral and Chen, Z. (2006) Observing a molecular
pathogens. Antimicrobial Agents and knife at work. Journal of the American Chemical
Chemotherapy 51, 41254132. Society 128, 27112714.
Bessalle, R., Kapitkovsky, A., Gorea, A., Shalit, I. Choi, S., Isaacs, A., Clements, D., Liu, D., Kim, H.,
and Fridkin, M. (1990) All-D-magainin: chirality, Scott, R.W., Winkler, J.D. and DeGrado, W.F.
antimicrobial activity and proteolytic resistance. (2009) De novo design and in vivo activity of
FEBS Letters 274, 151155. conformationally restrained antimicrobial
Bevins, C.L. and Zasloff, M. (1990) Peptides from arylamide foldamers. Proceedings of the
frog skin. Annual Review of Biochemistry 59, National Academy of Sciences of the USA 106,
395414. 69686973.
Blondelle, S.E. and Lohner, K. (2000) Combinatorial Chongsiriwatana, N.P., Patch, J.A., Czyzewski,
libraries: a tool to design antimicrobial and A.M., Dohm, M.T., Ivankin, A., Gidalevitz, D.,
antifungal peptide analogues having lytic Zuckermann, R.N. and Barron, A.E. (2008)
specificities for structureactivity relationship Peptoids that mimic the structure, function, and
studies. Biopolymers 55, 7487. mechanism of helical antimicrobial peptides.
Blondelle, S.E., Perez-Paya, E. and Houghten, R.A. Proceedings of the National Academy of
(1996) Synthetic combinatorial libraries: novel Sciences of the USA 105, 27942799.
discovery strategy for identification of Dartois, V., Sanchez-Quesada, J., Cabezas,E., Chi,
antimicrobial agents. Antimicrobial Agents and E., Dubbelde,C., Dunn,C., Granja, J., Gritzen,C.,
Chemotherapy 40, 10671071. Weinberger, D., Ghadiri, M.R. and Parr, R.T.
Boman, H.G. and Hultmark, D. (1987) Cell-free (2005) Systemic antibacterial activity of novel
immunity in insects. Annual Review of synthetic cyclic peptides. Antimicrobial Agents
Microbiology 41, 103126. and Chemotherapy 49, 33023310.
112 A. Mor
Deslouches, B., Islam, K., Craigo, K.J., Paranjape, Gwadz, R.W., Kaslow, D., Lee, J.Y., Maloy, W.L.,
M.S., Montelaro, C.R. and Mietzner, A.T. (2005) Zasloff, M. and Miller, L.H. (1989) Effects of
Activity of the de novo engineered antimicrobial magainins and cecropins on the sporogonic
peptide WLBU2 against Pseudomonas development of malaria parasites in mosquitoes.
aeruginosa in human serum and whole blood: Infection and Immunity 57, 26282633.
implications for systemic applications. Hancock, R.E. and Lehrer, R. (1998) Cationic
Antimicrobial Agents and Chemotherapy 49, peptides: a new source of antibiotics. Trends in
32083216. Biotechnology 16, 8288.
Easton, M.D., Nijnik, A., Mayer, L.M. and Hancock Held-Kuznetsov, V., Rotem, S., Assaraf, Y.G. and
R.E.W. (2009) Potential of immunomodulatory Mor, A. (2009) Host-defense peptide mimicry for
host defense peptides as novel anti-infectives. novel antitumor agents. FASEB Journal 23,
Trends in Biotechnology 27, 582590. 42994307.
Eichler, J. and Houghten, R.A. (1995) Generation Hoffmann, J.A. (1995) Innate immunity of insects.
and utilization of synthetic combinatorial Current Opinions in Immunology 7, 410.
libraries. Molecular Medicine Today 1, 174180. Ishitsuka, Y., Arnt, L., Majewski, J., Frey, S.,
Epand, R.F., Lehrer, R.I., Waring, A., Wang, W., Ratajczek, M., Kjaer, K., Tew, G.N. and Lee, K.Y.
Maget-Dana, R., Lelivre, D. and Epand, R.M. (2006) Amphiphilic poly(phenyleneethynylene)s
(2003) Direct comparison of membrane can mimic antimicrobial peptide membrane
interactions of model peptides composed of disordering effect by membrane insertion.
only Leu and Lys residues. Biopolymers 71, Journal of the American Chemical Society 128,
216. 1312313129.
Epand, R.M., Epand, R.F. and Savage, P.B. (2008a) Javadpour, M.M. and Barkley, M.D. (1997) Self-
Ceragenins (cationic steroid compounds), a assembly of designed antimicrobial peptides in
novel class of antimicrobial agents. Drug News solution and micelles. Biochemistry 36,
& Perspectives 21, 307311. 95409549.
Epand, R.M., Rotem, S., Mor, A., Berno, B., Epand, Jenssen, H., Hammill, P. and Hancock, R.E. (2006)
R.F. (2008b) Bacterial membranes as predictors Peptide antimicrobial agents. Clinical
of antimicrobial potency. Journal of the American Microbiology Reviews 19, 491511.
Chemical Society 130, 1434614352.
Jing, W., Hunter, H.N., Hagel, J. and Vogel, H.J.
Epand, R.F., Sarig, H., Mor, A. and Epand, R.M.
(2003) The structure of the antimicrobial peptide
(2009) Cell-wall interactions and the selective
Ac-RRWWRF-NH2 bound to micelles and its
bacteriostatic activity of a miniature oligo-acyl-
interactions with phospholipid bilayers. Journal
lysyl. Biophysical Journal 97, 22502257.
of Peptide Research 61, 219229.
Feder, R., Dagan, A. and Mor, A. (2000) Structure
Kirshenbaum, K., Barron, A.E., Goldsmith, R.A.,
activity relationship study of antimicrobial
Armand, P., Bradley, E.K., Truong, K.T.V., Dill,
dermaseptin S4 showing the consequences of
K.A., Cohen, F.E. and Zuckermann, R.N. (1998)
peptide oligomerization on selective cytotoxicity.
Sequence-specific polypeptoids: a diverse
Journal of Biological Chemistry 275, 4230
family of heteropolymers with stable secondary
4238.
structure. Proceedings of the National Academy
Ganz, T. (1999) Defensins and host defense.
Science 286, 420421. of Sciences of the USA 95, 43034308.
Goldman, M.J., Anderson, G.M., Stolzenberg, E.D., Kragol, G., Lovas, S., Varadi, G., Condie, B.A.,
Kari, U.P., Zasloff, M. and Wilson, J.M. (1997) Hoffmann, R. and Otvos, L. Jr (2001) The
Human -defensin-1 is a salt-sensitive antibiotic antibacterial peptide pyrrhocoricin inhibits the
in lung that is inactivated in cystic fibrosis. Cell ATPase actions of DnaK and prevents
88, 553560. chaperone-assisted protein folding. Biochemistry
Gordon, Y.J., Romanowski, E.G. and McDermott, 40, 30163026.
A.M. (2005) A review of antimicrobial peptides Krishna, S., Woodrow, C.J., Staines, H.M., Haynes,
and their therapeutic potential as anti-infective R.K. and Mercereau-Puijalon, O. (2006)
drugs. Current Eye Research 30, 505515. Re-evaluation of how artemisinins work in light
Guarna, M.M., Coulson, R. and Rubinchik, E. of emerging evidence of in vitro resistance.
(2006) Anti-inflammatory activity of cationic Trends in Molecular Medicine 12, 200205.
peptides: application to the treatment of acne Lai, X.Z., Feng, Y., Pollard, J., Chin, J.N., Rybak,
vulgaris. Fems Microbiology Letters 257, 16. M.J., Bucki, R., Epand, R.F., Epand, R.M. and
Gunn, J.S. (2008) The Salmonella PmrAB regulon: Savage, P.B. (2008) Ceragenins: cholic acid-
lipopolysaccharide modifications, antimicrobial based mimics of antimicrobial peptides.
peptide resistance and more. Trends in Accounts of Chemical Research 41,
Microbiology 16, 284290. 12331240.
Chemical Mimics with Systemic Efficacy 113
Latham, P.W. (1999) Therapeutic peptides revisited. Nicolas, P. (2009) Multifunctional host defense
Nature Biotechnology. 17, 755757. peptides: intracellular-targeting antimicrobial
Lee, I.H., Cho, Y. and Lehrer R.I. (1997) Effects of peptides. FEBS Journal 276, 64836496.
pH and salinity on the antimicrobial properties Nicolas, P. and Mor, A. (1995) Peptides as weapons
of clavanins. Infection and Immunity 65, against microorganisms in the chemical defense
28982903. system of vertebrates. Annual Reviews in
Levy, O., Ooi, C.E., Weiss, J., Lehrer, R.I. and Microbiology 49, 277304.
Elsbach, P. (1994) Individual and synergistic Oh, J.E., Hong, S.Y. and Lee, K.H. (1999) Design,
effects of rabbit granulocyte proteins on synthesis and characterization of antimicrobial
Escherichia coli. Journal of Clinical Investigation pseudopeptides corresponding to membrane-
94, 672682. active peptide. Journal of Peptide Research 54,
129136.
Liu, D., Choi, S., Chen, B., Doerksen, R.J.,
Otto, M. (2009) Bacterial sensing of antimicrobial
Clements, D.J., Winkler, J.D., Klein, M.L. and
peptides. Contributions to Microbiology 16,136
DeGrado, W.F. (2004) Nontoxic membrane-
149.
active antimicrobial arylamide oligomers.
Park, C.B., Yi, K.S., Matsuzaki, K., Kim, M.S. and
Angewandte Chemie (International ed. in
Kim, S.C. (2000) Structureactivity analysis of
English) 43, 11581162. buforin II, a histone H2A-derived antimicrobial
Livne, L., Kovachi, T., Sarig, H., Epand, R.F., peptide: the proline hinge is responsible for the
Zaknoon, F., Epand, R.M. and Mor, A. (2009) cell-penetrating ability of buforin II. Proceedings
Design and characterization of a broad- of the National Academy of Sciences of the
spectrum bactericidal acyl-lysyl oligomer. USA 97, 82458250.
Chemistry & Biology 16, 12501258. Patch, J.A. and Barron, A.E. (2002) Mimicry of
Makobongo, M.O., Kovachi, T., Gancz, H., Mor, A. bioactive peptides via non-natural, sequence
and Merrell, D.S. (2009) In vitro antibacterial specific peptidomimetic oligomers. Current
activity of acyl-lysyl oligomers against Opinion in Chemical Biology 6, 872877.
Helicobacter pylori. Antimicrobial Agents and Perron, G.G., Zasloff, M. and Bell, G. (2006)
Chemotherapy 53, 42314239. Experimental evolution of resistance to an
Marr, A.K., Gooderham, W.J. and Hancock, R.E. antimicrobial peptide. Proceedings of the Royal
(2006) Antibacterial peptides for therapeutic Society B Biological Sciences 273, 251256.
use: obstacles and realistic outlook. Current Radzishevsky, I.S., Rotem, S., Zaknoon, F.,
Opinion in Pharmacology 6, 468472. Gaidukov, L., Dagan, A. and Mor, A. (2005)
McInturff, J.E., Wang, S.J., Machleidt, T., Lin, T.R., Effects of acyl versus aminoacyl conjugation on
Oren, A., Hertz, C.J., Krutzik, S.R., Hart, S., the properties of antimicrobial peptides.
Zeh, K., Anderson, D.H., Gallo, R.L., Modlin, Antimicrobial Agents and Chemotherapy 49,
R.L. and Kim, J. (2005) Granulysin-derived 24122420.
peptides demonstrate antimicrobial and anti- Radzishevsky, I.S., Krugliak, M., Ginsburg, H., and
inflammatory effects against Propionibacterium Mor, A. (2007a) Antiplasmodial activity of lauryl-
acnes. Journal of Investigative Dermatology lysine oligomers. Antimicrobial Agents and
125, 256263. Chemotherapy 51, 17531759.
Radzishevsky, I.S., Rotem, S., Bourdetsky, D.,
Miller, S.M., Simon, R.J., Ng, S., Zuckermann, R.N.,
Navon-Venezia, S., Carmeli, Y. and Mor, A.
Kerr, J.M. and Moos, W.H. (1994) Proteolytic
(2007b) Improved antimicrobial peptides based
studies of homologous peptide and N-substituted
on acyl-lysine oligomers. Nature Biotechnology
glycine peptoid oligomers. Bioorganic &
25, 657659.
Medicinal Chemistry Letters 4, 26572662.
Radzishevsky, I.S., Kovachi, T., Porat, Y., Ziserman,
Minahk, C.J. and Morero, R.D. (2003) Inhibition of L., Zaknoon, F., Danino, D. And Mor, A. (2008)
enterocin CRL35 antibiotic activity by mono- Structureactivity relationships of antibacterial
and divalent ions. Letters in Applied Microbiology acyl-lysine oligomers. Chemistry & Biology 15,
37, 374379. 354362.
Mor, A. (2009) Multifunctional host defense Rivas, L., Luque-Ortega, J.R. and Andreu, D. (2008)
peptides: antiparasitic activities. FEBS Journal Amphibian antimicrobial peptides and protozoa:
276, 64746482. lessons from parasites. Biochimica et Biophysica
Mor, A., Hani, K. and Nicolas, P. (1994) The Acta 1788, 15701581.
vertebrate peptide antibiotics dermaseptins Robinson, J.A., Demarco, S., Gombert, F., Moehle,
have overlapping structural features but target K. and Obrecht, D. (2008) The design, structures
specific microorganisms. Journal of Biological and therapeutic potential of protein epitope
Chemistry 269, 3163531641. mimetics. Drug Discovery Today 13, 944951.
114 A. Mor
Rotem, S. and Mor, A. (2009) Antimicrobial peptide Riedel, K., Demarco, S.J. and Robinson, J.A.
mimics for improved therapeutic properties. (2010) Peptidomimetic antibiotics target outer-
Biochimica et Biophysica Acta 1788, membrane biogenesis in Pseudomonas
15821592. aeruginosa. Science 327, 10101013.
Rotem, S., Radzishevsky, I. and Mor, A. (2006) Szabo, D., Ostorhazi, E., Binas, A., Rozgonyi, F.,
Physicochemical properties that enhance Kocsis, B., Cassone, M., Wade, J.D., Nolte, O.
discriminative antibacterial activity of short and Otvos, L. Jr (2010) The designer proline-
dermaseptin derivatives. Antimicrobial Agents rich antibacterial peptide A3-APO is effective
and Chemotherapy 50, 26662672. against systemic Escherichia coli infections in
Rotem, S., Radzishevsky, I.S., Bourdetsky, D., different mouse models. International Journal of
Navon-Venezia, S., Carmeli, Y. and Mor, A. Antimicrobial Agents 35, 357361.
(2008) Analogous oligo-acyl-lysines with distinct Tang, H., Doerksen, R.J., Jones, T.V., Klein, M.L.
antibacterial mechanisms. FASEB Journal 22, and Tew, G.N. (2006) Biomimetic facially
26522661 amphiphilic antibacterial oligomers with
Rozek, A., Powers, J.P, Friedrich, C.L. and Hancock conformationally stiff backbones. Chemistry &
R.E. (2003) Structure-based design of an Biology 13, 427435.
indolicidin peptide analogue with increased Tew, G.N., Liu, D., Chen, B., Doerksen, R.J.,
protease stability. Biochemistry 42, 14130 Kaplan, J., Carroll, P.J., Klein, M.L. and DeGrado,
14138. W.F. (2002) De novo design of biomimetic
Rydlo, T., Rotem, S. and Mor, A. (2006) Antibacterial antimicrobial polymers. Proceedings of the
properties of dermaseptin S4 derivatives under National Academy of Sciences of the USA 99,
extreme incubation conditions. Antimicrobial 51105114.
Agents and Chemotherapy 50, 490497. Tew, G.N., Clements, D., Tang, H., Arnt, L. and
Sarig, H., Rotem, S., Ziserman, L., Danino, D. and Scott, R.W. (2006) Antimicrobial activity of an
Mor, A. (2008) Impact of self-assembly abiotic host defense peptidemimic. Biochimica
properties on antibacterial activity of short acyl- et Biophysica Acta 1758, 13871392.
lysine oligomers. Antimicrobial Agents and Tew, G.N., Scott, R.W., Klein, M.L. and Degrado
Chemotherapy 52, 43084314. W.F. (2010) De novo design of antimicrobial
Sarig, H., Livne, L., Held-Kuznetsov, V., Zaknoon, polymers, foldamers, and small molecules: from
F., Ivankin, A., Gidalevitz, D. and Mor, A. (2010) discovery to practical applications. Accounts of
A miniature mimic of host defense peptides with Chemical Research 43, 3039.
systemic antibacterial efficacy. FASEB Journal Tossi, A., Tarantino, C. and Romeo, D. (1997)
24, 19041913. Design of synthetic antimicrobial peptides
Scott, R.W., DeGrado, W.F. and Tew, G.N. (2008) based on sequence analogy and amphipathicity.
De novo designed synthetic mimics of European Journal of Biochemistry 250, 549
antimicrobial peptides. Current Opinion in 558.
Biotechnology 19, 620627. Tossi, A., Sandri, L. and Giangaspero, A. (2000)
Seo, J., Barron, A.E. and Zuckermann, R.N. (2010) Amphipathic, -helical antimicrobial peptides.
Novel peptoid building blocks: synthesis of Biopolymers 55, 430.
functionalized aromatic helix-inducing Uchida, M., McDermott, G., Wetzler, M., Le Gros,
submonomers. Organic Letters 12, 492495. M.A., Myllys, M., Knoechel, C., Barron, A.E. and
Shai, Y. (2002) Mode of action of membrane active Larabell, C.A. (2009) Soft X-ray tomography of
antimicrobial peptides. Biopolymers 66, 236 phenotypic switching and the cellular response
248. to antifungal peptoids in Candida albicans.
Shai, Y. and Oren, Z. (1996) Diastereoisomers of Proceedings of the National Academy of
cytolysins, a novel class of potent antibacterial Sciences of the USA 106, 1937519380.
peptides. Journal of Biological Chemistry 271, Van Bambeke, F., Mingeot-Leclercq, M.P.,
73057308. Struelens, M.J. and Tulkens, P.M. (2008) The
Som, A., Vemparala, S., Ivanov, I. and Tew, G.N. bacterial envelope as a target for novel anti-
(2008) Synthetic mimics of antimicrobial MRSA antibiotics. Trends in Pharmacological
peptides. Biopolymers 90, 8393. Sciences 29, 124134.
Srinivas, N., Jetter, P., Ueberbacher, B.J., Wade, D., Boman, A., Whlin, B., Drain, C.M.,
Werneburg, M., Zerbe, K., Steinmann, J., Van Andreu, D., Boman, H.G. and Merrifield, R.B.
der Meijden, B., Bernardini, F., Lederer, A., (1990) All-D amino acid-containing channel-
Dias, R.L., Misson, P.E., Henze, H., Zumbrunn, forming antibiotic peptides. Proceedings of the
J., Gombert, F.O., Obrecht, D., Hunziker, P., National Academy of Sciences of the USA 87,
Schauer, S., Ziegler, U., Kch, A., Eberl, L., 47614765.
Chemical Mimics with Systemic Efficacy 115
Wu, C.W., Sanborn, T.J., Huang, K., Zuckermann, Yang, L., Gordon, V.D., Trinkle, D.R., Schmidt, N.W.,
R.N. and Barron, A.E. (2001) Peptoid oligomers Davis, M.A., DeVries, C., Som, A., Cronan, J.E.
with -chiral, aromatic side chains: sequence Jr, Tew, G.N. and Wong, G.C. (2008) Mechanism
requirements for the formation of stable peptoid of a prototypical synthetic membrane-active
helices. Journal of the American Chemical antimicrobial: efficient hole-punching via
Society 123, 67786784. interaction with negative intrinsic curvature
Wu, C.W., Kirshenbaum, K., Sanborn, T.J., Patch, lipids. Proceedings of the National Academy of
J.A., Huang, K., Dill, K.A., Zuckermann, R.N. Sciences of the USA 105, 2059520600.
and Barron, A.E. (2003) Structural and Yeaman, M.R. and Yount, N.Y. (2003) Mechanisms
spectroscopic studies of peptoid oligomers with of antimicrobial peptide action and resistance.
-chiral aliphatic side chains. Journal of the Pharmacological Reviews 55, 2755.
American Chemical Society 125, 13525 Zaknoon, F., Sarig, H., Rotem, S., Livne, L., Ivankin,
13530. A., Gidalevitz, D. and Mor, A. (2009) Antibacterial
Xiong, Y.Q., Yeaman, M.R. and Bayer, A.S. (1999) properties and mode of action of a short acyl-
In vitro antibacterial activities of platelet lysyl oligomer. Antimicrobial Agents and
microbicidal protein and neutrophil defensin Chemotherapy 53, 34223429.
against Staphylococcus aureus are influenced Zasloff, M. (2002) Antimicrobial peptides of
by antibiotics differing in mechanism of action. multicellular organisms. Nature 415, 389395.
Antimicrobial Agents and Chemotherapy 43, Zuckermann, R.N., Kerr, J.M., Kent, S.B.H. and
11111117. Moos, W.H. (1992) Efficient method for the
Yang, L., Gordon, V.D., Mishra, A., Som, A., Purdy, preparation of peptoids [oligo(N-substituted
K.R., Davis, M.A., Tew, G.N. and Wong, G.C. glycines)] by submonomer solid-phase
(2007) Synthetic antimicrobial oligomers induce synthesis. Journal of the American Chemical
a composition-dependent topological transition Society 114, 1064610647.
in membranes. Journal of the American
Chemical Society 129, 1214112147.
7 Biophysical Analysis of Membrane-
targeting Antimicrobial Peptides:
Membrane Properties and the Design of
Peptides Specifically Targeting Gram-
negative Bacteria
Abstract
Gram-negative bacteria are more resistant to many antimicrobial agents than Gram-positive bacteria.
This is because drugs must pass through an additional barrier of the outer membrane in order to access
the cytoplasmic membrane or the interior of the cell. In spite of this fact, the presence of the outer
membrane also provides an alternative target for the action of antimicrobial agents. In addition, the
cytoplasmic membranes of Gram-negative bacteria are generally more enriched with the phospholipid
phosphatidylethanolamine than those of Gram-positive bacteria. In this chapter, we outline novel
strategies for using the interaction of antimicrobial agents with components of the membranes of Gram-
negative bacteria in order to design agents that have a higher toxicity for these bacteria.
* Corresponding author.
Fig. 7.1. Schematic diagram of the architecture of the membranes of Gram-negative and Gram-positive
bacteria (not to scale). (A) Gram-negative bacteria (from left to right): O-antigen depicted as hexagons on
the outer monolayer of the outer membrane; porins traversing the lipopolysaccharide (LPS) layer are
depicted as cylinders; the LPS layer is followed by lipoproteins; the peptidoglycan layer is shown as dots;
ovals represent peptides; the inner membrane is presented with transmembrane proteins. The complete
LPS structure is shown on top and the peptidoglycan structure to the right. G, N-acetylglucosamine; M,
N-acetylmuramic acid; DAP, diaminopipelic acid. (B) Gram-positive bacteria (from left to right): teichoic
acid (TA) and lipoteichoic acid (LTA) as long, thin structures reaching through the peptidoglycan layer,
which is depicted as dots; peptides are ovals; the membrane is shown with transmembrane proteins. The
LTA structure is shown on top and the peptidoglycan structure to the right.
118 R.M. Epand and R.F. Epand
layer (Fig. 7.1A). If it has a molecular mass components that cationic antimicrobial
larger than approximately 700 Da then it peptides (AMPs) are exposed to on their
crosses the lipopolysaccharide (LPS) layer, journey towards the cytoplasmic membrane,
which is about 8 nm thick. If it does not bind and the number of other components they
completely to the phosphate moieties of the come in contact with, they emerge as truly
lipid A portion in the LPS molecule then it amazing agents.
will continue its journey through a number In addition to the dierences described
of lipoproteins and a peptidoglycan layer. In above, bacterial species vary greatly in the
Gram-negative bacteria, the peptidoglycan phospholipid composition of their mem-
layer comprises only 10% of the dry weight branes. In general, the membranes of Gram-
of the cell and it is composed of a glycan negative bacteria have a far higher
backbone of alternating repeating sugar phosphatidylethanolamine (PE) content than
units of N-acetylglucosamine (G) and those of Gram-positive bacteria, although
N-acetylmuramic acid (M). These are cross- there are some exceptions.
linked directly by four amino acids (two l-
and two d-amino acids) on each layer, thus
forming a rigid mesh that allows facile
passage of peptides. The peptide molecules 7.2 Role of Bacterial Membrane Lipid
accumulate in the periplasmic space, Composition in Antimicrobial
achieving high concentrations that can be Sensitivity
many fold the bulk concentration of peptide.
From there they are attracted electrostatically Some antimicrobial agents target specific
to the outer leaflet of the inner membrane. At lipid components of bacterial membranes.
each step the peptide molecules are exposed An example of this is the AMP nisin, which
to a number of proteins that participate in binds specifically to lipid II (Breukink et al.,
specific biosynthetic and other functions of 1999). Duramycin and cinnamycin are two
the bacteria. other AMPs of the same lantibiotic class as
In trying to gain entry into a Gram- nisin that specifically target PE (Clejan et al.,
positive bacterium, a peptide encounters 1989). In addition to drugs that target specific
teichoic acid covalently bound to a sugar lipids, less specific interactions of anti-
backbone and lipoteichoic acid covalently microbials with membrane lipids, such as
bound to the membrane (Fig. 7.1B). It gains electrostatic or hydrogen-bonding inter-
direct access to the peptidoglycan layer, actions, can also influence the potency and
which is 50% of the dry weight of the cell mechanism of action of antimicrobial agents.
and contains a number of associated proteins. This can be particularly important because
The peptidoglycan layer in Gram-positive the lipid composition of bacterial membranes
bacteria diers from the one in Gram- varies widely, resulting in certain bacterial
negative bacteria in the linkage between the species being more susceptible to some
amino acids. This is not direct, but is rather antimicrobial agents than others. For
done through an amino acid bridge, which example, the selective toxicity of two
varies within species as a result of adaptation. isomeric /-peptides is dependent on the
This makes the peptidoglycan layer wider lipid phase preference of the lipids in the
and more accessible to the peptide, which bacterial membrane (Epand et al., 2005). The
can easily traverse it to reach the outer leaflet toxicity of a cationic steroid compound has
of the cytoplasmic membrane, in spite of the been shown to be dependent on the PE
fact that this layer is 2040 nm in length. content of the membrane (Epand et al., 2007).
Gram-positive bacteria have little or no Additionally, the toxicity of certain oligo-
periplasm; consequently, the cationic pep- acyl-lysines is most potent with bacteria that
tides accumulate directly on the anionic contain both anionic and either zwitterionic
lipids by electrostatic attraction, reaching or uncharged lipid in their membrane, so
threshold concentrations for membrane that these agents can promote the clustering
disruption. Given the number of anionic of anionic lipid (Epand, R.M. et al., 2008). The
Analysis of Membrane-targeting AMPs 119
Table 7.1. Phosphatidylethanolamine content and CSA-8 minimum inhibitory concentration for several
species of bacteria.
Minimum inhibitory Phosphatidylethanolamine
Bacteria concentration (g ml1) content (%)
Gram-negative bacteria
Proteus mirabilis 500 80
Escherichia coli 36 80
Pseudomonas aeruginosa 20 60
Caulobacter crescentus 5 ~0
Gram-positive bacteria
Bacillus polymyxa 35 60
Bacillus anthracis 20 43
Bacillus cereus 50 43
Bacillus subtilis 0.5 12
Staphylococcus aureus 2 ~0
Streptococcus pyogenes 0.5 ~0
variation in PE content for several species of 7.3 Antimicrobial Agents that Target
bacteria is shown in Table 7.1. the Outer Membrane of Gram-
Table 7.1 also shows some exceptions to negative Bacteria
the general rule. For example, the Gram-
negative bacterium Caulobacter crescentus has In addition to damage to the cytoplasmic
a low PE content while three related Gram- membrane that may lead to toxicity, one must
positive bacteria, Bacillus polymyxa, Bacillus also consider a role for the outer membrane
anthracis and Bacillus cereus, have a higher of Gram-negative bacteria. There are several
content of PE. A variable PE content can also ways in which the outer membrane can aect
be found in the genus Clostridium. bacterial viability. The outer membrane and
The relationship of PE content to the cell wall provide structural rigidity to protect
antimicrobial action of the cationic sterol the cell against damage caused, for example,
compound CSA-8 illustrates the importance by osmotic stress. Several classical antibiotics
of membrane phospholipid content to the act by aecting the synthesis of cell wall or
potency of this antimicrobial agent (Epand et outer membrane components.
al., 2007). This conclusion was further Another role of the outer membrane can
confirmed using a mutant form of Escherichia be to act as a barrier for the passage of
coli that was unable to synthesize PE (Epand antimicrobial agents to the plasma mem-
et al., 2007). This study demonstrates that the brane. The interaction of negatively charged
lipid composition of the bacterial membrane LPS of the outer membrane with cationic
can be more important in determining antimicrobial agents can prevent the toxicity
sensitivity to an antimicrobial agent than the of these agents by inhibiting access to the cell
presence or absence of an outer membrane. membrane. An interesting example of this is
This is an example of the more common the blockage of penetration of the small
situation in which Gram-positive bacteria are cationic peptides, the temporins (Mangoni et
more sensitive to the action of an al., 2008). This study showed the dependence
antimicrobial agent than Gram-negative of the barrier function of the outer membrane
bacteria. In the next paragraphs, we describe on the length of the polysaccharide chain of
other cases in which antimicrobial agents can LPS. In addition, synergistic action between
be more toxic to bacteria with a high PE pairs of temporin molecules was demon-
content. strated.
120 R.M. Epand and R.F. Epand
acquire well-resolved spectra using high one lipid from another in a mixture using
molecular weight membrane samples. A Fourier transform infrared spectroscopy
convenient isotope to use for the study of (Arouri et al., 2009). Deuteration is performed
membranes is 31P. There are few phosphates, to separate the absorption frequencies of the
usually only one, in most phospholipids. The deuterated and protonated components. The
paramagnetic isotope of phosphorous, 31P, has CH2 stretching vibrations are sensitive to the
a natural abundance of almost 100% and 31P conformation of the lipid acyl chains, and can
has a spin quantum number of , resulting in be used to follow the changes in the trans to
it not being broadened by nuclear-quadrupole gauche ratio. For the protonated component,
coupling. There have been two applications of the frequencies of the symmetric and
31P MAS/NMR to test the formation of anionic antisymmetric CH2 stretching vibrations are
lipid clusters with antimicrobial agents at approximately 2850 cm1 and 2929 cm1,
(Epand, R.M. et al., 2008; Epand et al., 2009b). respectively. With this method, the
Even in lipid mixtures that appear to mix antimicrobial cationic peptide RRWWRF
homogeneously, 31P MAS/NMR still allows has been shown to cause segregation of
the separate identification of each component anionic and zwitterionic lipids only in the gel
of the mixture if they have suciently phase with dimyristoyl-phosphatidylcholine
dierent chemical shifts. Although the (DMPC)/dipalmitoyl-phosphatidylglycerol
chemical shift of each lipid component will be (DPPG) mixtures, while with dipalmitoyl-
towards each other as a result of the presence phosphatidylethanolamine (DPEE)/DPPG a
of the other lipid in the environment, the two lipid mixture more representative of bacterial
peaks are still resolvable (Epand, R.M. et al., membranes phase separation was observed
2008; Epand et al., 2009b). If clustering of in both liquid and solid phases (Arouri et al.,
anionic lipid occurs, the isotropic chemical 2009).
shift of the zwitterionic component will be All of the above methods have their
more towards that of the pure zwitterionic advantages and limitations. However, they
component. The line width of each peak is a are all indirect with regard to specifying the
second independent parameter that can also morphological properties of the samples.
be used to test for demixing. The line width of Some imaging methods have also been
the anionic component increases as a result of applied that give information about the size
slower motion after binding the cationic anti- and properties of the domains. A recent study
microbial agent, while that of the zwitterionic using atomic force microscopy in combination
component changes from its value in the lipid with polarized fluorescence microscopy
mixture to that found for the pure zwitterionic showed the formation of domains in model
component. This analysis has also been membranes using a small cationic AMP,
performed as a function of temperature to PFWRIRIRR-amide (Oreopoulos et al., 2010).
show the generality of the result when both This study showed the time evolution of the
components are in the liquid crystalline formation of these domains. In addition,
phase. freeze-fracture electron microscopy has
Another NMR approach makes use of provided evidence for the formation of
2H-NMR to measure order parameters of domain structures with this same cationic
deuterated lipids (Jean-Francois et al., 2008). AMP (Epand, R.M. et al., 2010). There is thus
Anionic and zwitterionic lipid components direct evidence from imaging studies, for the
can be separately studied by deuteration of peptide PFWRIRIRR-amide, that membrane
one of the components of the pair. The results domains are formed.
suggest that the zwitterionic component
experiences two dierent environments that
are in slow exchange. One of the components
resembles that of the pure zwitterionic 7.4.2 Bacterial species specificity of
component. These results are also in accord toxicity
with the clustering of anionic lipid to produce
a domain enriched in zwitterionic lipid. The ability of certain antimicrobial agents to
Deuteration can also be used to distinguish cluster anionic lipids is of particular interest
122 R.M. Epand and R.F. Epand
because it explains the selective toxicity of principal one being sphingosine, bacteria can
these agents towards certain bacterial have large amounts of cationic lipids. The
species. If an antimicrobial agent has greater separation of cationic and anionic lipids by
anity for one lipid species over another, cationic antimicrobial agents would not be
this is sucient to promote segregation of very ecient because the cationic lipid
lipid components. This can happen even would prevent the cationic peptide from
with a mixture of two lipids with dierent binding to the membrane. A cationic lipid
anionic headgroups, as we have shown with that is frequently found in bacterial
cationic amphipathic helical peptides that membranes is lysyl-PG. It has been shown
can segregate CL from phosphatidylglycerol that the presence of cationic lipids is a factor
(PG) (Epand, R.F. et al., 2010); however, this in increasing the resistance of bacteria to
phenomenon is not associated with the cationic antimicrobial agents (Peschel et al.,
specificity of toxicity for dierent bacterial 2001; Camargo et al., 2008). However, at least
species. In contrast, when an anionic lipid is in the case of Staphylococcus aureus, most of
clustered away from lipids that are this lipid is found on the cytoplasmic surface
zwitterionic or have no overall charge, the of the plasma membrane. Resistance to
result is a bacteriostatic action. The reason antimicrobial agents occurs in those strains
why these two dierent kinds of lipid of S. aureus in which the lipid is translocated
clustering have dierent consequences for to the cell exterior by a mechanism facilitated
bacterial toxicity is not known, but may by a membrane protein (Ernst et al., 2009;
reflect dierences in the size of the domains Mishra et al., 2009). Although the membrane-
that are formed or the nature of the phase sidedness of lysyl-PG in S. aureus has been
boundary between domains. determined, in general there is little infor-
The sequestering of anionic lipids away mation about the sidedness of membrane
from ones with no overall charge will occur lipids in bacteria.
only in bacterial species that contain We will now summarize the ratio of the
significant amounts of zwitterionic or minimal inhibitory concentration (MIC)
uncharged lipids. All bacterial species contain against S. aureus and E. coli. These two
large fractions of anionic lipids, generally in bacterial species represent a species with a
the form of PG and CL. However, their low content of neutral and zwitterionic
content of zwitterionic or uncharged lipids lipids (S. aureus) and another with a high
varies widely, from close to 0% to about 85%. content of the zwitterionic lipid PE (E. coli;
The most common zwitterionic lipid is PE. In see Table 7.1). We have chosen these two
addition, some bacteria have species of species as representative because they are
aminoacyl-PG, most of which are zwitterionic, commonly used and there is more
as well as zwitterionic lysyl-CL. Uncharged information about MICs with these bacteria.
glycosyl-diacylglycerols may also be present. We recognize that there is a potential
However, few bacterial species have a large complication with S. aureus because some
amount of this lipid as a free polar lipid strains contain lysine-PG, but since this lipid
component of the plasma membrane, an is generally on the cytoplasmic side of the
exception being Streptococcus pyogenes in membrane, we assume it will not greatly
which some of the membrane lipids are free aect the measured values of the MIC. Thus,
glycosyl-diglycerides (Shaw, 1970; Rosch et S. aureus represents a bacteria that cannot
al., 2007). It should also be pointed out that segregate anionic from zwitterionic or
glycosyl-diglycerides are also components of charged lipids, while E. coli can segregate
lipoteichoic acid (LTA), a major component of these two types of lipids. An antimicrobial
the cell wall of Gram-positive bacteria. agent that acts largely by the lipid clustering
However, this glycosyl-diglyceride is not mechanism will thus be more toxic to E. coli
usually free as a phospholipid component of than to S. aureus and hence have a high ratio
the cell membrane, but rather is a covalently of S. aureus MIC to E. coli MIC. These two
linked moiety of the cell wall. bacteria also dier in that E. coli is a Gram-
Unlike mammalian membranes, which negative bacterium with an outer membrane,
have very low amounts of cationic lipids, the while S. aureus is a Gram-positive bacterium.
Analysis of Membrane-targeting AMPs 123
Another factor lowering the ratio of S. aureus S. aureus/MIC E. coli is >1 for all of the other
MIC to E. coli MIC may be a result of the MSI peptides.
outer membrane of E. coli not allowing KR-12, GF-17 (Table 4.2, Chapter 4) and
passage of the antimicrobial agent. GF-17 D3 are fragments of the human
The MSI peptides are examples of cathelicidin peptide LL-37. KR-12 is the
cationic amphipathic helical AMPs (Table shortest fragment of LL-37 possessing
7.2). They are representative of a large group antimicrobial activity (Wang, 2008). It is
of AMPs. MSI-103 and MSI-469 have capable of clustering anionic lipids and
identical sequences, but MSI-469 also has an shows specificity for E. coli relative to S.
N-terminal octyl group. The results indicate aureus (Table 7.2). The longer LL-37 fragment,
that the octyl group does not have a major GF-17, does not have this specificity in
eect on antimicrobial activity. MSI-843 and microbial toxicity. This is because GF-17 is
MSI-1254 are also N-octyl peptides, the more potent against S. aureus and not
dierence between them being that MSI-843 because it is less potent against E. coli. The
has ornithine as its cationic residue, while lack of selective toxicity suggests that GF-17
MSI-1254 has diaminobutyric acid. We have is not acting principally by a charge cluster
shown that MSI-1254 does not cross the outer mechanism. This is supported by our
membrane of E. coli (Epand, R.F. et al., 2010). findings that GF-17, but not KR-12, can
This explains the relatively high MIC for this promote the release of entrapped dye from
bacteria among the MSI peptides and also liposomes (Epand et al., 2009b). Interestingly,
the low ratio of 1 for the MICs against the when three d-amino acid residues are
two dierent bacteria. The ratio of MIC introduced at dierent locations in GF-17,
the new peptide, GF-17 D3, acquires bacterial 2008) and it has a very high ratio of S. aureus
species specificity of its antimicrobial action. MIC/E. coli MIC (Table 7.2). In contrast, a
This substitution destroys the amphipathic shorter oligo-acyl-lysine, C12(7)K-12, does
helix and GF-17 D3 no longer promotes dye not cluster anionic lipids and has a low ratio
release from liposomes and consequently of S. aureus MIC/E. coli MIC because it
loses its toxicity against S. aureus. We have deposits on the LPS layer of Gram-negative
suggested that, in general, an increase in the bacteria (Epand et al., 2009a).
conformational flexibility of a peptide
facilitates the charge cluster mechanism
(Epand and Epand, 2009). This is in accord 7.4.3 Putative mechanism of action
with the observation that the charge cluster
mechanism is more important for GF-17 D3 It would be expected that the recruitment of
than for GF-17. NMR studies also show that anionic lipids to a region of the bacterial
the amphipathic helix of GF-17 D3 is largely membrane would result in the concentration
destroyed (Li et al., 2006). of negative charge in a domain to which
The peptides PR-9, RR-9 and PI-9 are all cationic peptides would congregate, possibly
cationic nonapeptides. The three have been causing the formation of a pore. However,
shown by DSC to cluster anionic lipids the pore that is formed is not very large.
(Epand, R.M. et al., 2010) and the resulting Antimicrobial agents that cause clustering do
domains that are formed with PR-9 have not cause leakage of dyes from membrane
been imaged by freeze-fracture electron mimetic liposomes of bacterial cytoplasmic
microscopy (Epand, R.M. et al., 2010) as well membranes and are not toxic to S. aureus.
as by atomic force microscopy and polarized However, they may breach the membrane
total internal reflection fluorescence barrier to a limited extent, causing slow
microscopy (Oreopoulos et al., 2010). From leakage of contents and/or depolarization of
circular dichroism (Epand, R.M. et al., 2010) the membrane. Evidence for this comes from
and NMR (Zorko et al., 2009) measurements, studies of C12K-78, one of the best examples
as well as the short length of the peptides, it of an antimicrobial agent causing clustering.
is likely that they are conformationally It has been shown with C12K-78 that, in the
flexible even when bound to a membrane. presence of EDTA added to allow the dye
These peptides were shown to bind to the DiSC3(5) to reach the inner membrane, the
LPS layer in Gram-negative bacteria. cytoplasmic membrane of E. coli is
Therefore, the charge cluster mechanism was depolarized (Radzishevsky et al., 2007) and
assessed in Gram-positive bacteria containing there is even some slow influx across the
mostly anionic lipids in their cytoplasmic cytoplasmic membrane of E. coli ML-35 of
membrane versus those containing large small organic molecules (Rotem et al., 2008).
amounts of PE or uncharged lipids (Table This breach of the membrane barrier may be
7.2). The ratio of the MICs for two bacterial sucient to cause the bactericidal eect.
genera within the same species was found to Furthermore, in addition to concentrat-
be dramatically high, reflecting the absence ing the cationic antimicrobial agent, the
of an outer membrane. domain of anionic lipids would be sur-
Cateslytin has been shown by 2H-NMR rounded by an interface with the rest of the
to cluster anionic lipids (Jean-Francois et al., membrane that would be under line tension
2008) and this peptide also exhibits specificity and be less stable. Of course, it is likely that
in its antimicrobial action. there are always domains in bacterial
C12K-78 is an oligo-acyl-lysine, a large membranes (Matsumoto et al., 2006) that
group of antimicrobial agents, many of have phase boundary defects. However,
which exhibit significant selective toxicity those that form in the presence of lipid-
towards certain bacteria (Livne et al., 2009; clustering antimicrobial agents form very
Rotem and Mor, 2009; Zaknoon et al., 2009; rapidly (Oreopoulos et al., 2010) and the
Sarig et al., 2010). C12K-78 has been shown bacteria would not have time to compensate
to cluster anionic lipids (Epand, R.M. et al., for this rearrangement.
Analysis of Membrane-targeting AMPs 125
In addition to this mechanism, there can ation in the presence of anionic lipids. This
also be damage to the bacteria as a result of transformation provides 0.4 kcal mol1
the redistribution of lipids in the membrane. residue of free energy. If the process of helix
This may disrupt existing natural domains in formation were to be coupled with lipid
the membrane or decrease the available phase segregation, the energy provided by
anionic lipid required for specific protein helix formation could contribute to
functions. overcoming the unfavourable energy of
mixing required to segregate the lipid
components. In addition, insertion of
7.4.4 Properties of antimicrobial agents aromatic residues or an acyl chain of a
that favour clustering lipopeptide can also supply the necessary
free energy for cluster formation and
Some antimicrobial agents are more eective stabilization. In general, the formation of any
than others in promoting the clustering of kind of non-covalent bonding, either within
anionic lipids. A sucient number and the peptide or lipid or between the lipid and
density of positive charges are required. By peptide as a consequence of the interaction
density of positive charges, we are referring of the peptide with lipid, could be coupled
to the net number of positive charges divided with the unfavourable energy of lipid
by the total number of residues. Magainin 2 clustering. Non-covalent interactions could
is not eective in clustering anionic lipids, include an increase in the number of
although a very similar peptide, MSI-78, can hydrogen bonds in addition to those
induce clustering (Epand, R.F. et al., 2010). accounted for by helix formation or salt
We suggest that the dierence between these bridges, such as those between arginine
two peptides is a consequence of the much residues and the phosphate groups in the
greater density of positive charges on MSI-78 associated lipids. The relative importance of
compared with magainin. these various forces will vary from one
Another important property is the ability system to another.
to pass the outer membrane of Gram-negative
bacteria. This allows for a wider range of
bacterial species to be tested for the correlation 7.5 Concluding Remarks and
between in vitro clustering of anionic lipids Perspectives
and increased toxicity to bacteria that are rich
in both anionic and zwitterionic lipids, as are The mechanism of clustering anionic lipids is
most Gram-negative bacteria that have a high a component of the mechanism of action of
content of PE (with some exceptions, such as many antimicrobial agents, including oligo-
C. crescentus). More hydrophobic compounds, acyl-lysines, some amphipathic helical
such as the oligo-acyl-lysines, appear more peptides and small, arginine and lysine-rich
capable of crossing the outer membrane than peptides. This phenomenon is more
short, arginine-rich peptides (Epand, R.M. important for antimicrobial agents that have
et al., 2010). a high number and density of positive
Conformational flexibility is another charges, that are conformationally flexible
property associated with an increased ability and that are suciently hydrophobic to
to cluster anionic lipids. This has been penetrate the outer membrane of Gram-
discussed above in comparing the anti- negative bacteria. Several factors contribute
microbial activity of GF-17 with GF-17 D3. to the importance of this mechanism in the
design of agents for clinical application.
Optimizing the potency and eectiveness of
7.4.5 Sources of energy to account for agents that promote this phenomenon will
clustering and entropy of demixing allow targeting of antimicrobial agents to
specific strains of bacteria. In addition, it may
Many cationic AMPs are not structured in provide a mechanism of bactericidal action
solution, but acquire an -helical conform- that does not involve the lysis and disruption
126 R.M. Epand and R.F. Epand
of bacteria and may therefore have fewer a cationic sequence-random copolymer that
toxic side eects when applied to humans, mimics host-defense antimicrobial peptides.
allowing for faster clearance of bacteria by Journal of Molecular Biology 379, 3850.
Epand, R.F., Sarig, H., Mor, A. and Epand, R.M.
macrophages.
(2009a) Cell-wall interactions and the selective
Further studies are required to fully bacteriostatic activity of a miniature oligo-acyl-
elucidate how lipid clustering leads to lysyl. Biophysical Journal 97, 22502257.
bacteriostatic eects, as well as to enhance Epand, R.F., Wang, G., Berno, B. and Epand, R.M.
our understanding of the relationship (2009b) Lipid segregation explains selective
between membrane domains normally toxicity of a series of fragments derived from the
present in bacteria and those that are formed human cathelicidin LL-37. Antimicrobial Agents
as a result of the interaction of an anti- and Chemotherapy 53, 37053714.
microbial agent with a bacterial membrane. Epand, R.F., Maloy, L., Ramamoorthy, A. and
Epand, R.M. (2010) Probing the charge cluster
In addition, there has been interest in cationic
mechanism in amphipathic helical cationic
antimicrobial agents that can function as antimicrobial peptides. Biochemistry 49,
anticancer agents. The role of clustering of 40764084.
exposed phosphatidylserine in cancer cells by Epand, R.M. (2007) Detecting the presence of
antimicrobial agents should be investigated. membrane domains using DSC. Biophysical
Chemistry 126, 197200.
Epand, R.M. and Epand, R.F. (2009) Lipid domains
in bacterial membranes and the action of
References
antimicrobial agents. Biochimica et Biophysica
Acta 1788, 289294.
Arouri, A., Dathe, M. and Blume, A. (2009) Peptide
Epand, R.M., Rotem, S., Mor, A., Berno, B. and
induced demixing in PG/PE lipid mixtures: a
Epand, R.F. (2008) Bacterial membranes as
mechanism for the specificity of antimicrobial
predictors of antimicrobial potency. Journal of
peptides towards bacterial membranes?
the American Chemical Society 130,
Biochimica et Biophysica Acta 1788, 650659. 1434614352.
Breukink, E., Wiedemann, I., van Kraaij, C., Kuipers, Epand, R.M., Epand, R.F., Arnusch, C.J.,
O.P., Sahl, H.G. and de Kruijff, B. (1999) Use of Papahadjopoulos-Sternberg, B., Wang, G. and
the cell wall precursor lipid II by a pore-forming Shai, Y. (2010) Lipid clustering by three
peptide antibiotic. Science 286, 23612364. homologous arginine-rich antimicrobial peptides
Camargo, I.L., Neoh, H.M., Cui, L. and Hiramatsu, is insensitive to amino acid arrangement.
K. (2008) Serial daptomycin selection generates Biochimica et Biophysica Acta 1798,
daptomycin-nonsusceptible Staphylococcus 12721280.
aureus strains with a heterogeneous vancomycin- Ernst, C.M., Staubitz, P., Mishra, N.N., Yang, S.J.,
intermediate phenotype. Antimicrobial Agents Hornig, G., Kalbacher, H., Bayer, A.S., Kraus, D.
and Chemotherapy 52, 42894299. and Peschel, A. (2009) The bacterial defensin
Clejan, S., Guffanti, A.A., Cohen, M.A. and Krulwich, resistance protein MprF consists of separable
T.A. (1989) Mutation of Bacillus firmus OF4 to domains for lipid lysinylation and antimicrobial
duramycin resistance results in substantial peptide repulsion. PLoS Pathogens 5,
replacement of membrane lipid phospha- e1000660.
tidylethanolamine by its plasmalogen form. Jean-Francois, F., Castano, S., Desbat, B., Odaert,
Journal of Bacteriology 171, 17441746. B., Roux, M., Metz-Boutigue, M.H. and Dufourc,
Epand, R.F., Schmitt, M.A., Gellman, S.H., Sen, A., E.J. (2008) Aggregation of cateslytin -sheets
Auger, M., Hughes, D.W. and Epand, R.M. on negatively charged lipids promotes rigid
(2005) Bacterial species selective toxicity of two membrane domains. A new mode of action for
isomeric /-peptides: role of membrane lipids. antimicrobial peptides? Biochemistry 47,
Molecular Membrane Biology 22, 457469. 63946402.
Epand, R.F., Savage, P.B. and Epand, R.M. (2007) Lehrer, R.I., Barton, A. and Ganz, T. (1988)
Bacterial lipid composition and the antimicrobial Concurrent assessment of inner and outer
efficacy of cationic steroid compounds membrane permeabilization and bacteriolysis in
(ceragenins). Biochimica et Biophysica Acta E. coli by multiple-wavelength spectrophoto-
1768, 25002509. metry. Journal of Immunological Methods 108,
Epand, R.F., Mowery, B.P., Lee, S.E., Stahl, S.S., 153158.
Lehrer, R.I., Gellman, S.H. and Epand, R.M. Li, X., Li, Y., Han, H., Miller, D.W. and Wang, G.
(2008) Dual mechanism of bacterial lethality for (2006) Solution structures of human LL-37
Analysis of Membrane-targeting AMPs 127
fragments and NMR-based identification of a OConnor, D.T. and Gallo, R.L. (2008) The
minimal membrane-targeting antimicrobial and neuroendocrine peptide catestatin is a
anticancer region. Journal of the American cutaneous antimicrobial and induced in the skin
Chemical Society 128, 57765785. after injury. Journal of Investigative Dermatology
Livne, L., Kovachi, T., Sarig, H., Epand, R.F., 128, 15251534.
Zaknoon, F., Epand, R.M. and Mor, A. (2009) Radzishevsky, I.S., Rotem, S., Bourdetsky, D.,
Design and characterization of a broad- Navon-Venezia, S., Carmeli, Y. and Mor, A.
spectrum bactericidal acyl-lysyl oligomer. (2007) Improved antimicrobial peptides based
Chemistry & Biology 16, 12501258. on acyl-lysine oligomers. Nature Biotechnology
Mangoni, M.L., Epand, R.F., Rosenfeld, Y., Peleg, 25, 657659.
A., Barra, D., Epand, R.M. and Shai, Y. (2008) Rosch, J.W., Hsu, F.F. and Caparon, M.G. (2007)
Lipopolysaccharide, a key molecule involved in Anionic lipids enriched at the ExPortal of
the synergism between temporins in inhibiting Streptococcus pyogenes. Journal of Bacteriology
bacterial growth and in endotoxin neutralization. 189, 801806.
Journal of Biological Chemistry 283, Rotem, S. and Mor, A. (2009) Antimicrobial peptide
2290722917. mimics for improved therapeutic properties.
Matsumoto, K., Kusaka, J., Nishibori, A. and Hara, Biochimica et Biophysica Acta Biomembranes
H. (2006) Lipid domains in bacterial membranes. 1788, 15821592.
Molecular Microbiology 61, 11101117. Rotem, S., Radzishevsky, I.S., Bourdetsky, D.,
Mishra, N.N., Yang, S.J., Sawa, A., Rubio, A., Nast, Navon-Venezia, S., Carmeli, Y. and Mor, A.
C.C., Yeaman, M.R. and Bayer, A.S. (2009) (2008) Analogous oligo-acyl-lysines with distinct
Analysis of cell membrane characteristics of in antibacterial mechanisms. FASEB Journal 22,
vitro-selected daptomycin-resistant strains of 26522661.
methicillin-resistant Staphylococcus aureus. Sarig, H., Livne, L., Held-Kuznetsov, V., Zaknoon,
Antimicrobial Agents and Chemotherapy 53, F., Ivankin, A., Gidalevitz, D. and Mor, A. (2010)
23122318. A miniature mimic of host defense peptides with
Oreopoulos, J., Epand, R.F., Epand, R.M. and Yip, systemic antibacterial efficacy. FASEB Journal
C.M. (2010) Peptide-induced domain formation 24, 19041913.
in supported lipid bilayers: direct evidence by Shaw, N. (1970) Bacterial glycolipids. Bacteriological
combined atomic force and polarized total Reviews 34, 365377.
internal reflection fluorescence microscopy. Wang, G. (2008) Structures of human host defense
Biophysical Journal 98, 815823. cathelicidin LL-37 and its smallest antimicrobial
Peschel, A., Jack, R.W., Otto, M., Collins, L.V., peptide KR-12 in lipid micelles. Journal of
Staubitz, P., Nicholson, G., Kalbacher, H., Biological Chemistry 283, 3263732643.
Nieuwenhuizen, W.F., Jung, G., Tarkowski, A., Zaknoon, F., Sarig, H., Rotem, S., Livne, L., Ivankin,
van Kessel, K.P. and van Strijp, J.A. (2001) A., Gidalevitz, D. and Mor, A. (2009) Antibacterial
Staphylococcus aureus resistance to human properties and mode of action of a short acyl-
defensins and evasion of neutrophil killing via lysyl oligomer. Antimicrobial Agents and
the novel virulence factor MprF is based on Chemotherapy 53, 34223429.
modification of membrane lipids with L-lysine. Zorko, M., Japelj, B., Hafner-Bratkovic, I. and Jerala,
Journal of Experimental Medicine 193, R. (2009) Expression, purification and structural
10671076. studies of a short antimicrobial peptide.
Radek, K.A., Lopez-Garcia, B., Hupe, M., Niesman, Biochimica et Biophysica Acta Biomembranes
I.R., Elias, P.M., Taupenot, L., Mahata, S.K., 1788, 314323.
8
Non-membrane Targets of
Antimicrobial Peptides: Novel Therapeutic
Opportunities?
Abstract
The emergence and rapid spread of multiresistant bacteria necessitates every eort to develop new
classes of antibiotics with novel targets and modes of action. One potential source of novel antibiotics is
the cationic antimicrobial peptides (AMPs), which constitute an important component of the innate
immune system in a variety of organisms. Most AMPs exert their activity by interacting with bacterial
membranes, thus perturbing their permeability. However, an increasing number of peptides are being
described that translocate across the bacterial membranes and act on intracellular targets in bacteria.
These non-membrane-active AMPs have been shown to bind and inactivate intracellular biopolymers
such as nucleic acids and proteins without destroying or remaining attached to the bacterial membranes.
As such, they have emerged as viable candidates for the treatment of human infections. In this chapter,
we focus on the six well-characterized, non-membrane-active AMPs (buforin II, PR-39, indolicidin,
apidaecin, drosocin and pyrrhocoricin) and discuss whether binding of these peptides to their
intracellular targets correlates with bacterial cell death. The potential exploitation of these peptides as
human therapeutics is also discussed.
* Corresponding author.
exclude some type of interaction with the microbial action of buforin II proved to be
bacterial membrane, and supports a model in dierent from those of AMPs that function
which penetration across the membrane is by membrane permeabilization. Using
followed by binding to and inhibition of fluorescein isothiocyanate-labelled buforin II
functional intracellular end molecules (Otvos, and gel-retardation experiments, it was
2002). In this chapter, we focus on the six well- revealed that buforin II kills bacteria without
characterized, non-membrane-active AMPs cell lysis and has a strong anity for DNA
(buforin II, PR-39, indolicidin, apidaecin, and RNA in vitro (Park et al., 1998). Kobayashi
drosocin and pyrrhocoricin) and discuss et al. investigated the interaction of buforin II
whether binding of these peptides to their with phospholipid membranes and compared
intracellular targets correlates with bacterial these results with those of similar experiments
cell death. The potential exploitation of these with magainin 2 (Kobayashi et al., 2000). These
peptides as human therapeutics is also researchers used equipotent tryptophan-
reviewed. substituted peptides to fluorometrically
monitor peptidelipid interactions. Control
circular dichroism (CD) studies showed that,
8.2 Buforin II: -Helical AMP with like magainin 2, buforin II binds selectively to
Single Proline Residue Binding to liposomes composed of acidic phospholipids.
Nucleic Acids However, the fluorometric experiments
revealed that, in contrast to magainin 2,
Buforins are -helical AMPs isolated from the buforin II translocates across liposome
stomach tissue of the Asian toad Bufo bufo membranes eciently without inducing sig-
gargarizans. They display a broad spectrum of nificant membrane permeabilization or lipid
antimicrobial activity against bacteria and flip-flop. Furthermore, the Pro11 residue,
fungi (Park et al., 1996). Buforin I is a 39-amino which induces a kink in the buforin II -helix,
acid peptide with complete sequence identity is the key structural feature required for
to the N-terminal region of histone H2A, buforin IIs unique properties.
which specifies the proteins DNA-binding A subsequent study by Kobayashi et al.
activity. Buforin II is a 21-residue peptide that (2004) revealed that buforin II crosses lipid
is produced from buforin I by treatment with bilayers in a manner similar to that of
the endoproteinase Lys-C. Buforin II magainin 2 via the transient formation
represents an interesting example of an of a peptidelipid supramolecular complex
-helical AMP because it employs a unique (toroidal) pore. However, the presence of
mechanism of activity that does not involve Pro11 distorts the helical structure of buforin
membrane perturbation (Haney et al., 2009). II, concentrating five basic amino acid
The structure of buforin II was deter- residues in a limited amphipathic region
mined using nuclear magnetic resonance (Arg5Lys21); this structure destabilizes the
(NMR) spectroscopy and restrained molecular pore by enhanced electrostatic repulsion and
dynamics (Yi et al., 1996). Buforin II adopts a enables ecient translocation of buforin II
helix-hinge-helix structure in 50% tri- into the cell. The importance of the Pro11
fluoroethanol; the N-terminal extended residue was also demonstrated in a systematic
-helix (residues 510) and the C-terminal structureactivity relationship study (Park et
-helix (residues 1221) are separated by a al., 2000). In this study, antimicrobial
proline residue at amino acid position 11. The potencies, secondary structures and mechan-
four N-terminal residues are relatively isms of bacterial killing action were assessed
unstructured. Interestingly, the Pro11 residue for a series of structurally altered synthetic
distorts the C-terminal helix in such a way as buforin II analogues. The results revealed
to maximize the amphipathic nature of the that the proline hinge (Pro11) is a key
C-terminal helix. This originally led structural factor for the cell-penetrating
researchers to postulate that buforin II property without permanent membrane
interacts with bacterial membranes in a association, while the cell-penetrating
similar fashion to other amphiphatic -helical eciency, which depends on -helical con-
AMPs. However, the mechanism of anti- tent, is a critical factor for determining the
Non-membrane Targets of AMPs 131
antimicrobial potency of buforin II. Indeed, from having another as yet unidentified
these experiments showed that only a single intracellular target.
amino acid substitution at the Pro11 position With its primarily intracellular target
changes buforin II into a membrane-active inhibition and ability to move between
magainin-like peptide. Conversely, insertion various modes of action, buforin II derivatives
of a proline hinge region (Arg5Gly11) into are ideal candidates for antimicrobial drug
the N-terminal helix of magainin 2 switches development. However, poor protease
this AMP from a membrane-permeabilizing stability of short AMPs such as buforin II
peptide to a cell-penetrating one. severely limits their therapeutic value.
Because buforin II was shown to bind Recently, Meng and Kumar (2007) reported
nucleic acids in vitro, it has been hypothesized that incorporation of hexafluoroleucine at
that buforin II kills bacteria by interacting selected sites (Leu18 and Leu19) of buforin II
with their nucleic acids after translocation results in a simultaneous increase in potency
across the cell membrane (Park et al., 1998). and resistance to protease degradation. This
Although the proposed mechanism is quite observation suggests that fluorination may be
intriguing, many questions remain to be an important strategy for increasing the
answered. The connection between nucleic metabolic stability of buforin II. In addition to
acid binding and antimicrobial activity has antimicrobial activity, buforin II and buforin
not been demonstrated directly, and it is IIb a synthetic analogue of buforin II that
unclear whether buforin II and nucleic acids contains a proline hinge between the two
interact in a specific manner or whether they -helices and a model -helical sequence at
only bind to each other because of their the C-terminus (3RLLR) selectively targets
opposite net charges. Uyterhoeven et al. cancer cells through interaction with the cell-
(2008) recently characterized the nucleic acid surface gangliosides. Buforin IIb then
binding properties of buforin II using mol- traverses cancer cell membranes without
ecular modelling and a fluorescent inter- damaging them and induces mitochondria-
calator displacement assay. These researchers dependent apoptosis (Lee et al., 2008). Buforin
observed that, in addition to non-specific IIb also displays powerful cytotoxic activity
electrostatic attractions between a cationic when injected into solid tumours in p53-
peptide and nucleic acids, specific side chains deficient mice (Cho et al., 2009). These results
(Arg2 and Arg20) of buforin II form inter- suggest that buforin IIb may be developed
actions with DNA that are stronger than the into a novel therapeutic agent for the treat-
non-specific electrostatic ones. They also ment of cancers.
showed that disruption of the buforin IIDNA
interactions by substituting basic residues of
buforin II with alanine generally decreases the
antimicrobial activity of buforin II. Moreover, 8.3 PR-39 and Indolicidin: Mammalian
we recently found that buforin II dose- Proline-rich Peptides Inhibiting
dependently inhibits the transcription and Macromolecule Synthesis and Cell
translation of the lacZ gene of pUC19 in Division
vitro (unpublished data, 2010). In addition,
when E. coli harbouring pUC19 is treated PR-39 is a 39-amino acid cathelicidin-derived
with buforin II at concentrations below the AMP isolated from porcine small intestine
MIC, the amount of lacZ expressed, which and neutrophil azurophilic granules. It is rich
is determined by -galactosidase assay, in proline (49%) and arginine (24%)
decreases as the concentration of buforin II (Agerberth et al., 1993; Shi et al., 1994). Within
increases, suggesting that buforin II inhibits its primary sequence, PR-39 is characterized
transcription or translation in bacteria. Taken by seven repeats of Xaa-Pro-Pro-Xaa. CD and
together, these results support the assertion Fourier-transform infrared spectroscopy
that buforin II kills bacteria by translocation performed with PR-39, either in aqueous
into the cell and inhibition of transcription or solution or in the presence of lipid vesicles,
translation through interaction with nucleic have suggested that they adopt a disordered
acids, although it does not preclude buforin II left-handed polyproline II helix structure
132 J.H. Cho and S.C. Kim
because of the proline residues disposed (Gennaro et al., 2002; Lehrer and Ganz, 2002;
consecutively in the linear peptide chain Zanetti, 2004; Sang and Blecha, 2009). Taken
(Cabiaux et al., 1994). PR-39 has antibacterial together, it is not clear whether bacterial cell
activity against several strains of both Gram- filamentation caused by PR-39 and PR-26 is
positive and Gram-negative bacteria (Bevins, due to the blocking of DNA replication
1994). The mechanism of PR-39 bactericidal through DNA binding or the inhibition of
activity has been investigated by isotope membrane proteins involved in septum
incorporation experiments (Boman et al., formation.
1993). In contrast to the killing by membrane In addition to antimicrobial properties,
disruption seen with most other AMPs, PR-39 exerts multiple and diverse actions on
Boman and colleagues found that bacteria are mammalian cells. PR-39 has been found to
killed by PR-39 through a mechanism that induce syndecan expression in mesenchymal
stops protein and DNA synthesis. PR-39 cells, and to influence cell motility and
requires a lag period of about 8 min to metastatic potential in wound repair (Gallo et
penetrate the outer membrane of wild-type E. al., 1994). The peptide binds to the cytosolic
coli, while subsequent killing is quite fast. component of NADPH oxidase complex
Kinetic data suggest induced proteolysis in protein p47phox (Shi et al., 1996b) and a
the target bacteria may be important for signalling adaptor protein p130Cas (Chan and
bactericidal activity. Consistent with this Gallo, 1998), suggesting an important role for
suggestion is the finding that actively growing this peptide in inflammation. PR-39 also plays
cells are killed more rapidly than non-growing a critical role in limiting cardiac injury
cells. In addition, Shi et al. (1996a) found from through induction of angiogenesis (Li et al.,
a scanning electron micrographic study that 2000; Bao et al., 2001; Gaczynska et al., 2003)
PR-39 and its N-terminal 126 fragment, and inhibition of apoptosis in hypoxic
PR-26, induce filamentation of Salmonella endothelial cells (Ramanathan et al., 2004; Wu
typhimurium without forming pores on the et al., 2004). These results underscore the
bacterial outer membrane. Cells exposed to therapeutic potential of PR-39 in a number of
these peptides have an extremely elongated diseases, including inflammation, ischemia
morphology, which indicates that the peptide- reperfusion injury and heart diseases.
treated cells are unable to undergo cell Indolicidin is isolated from the
division. cytoplasmic granules of bovine neutrophils. It
Unlike with buforin IIb and other cell- has a unique composition consisting of 39%
penetrating AMPs, the translocation of PR-39 tryptophan and 23% proline, and the native
into the bacterial cell and its subsequent peptide is amidated at the C-terminus (Selsted
interaction with intracellular target molecules et al., 1992). Indolicidin is the smallest of the
such as nucleic acids have not yet been known naturally occurring linear AMPs,
confirmed. In addition, a study on the contains the highest percentage of tryptophan
secondary structure and membrane inter- of any known protein and consists of only six
action of PR-39 showed that its secondary dierent amino acids. Owing to the presence
structure is not altered upon incubation of the of tryptophan residues interspersed with
peptide with negatively charged vesicles and proline residues throughout the sequence, it
that nearly all of the added peptide is probably assumes a structure distinct from
membrane bound (Cabiaux et al., 1994). Based the well-described -helical and -structured
on these results, Shi et al. (1996a) suggested peptides. Solution structures of indolicidin in
that PR-39 and PR-26 bind to molecules dierent environments have been determined
anchored on the outer membrane of Gram- by fluorescence, CD and NMR spectroscopy.
negative bacteria and that these molecules are However, the proposed structures of
necessary for formation of the septum. indolicidin on lipid membranes have been
However, multiple cellular targets, including somewhat controversial. It has been suggested
proteins containing PR-39-binding domains, that indolicidin adopts a polyproline helix
have been identified in eukaryotic cells, and structure (Falla et al., 1996) or a turn structure
PR-39 has been shown to enter eukaryotic (Ladokhin et al., 1997, 1999). However, Rozek
cells and regulate many biological processes et al. (2000) showed that indolicidin adopts a
in which these targets are directly involved wedge-shaped structure with hydrophobic
Non-membrane Targets of AMPs 133
tryptophan residues in the trough, flanked by integrase (Marchand et al., 2006). Indolicidin
two positively charged regions (see Fig. 9.6A, also interferes with topoisomerase-I-mediated
Chapter 9), which appears ideal for inter- DNA relaxation without unwinding DNA,
calation between the lipid molecules of the suggesting that indolicidin may inhibit a
lipid bilayer. Moreover, a study by Hsu et al. large variety of DNA processing enzymes
(2005) revealed that indolicidin adopts through DNA binding. Taken together,
multiple conformations in aqueous solutions indolicidin is thought to cross the membranes
and membrane-mimicking environments, into the cytoplasm at concentrations above
suggesting that the structural plasticity the MIC but below the minimal lytic con-
accounts for indolicidins eects. centration, and kills bacteria by multiple
Indolicidin exhibits significant activity actions at the level of DNA.
against a wide range of targets, including The short sequence and broad spectrum
bacteria, fungi, protozoa and human immuno- of activity are features that make indolicidin
deficiency virus (HIV)-1 (Zanetti et al., 2002; appealing as a putative anti-infective agent.
Chan et al., 2006). Like other membrane- Although indolicidin is an eective AMP,
permeabilizing peptides, the antimicrobial various attempts have been made to find
action of indolicidin was thought to increase derivatives that are more antibacterial and
the permeability of the cytoplasmic mem- less cytotoxic. Omiganan pentahydrochloride,
branes of target microorganisms. However, a synthetic analogue of indolicidin, is
the complete mode of action of this peptide currently under clinical development for the
has not been fully elucidated and is still prevention of catheter-related local and
under debate. Although indolicidin binding bloodstream infections as well as for the
to bacterial membranes results in membrane treatment of acne and rosacea (Melo et al.,
permeabilization (Falla et al., 1996; Zhao et al., 2006; Vaara, 2009). It has been reported that
2001; Schibli et al., 2002; Nan et al., 2009) or indolicidin binds abasic-site-containing DNA
thinning (Shaw et al., 2006; Hsu and Yip, (Marchand et al., 2006). Abasic sites are
2007), it does not cause cell lysis at considered to be the most frequent DNA
concentrations four times the MIC of the lesions in mammalian cells, with an estimated
peptide (Subbalakshmi and Sitaram, 1998; frequency of 10,000 events per day per cell
Wu et al., 1999). Thus, it is conceivable that (Boiteux and Guillet, 2004). Therefore, further
indolicidin uses its membrane-anity investigations on the potential interference
property to enter the cytoplasm and exerts its of indolicidin with DNA repair mechanisms
antibacterial activity by attacking other are needed to use this peptide in human
targets, similarly to PR-39. In fact, Sub- therapeutics.
balakshmi and Sitaram (1998) demonstrated
that indolicidin induces filamentation in E.
coli cells as a result of preferential inhibition
of DNA synthesis, rather than RNA and 8.4 Apidaecin, Drosocin and
protein synthesis. Moreover, Hsu et al. (2005) Pyrrhocoricin: Short, Insect Proline-
confirmed that indolicidin binds DNA in gel- rich Peptides Binding to Heat-shock
retardation and fluorescence quenching Protein DnaK
experiments. These researchers also demon-
strated that indolicidin prefers to bind duplex Apidaecin, drosocin and pyrrhocoricin are
DNA rather than single-stranded DNA, using short, proline-rich AMPs isolated from insects
surface plasmon resonance. It is thought that (Apis mellifera, Drosophila melanogaster and
indolicidin prefers to bind strongly to Pyrrhocoris apterus, respectively). They consist
phosphate groups via its positively charged of 1820 amino acid residues (Casteels et al.,
amino acids, and the tryptophan residues 1989; Bulet et al., 1993; Cociancich et al., 1994).
stack between the nucleotide bases or The names of these peptides reflect their
deoxyriboses in each strand of the DNA origin rather than a subdivision among the
duplex. A subsequent study revealed that individual sequences. Apidaecin, drosocin
indolicidin interferes with the formation of and pyrrhocoricin are remarkably similar in
the catalytic HIV-1 integraseDNA complex amino acid composition and sequence motif
by directly binding to DNA and not to pattern (Li et al., 2006). In addition to being
134 J.H. Cho and S.C. Kim
human equivalent Hsp70. These peptides activity spectrum of drosocin can be 100%
also interact with lipopolysaccharide, which correlated with the homology to E. coli DnaK
could be responsible for their initial binding at the D-E helix region in the 31 studied
to the bacterial surface, and in a non-specific bacterial species. However, there is some
manner with the bacterial chaperonin GroEL. disagreement as to the specific binding site of
In addition, an inactive pyrrhocoricin DnaK. Chesnokova et al. (2004) studied the
analogue, made of all-d amino acids, does chemistry of pyrrhocoricin binding to DnaK
not interact with DnaK. This suggests that using complementary pre-state kinetic and
inhibition of DnaKs function by these single-turnover ATPase assays, and showed
peptides might be responsible for bacterial that pyrrhocoricin binds to and stimulates the
killing. ATPase activity of both wild-type and lidless
A subsequent study by Kragol et al. variants of DnaK. In addition, a study of CD
(2001) revealed that pyrrhocoricin and curves by Zhou et al. (2008) showed that
drosocin aect DnaKs two major functions: obvious spectral alteration is detected when
ATPase activity and refolding of misfolded apidaecin is added to lidless DnaK, while no
proteins. In this study, they found that alteration is observed when it is added to
biologically active l-pyrrhocoricin diminishes DnaK D-E helix fragments. Thus, these
the ATPase activity of recombinant DnaK, researchers suggested that pyrrhocoricin
while the inactive d-pyrrhocoricin analogue binds primarily to the conventional substrate-
and membrane-active AMPs such as cecropin binding site of DnaK, much like other
A or magainin 2 fail to inhibit ATPase activity substrates, and eectively decreases the
of DnaK. In addition, both alkaline cellular concentration of DnaK by competing
phosphatase and -galactosidase activities with natural substrates.
(reflecting DnaKs function of refolding Ideally, a protein present only in bacteria
misfolded proteins) of live bacteria are that carriers a significant function, but is
reduced upon incubation with l-pyrrhocoricin absent or non-homologous in human cells,
or drosocin. In contrast, d-pyrrhocoricin, may form the basis for the rational design of
magainin 2 or buforin II have only a species-specific antibacterial peptides and
negligible eect. According to fluorescence peptidomimetics (Casteels and Tempst, 1994).
polarization and dot-blot analysis of synthetic DnaK shares only 50% sequence homology
DnaK fragments and labelled pyrrhocoricin with eukaryotic Hsp70 (Bardwell and Craig,
analogues, the peptide binds to the hinge 1984), with some well-conserved N-terminal
region around the C-terminal helices D and E regions and some less conserved C-terminal
of DnaK, located just above the conventional ones (Karlin and Brocchieri, 1998). Therefore,
substrate-binding site. Structureactivity the variable sequence domain of DnaK
relationship studies of pyrrhocoricin identi- appears to oer a sensible target for
fied that the N-terminal half (residues 210) is antimicrobial drug development, and
responsible for inhibition of the ATPase apidaecin, drosocin and pyrrhocoricin are an
activity of DnaK, while the C-terminal half excellent starting point for these species-
(residues 1120) is responsible for membrane specific drug development eorts. It is
penetration (Kragol et al., 2002). Overall, these conceivable that drugs that act on a specific
data suggest that the binding of pyrrhocoricin target protein likely have the disadvantage of
and drosocin to DnaK prevents the frequent an easier selection of drug-resistant strains
opening and closing of the multihelical lid through mutations at the target level,
over the peptide-binding pocket of DnaK, similarly to conventional antibiotics, which
permanently closes the cavity and inhibits generally act by binding to a specific target
chaperone-assisted protein folding. The (Gennaro et al., 2002). However, Cassone et al.
sequence of the D-E helix is remarkably (2009) recently showed that induced
dissimilar in various bacterial and resistance to the designer proline-rich peptide
mammalian DnaK proteins. This may explain dimer A3-APO does not involve changes in
the selectivity and relatively restricted the intracellular target DnaK, thus dispelling
spectrum of activity of these peptides and the concern of target modification and
their lack of toxicity to eukaryotic cells. In arguing for focused research for novel
fact, Bikker et al. (2006) demonstrated that the bacterial targets.
136 J.H. Cho and S.C. Kim
08N4800-31410) from the Ministry of Bulet, P. and Stocklin, R. (2005) Insect antimicrobial
Education, Science and Technology, the Korea peptides: structures, properties and gene
Science & Engineering Foundation Grant regulation. Protein and Peptide Letters 12, 311.
Bulet, P., Dimarcq, J.L., Hetru, C., Lagueux, M.,
(R01-2008-000-20559-0), the 21C Frontier Charlet, M., Hegy, G., Van Dorsselaer, A. and
Program of Microbial Genomics and Hoffmann, J.A. (1993) A novel inducible
Applications (MG08-0204-1-0) and a grant antibacterial peptide of Drosophila carries an
from the Korea Healthcare Technology R&D O-glycosylated substitution. Journal of Biological
Project, Ministry for Health, Welfare and Chemistry 268, 1489314897.
Family Aairs, Republic of Korea (A084115). Bulet, P., Urge, L., Ohresser, S., Hetru, C. and Otvos,
L. Jr (1996) Enlarged scale chemical synthesis
and range of activity of drosocin, an O-
glycosylated antibacterial peptide of Drosophila.
European Journal of Biochemistry 238, 6469.
References Cabiaux, V., Agerberth, B., Johansson, J., Homble,
F., Goormaghtigh, E. and Ruysschaert, J.M.
Agerberth, B., Boman, A., Andersson, M., Jornvall, (1994) Secondary structure and membrane
H., Mutt, V. and Boman, H.G. (1993) Isolation of interaction of PR-39, a Pro+Arg-rich antibacterial
three antibacterial peptides from pig intestine: peptide. European Journal of Biochemistry 224,
gastric inhibitory polypeptide (742), diazepam- 10191027.
binding inhibitor (3286) and a novel factor, Cassone, M., Frith, N., Vogiatzi, P., Wade, J.D. and
peptide 3910. European Journal of Biochemistry Otvos, L. Jr (2009) Induced resistance to the
216, 623629. designer proline-rich antimicrobial peptide
Bao, J., Sato, K., Li, M., Gao, Y., Abid, R., Aird, W., A3-APO does not involve changes in the
Simons, M. and Post, M.J. (2001) PR-39 and intracellular target DnaK. International Journal of
PR-11 peptides inhibit ischemia-reperfusion Peptide Research and Therapeutics 15,
injury by blocking proteasome-mediated I B 121128.
degradation. American Journal of Physiology: Casteels, P. and Tempst, P. (1994) Apidaecin-type
Heart and Circulatory Physiology 281, peptide antibiotics function through a non-
H2612H2618. poreforming mechanism involving stereo-
Bardwell, J.C. and Craig, E.A. (1984) Major heat specificity. Biochemical and Biophysical Research
shock gene of Drosophila and the Escherichia Communications 199, 339345.
coli heat-inducible dnaK gene are homologous. Casteels, P., Ampe, C., Jacobs, F., Vaeck, M. and
Proceedings of the National Academy of Tempst, P. (1989) Apidaecins: antibacterial
Sciences of the USA 81, 848852. peptides from honeybees. EMBO Journal 8,
Bevins, C.L. (1994) Antimicrobial peptides as agents 23872391.
of mucosal immunity. Ciba Foundation Castle, M., Nazarian, A., Yi, S.S. and Tempst, P.
Symposium 186, 250260. (1999) Lethal effects of apidaecin on Escherichia
Bikker, F.J., Kaman-van Zanten, W.E., de Vries-van coli involve sequential molecular interactions with
de Ruit, A.M., Voskamp-Visser, I., van Hooft, diverse targets. Journal of Biological Chemistry
P.A., Mars-Groenendijk, R.H., de Visser, P.C. and 274, 3255532564.
Noort, D. (2006) Evaluation of the antibacterial Chan, D.I., Prenner, E.J. and Vogel, H.J. (2006)
spectrum of drosocin analogues. Chemical Tryptophan- and arginine-rich antimicrobial pep-
Biology & Drug Design 68, 148153. tides: structures and mechanisms of action.
Boiteux, S. and Guillet, M. (2004) Abasic sites in Biochimica et Biophysica Acta 1758,
DNA: repair and biological consequences in 11841202.
Saccharomyces cerevisiae. DNA Repair 3, Chan, Y.R. and Gallo, R.L. (1998) PR-39, a
112. syndecan-inducing antimicrobial peptide, binds
Boman, H.G. (1995) Peptide antibiotics and their and affects p130Cas. Journal of Biological
role in innate immunity. Annual Review of Chemistry 273, 2897828985.
Immunology 13, 6192. Chesnokova, L.S., Slepenkov, S.V. and Witt, S.N.
Boman, H.G., Agerberth, B. and Boman, A. (1993) (2004) The insect antimicrobial peptide,
Mechanisms of action on Escherichia coli of L-pyrrhocoricin, binds to and stimulates the
cecropin P1 and PR-39, two antibacterial ATPase activity of both wild-type and lidless
peptides from pig intestine. Infection and DnaK. FEBS Letters 565, 6569.
Immunity 61, 29782984. Cho, J.H., Sung, B.H. and Kim, S.C. (2009) Buforins:
Brogden, K.A. (2005) Antimicrobial peptides: pore histone H2A-derived antimicrobial peptides from
formers or metabolic inhibitors in bacteria? toad stomach. Biochimica et Biophysica Acta
Nature Reviews Microbiology 3, 238250. 1788, 15641569.
Bukau, B. and Horwich, A.L. (1998) The Hsp70 and Coates, A., Hu, Y., Bax, R. and Page, C. (2002) The
Hsp60 chaperone machines. Cell 92, 351366. future challenges facing the development of new
138 J.H. Cho and S.C. Kim
antimicrobial drugs. Nature Reviews Drug Hsu, C.H., Chen, C., Jou, M.L., Lee, A.Y., Lin, Y.C.,
Discovery 1, 895910. Yu, Y.P., Huang, W.T. and Wu, S.H. (2005)
Cociancich, S., Dupont, A., Hegy, G., Lanot, R., Structural and DNA-binding studies on the bovine
Holder, F., Hetru, C., Hoffmann, J.A. and Bulet, P. antimicrobial peptide, indolicidin: evidence for
(1994) Novel inducible antibacterial peptides multiple conformations involved in binding to
from a hemipteran insect, the sap-sucking bug membranes and DNA. Nucleic Acids Research
Pyrrhocoris apterus. Biochemical Journal 300, 33, 40534064.
567575. Hsu, J.C. and Yip, C.M. (2007) Molecular dynamics
Cudic, M. and Otvos, L. Jr (2002) Intracellular targets simulations of indolicidin association with model
of antibacterial peptides. Current Drug Targets 3, lipid bilayers. Biophysical Journal 92, L100L102.
101106. Huang, H.W. (2000) Action of antimicrobial peptides:
Dutta, R.C., Nagpal, S. and Salunke, D.M. (2008) two-state model. Biochemistry 39, 83478352.
Functional mapping of apidaecin through Huang, H.W. (2006) Molecular mechanism of
secondary structure correlation. International antimicrobial peptides: the origin of cooperativity.
Journal of Biochemistry & Cell Biology 40, Biochimica et Biophysica Acta 1758,
10051015. 12921302.
Falla, T.J., Karunaratne, D.N. and Hancock, R.E. Huang, H.W., Chen, F.Y. and Lee, M.T. (2004)
(1996) Mode of action of the antimicrobial peptide Molecular mechanism of peptide-induced pores
indolicidin. Journal of Biological Chemistry 271, in membranes. Physical Review Letters 92,
1929819303. 198304.
Gaczynska, M., Osmulski, P.A., Gao, Y., Post, M.J. Jang, S.A., Sung, B.H., Cho, J.H. and Kim, S.C.
and Simons, M. (2003) Proline- and arginine-rich (2009) Direct expression of antimicrobial peptides
peptides constitute a novel class of allosteric in an intact form by a translationally coupled two-
inhibitors of proteasome activity. Biochemistry cistron expression system. Applied and
42, 86638670. Environmental Microbiology 75, 39803986.
Gallo, R.L., Ono, M., Povsic, T., Page, C., Eriksson, Karlin, S. and Brocchieri, L. (1998) Heat shock
E., Klagsbrun, M. and Bernfield, M. (1994) protein 70 family: multiple sequence comparisons,
Syndecans, cell surface heparan sulfate function, and evolution. Journal of Molecular
proteoglycans, are induced by a proline-rich Evolution 47, 565577.
antimicrobial peptide from wounds. Proceedings Kobayashi, S., Takeshima, K., Park, C.B., Kim, S.C.
of the National Academy of Sciences of the USA and Matsuzaki, K. (2000) Interactions of the
91, 1103511039. novel antimicrobial peptide buforin 2 with lipid
Gennaro, R., Zanetti, M., Benincasa, M., Podda, E. bilayers: proline as a translocation promoting
and Miani, M. (2002) Pro-rich antimicrobial factor. Biochemistry 39, 86488654.
peptides from animals: structure, biological Kobayashi, S., Chikushi, A., Tougu, S., Imura, Y.,
functions and mechanism of action. Current Nishida, M., Yano, Y. and Matsuzaki, K. (2004)
Pharmaceutical Design 8, 763778. Membrane translocation mechanism of the
Gordon, Y.J., Romanowski, E.G. and McDermott, antimicrobial peptide buforin 2. Biochemistry 43,
A.M. (2005) A review of antimicrobial peptides 1561015616.
and their therapeutic potential as anti-infective Kragol, G., Lovas, S., Varadi, G., Condie, B.A.,
drugs. Current Eye Research 30, 505515. Hoffmann, R. and Otvos, L. Jr (2001) The
Hancock, R.E. and Sahl, H.G. (2006) Antimicrobial antibacterial peptide pyrrhocoricin inhibits the
and host-defense peptides as new anti-infective ATPase actions of DnaK and prevents
therapeutic strategies. Nature Biotechnology 24, chaperone-assisted protein folding. Biochemistry
15511557. 40, 30163026.
Hancock, R.E. and Scott, M.G. (2000) The role of Kragol, G., Hoffmann, R., Chattergoon, M.A.,
antimicrobial peptides in animal defenses. Lovas, S., Cudic, M., Bulet, P., Condie, B.A.,
Proceedings of the National Academy of Rosengren, K.J., Montaner, L.J. and Otvos, L. Jr
Sciences of the USA 97, 88568861. (2002) Identification of crucial residues for the
Haney, E.F., Hunter, H.N., Matsuzaki, K. and Vogel, antibacterial activity of the proline-rich peptide,
H.J. (2009) Solution NMR studies of amphibian pyrrhocoricin. European Journal of Biochemistry
antimicrobial peptides: linking structure to 269, 42264237.
function? Biochimica et Biophysica Acta 1788, Ladokhin, A.S., Selsted, M.E. and White, S.H. (1997)
16391655. Bilayer interactions of indolicidin, a small
Hartl, F.U. and Hayer-Hartl, M. (2002) Molecular antimicrobial peptide rich in tryptophan, proline,
chaperones in the cytosol: from nascent chain to and basic amino acids. Biophysical Journal 72,
folded protein. Science 295, 18521858. 794805.
Hoffmann, R., Bulet, P., Urge, L. and Otvos, L. Jr Ladokhin, A.S., Selsted, M.E. and White, S.H. (1999)
(1999) Range of activity and metabolic stability of CD spectra of indolicidin antimicrobial peptides
synthetic antibacterial glycopeptides from insects. suggest turns, not polyproline helix. Biochemistry
Biochimica et Biophysica Acta 1426, 459467. 38, 1231312319.
Non-membrane Targets of AMPs 139
Lee, H.S., Park, C.B., Kim, J.M., Jang, S.A., Park, fluorinated amino acids. Journal of the American
I.Y., Kim, M.S., Cho, J.H. and Kim, S.C. (2008) Chemical Society 129, 1561515622.
Mechanism of anticancer activity of buforin IIb, a Mookherjee, N. and Hancock, R.E. (2007) Cationic
histone H2A-derived peptide. Cancer Letters host defence peptides: innate immune regulatory
271, 4755. peptides as a novel approach for treating
Lehrer, R.I. and Ganz, T. (2002) Cathelicidins: a infections. Cellular and Molecular Life Sciences
family of endogenous antimicrobial peptides. 64, 922933.
Current Opinion in Hematology 9, 1822. Mygind, P.H., Fischer, R.L., Schnorr, K.M., Hansen,
Li, J., Post, M., Volk, R., Gao, Y., Li, M., Metais, C., M.T., Sonksen, C.P., Ludvigsen, S., Raventos, D.,
Sato, K., Tsai, J., Aird, W., Rosenberg, R.D., Buskov, S., Christensen, B., De Maria, L.,
Hampton, T.G., Sellke, F., Carmeliet, P. and Taboureau, O., Yaver, D., Elvig-Jorgensen, S.G.,
Simons, M. (2000) PR39, a peptide regulator of Sorensen, M.V., Christensen, B.E., Kjaerulff, S.,
angiogenesis. Nature Medicine 6, 4955. Frimodt-Moller, N., Lehrer, R.I., Zasloff, M. and
Li, W.F., Ma, G.X. and Zhou, X.X. (2006) Apidaecin- Kristensen, H.H. (2005) Plectasin is a peptide
type peptides: biodiversity, structurefunction antibiotic with therapeutic potential from a
relationships and mode of action. Peptides 27, saprophytic fungus. Nature 437, 975980.
23502359. Nan, Y.H., Bang, J.K. and Shin, S.Y. (2009) Design of
Ludtke, S., He, K. and Huang, H. (1995) Membrane novel indolicidin-derived antimicrobial peptides
thinning caused by magainin 2. Biochemistry 34, with enhanced cell specificity and potent anti-
1676416769. inflammatory activity. Peptides 30, 832838.
Makovitzki, A., Avrahami, D. and Shai, Y. (2006) Oren, Z. and Shai, Y. (1998) Mode of action of linear
Ultrashort antibacterial and antifungal amphipathic -helical antimicrobial peptides.
lipopeptides. Proceedings of the National Biopolymers 47, 451463.
Academy of Sciences of the USA 103, Otvos, L. Jr (2002) The short proline-rich antibacterial
1599716002. peptide family. Cellular and Molecular Life
Marchand, C., Krajewski, K., Lee, H.F., Antony, S., Sciences 59, 11381150.
Johnson, A.A., Amin, R., Roller, P., Kvaratskhelia, Otvos, L. Jr (2005) Antibacterial peptides and
M. and Pommier, Y. (2006) Covalent binding of proteins with multiple cellular targets. Journal of
the natural antimicrobial peptide indolicidin to
Peptide Science 11, 697706.
DNA abasic sites. Nucleic Acids Research 34,
Otvos, L. Jr, Bokonyi, K., Varga, I., Otvos, B.I.,
51575165.
Hoffmann, R., Ertl, H.C., Wade, J.D., McManus,
Markossian, K.A., Zamyatnin, A.A. and Kurganov,
A.M., Craik, D.J. and Bulet, P. (2000a) Insect
B.I. (2004) Antibacterial proline-rich oligopeptides
peptides with improved protease-resistance
and their target proteins. Biochemistry (Moscow)
protect mice against bacterial infection. Protein
69, 10821091.
Science 9, 742749.
Marr, A.K., Gooderham, W.J. and Hancock, R.E.
Otvos, L. Jr, O, I., Rogers, M.E., Consolvo, P.J.,
(2006) Antibacterial peptides for therapeutic use:
obstacles and realistic outlook. Current Opinion Condie, B.A., Lovas, S., Bulet, P. and Blaszczyk-
in Pharmacology 6, 468472. Thurin, M. (2000b) Interaction between heat
Matsuzaki, K. (1999) Why and how are peptidelipid shock proteins and antimicrobial peptides.
interactions utilized for self-defense? Magainins Biochemistry 39, 1415014159.
and tachyplesins as archetypes. Biochimica et Park, C.B., Kim, M.S. and Kim, S.C. (1996) A novel
Biophysica Acta 1462, 110. antimicrobial peptide from Bufo bufo gargarizans.
McManus, A.M., Otvos, L. Jr, Hoffmann, R. and Biochemical and Biophysical Research Com-
Craik, D.J. (1999) Conformational studies by munications 218, 408413.
NMR of the antimicrobial peptide, drosocin, and Park, C.B., Kim, H.S. and Kim, S.C. (1998)
its non-glycosylated derivative: effects of Mechanism of action of the antimicrobial peptide
glycosylation on solution conformation. buforin II: buforin II kills microorganisms by
Biochemistry 38, 705714. penetrating the cell membrane and inhibiting
McPhee, J.B., Scott, M.G. and Hancock, R.E. (2005) cellular functions. Biochemical and Biophysical
Design of host defence peptides for antimicrobial Research Communications 244, 253257.
and immunity enhancing activities. Combinatorial Park, C.B., Yi, K.S., Matsuzaki, K., Kim, M.S. and
Chemistry & High Throughput Screening 8, Kim, S.C. (2000) Structureactivity analysis of
257272. buforin II, a histone H2A-derived antimicrobial
Melo, M.N., Dugourd, D. and Castanho, M.A. (2006) peptide: the proline hinge is responsible for the
Omiganan pentahydrochloride in the front line of cell-penetrating ability of buforin II. Proceedings
clinical applications of antimicrobial peptides. of the National Academy of Sciences of the USA
Recent Patents on Anti-infective Drug Discovery 97, 82458250.
1, 201207. Ramanathan, B., Wu, H., Ross, C.R. and Blecha, F.
Meng, H. and Kumar, K. (2007) Antimicrobial activity (2004) PR-39, a porcine antimicrobial peptide,
and protease stability of peptides containing inhibits apoptosis: involvement of caspase-3.
140 J.H. Cho and S.C. Kim
Developmental and Comparative Immunology and Otvos, L. Jr (2010) The designer proline-rich
28, 163169. antibacterial peptide A3-APO is effective against
Rozek, A., Friedrich, C.L. and Hancock, R.E. (2000) systemic Escherichia coli infections in different
Structure of the bovine antimicrobial peptide mouse models. International Journal of
indolicidin bound to dodecylphosphocholine and Antimicrobial Agents 35, 357361.
sodium dodecyl sulfate micelles. Biochemistry Uyterhoeven, E.T., Butler, C.H., Ko, D. and Elmore,
39, 1576515774. D.E. (2008) Investigating the nucleic acid
Sang, Y. and Blecha, F. (2009) Porcine host defense interactions and antimicrobial mechanism of
peptides: expanding repertoire and functions. buforin II. FEBS Letters 582, 17151718.
Developmental and Comparative Immunology
Vaara, M. (2009) New approaches in peptide
33, 334343.
antibiotics. Current Opinion in Pharmacology 9,
Schibli, D.J., Epand, R.F., Vogel, H.J. and Epand,
571576.
R.M. (2002) Tryptophan-rich antimicrobial
peptides: comparative properties and membrane Williamson, M.P. (1994) The structure and function
interactions. Biochemistry and Cell Biology 80, of proline-rich regions in proteins. Biochemical
667677. Journal 297, 249260.
Selsted, M.E., Novotny, M.J., Morris, W.L., Tang, Wimley, W.C., Selsted, M.E. and White, S.H. (1994)
Y.Q., Smith, W. and Cullor, J.S. (1992) Indolicidin, Interactions between human defensins and lipid
a novel bactericidal tridecapeptide amide from bilayers: evidence for formation of multimeric
neutrophils. Journal of Biological Chemistry 267, pores. Protein Science 3, 13621373.
42924295. Wu, J., Parungo, C., Wu, G., Kang, P.M., Laham,
Shai, Y. (2002) Mode of action of membrane active R.J., Sellke, F.W., Simons, M. and Li, J. (2004)
antimicrobial peptides. Biopolymers 66, PR39 inhibits apoptosis in hypoxic endothelial
236248. cells: role of inhibitor apoptosis protein-2.
Shaw, J.E., Alattia, J.R., Verity, J.E., Prive, G.G. and Circulation 109, 16601667.
Yip, C.M. (2006) Mechanisms of antimicrobial Wu, M., Maier, E., Benz, R. and Hancock, R.E.
peptide action: studies of indolicidin assembly at (1999) Mechanism of interaction of different
model membrane interfaces by in situ atomic classes of cationic antimicrobial peptides with
force microscopy. Journal of Structural Biology planar bilayers and with the cytoplasmic
154, 4258.
membrane of Escherichia coli. Biochemistry 38,
Shi, J., Ross, C.R., Chengappa, M.M. and Blecha, F.
72357242.
(1994) Identification of a proline-arginine-rich
antibacterial peptide from neutrophils that is Yeaman, M.R. and Yount, N.Y. (2003) Mechanisms of
analogous to PR-39, an antibacterial peptide antimicrobial peptide action and resistance.
from the small intestine. Journal of Leukocyte Pharmacological Reviews 55, 2755.
Biology 56, 807811. Yi, G.S., Park, C.B., Kim, S.C. and Cheong, C. (1996)
Shi, J., Ross, C.R., Chengappa, M.M., Sylte, M.J., Solution structure of an antimicrobial peptide
McVey, D.S. and Blecha, F. (1996a) Antibacterial buforin II. FEBS Letters 398, 8790.
activity of a synthetic peptide (PR-26) derived Zanetti, M. (2004) Cathelicidins, multifunctional
from PR-39, a proline-arginine-rich neutrophil peptides of the innate immunity. Journal of
antimicrobial peptide. Antimicrobial Agents and Leukocyte Biology 75, 3948.
Chemotherapy 40, 115121. Zanetti, M., Gennaro, R., Skerlavaj, B., Tomasinsig,
Shi, J., Ross, C.R., Leto, T.L. and Blecha, F. (1996b) L. and Circo, R. (2002) Cathelicidin peptides as
PR-39, a proline-rich antibacterial peptide that candidates for a novel class of antimicrobials.
inhibits phagocyte NADPH oxidase activity by Current Pharmaceutical Design 8, 779793.
binding to Src homology 3 domains of p47 phox. Zasloff, M. (2002) Antimicrobial peptides of multi-
Proceedings of the National Academy of cellular organisms. Nature 415, 389395.
Sciences of the USA 93, 60146018. Zhang, L., Benz, R. and Hancock, R.E. (1999)
Slepenkov, S.V. and Witt, S.N. (2002) The unfolding
Influence of proline residues on the antibacterial
story of the Escherichia coli Hsp70 DnaK: is
and synergistic activities of -helical peptides.
DnaK a holdase or an unfoldase? Molecular
Biochemistry 38, 81028111.
Microbiology 45, 11971206.
Steiner, H., Andreu, D. and Merrifield, R.B. (1988) Zhao, H., Mattila, J.P., Holopainen, J.M. and
Binding and action of cecropin and cecropin Kinnunen, P.K. (2001) Comparison of the
analogues: antibacterial peptides from insects. membrane association of two antimicrobial
Biochimica et Biophysica Acta 939, 260266. peptides, magainin 2 and indolicidin. Biophysical
Subbalakshmi, C. and Sitaram, N. (1998) Mechanism Journal 81, 29792991.
of antimicrobial action of indolicidin. FEMS Zhou, X.X., Li, W.F. and Pan, Y.J. (2008) Functional
Microbiology Letters 160, 9196. and structural characterization of apidaecin and
Szabo, D., Ostorhazi, E., Binas, A., Rozgonyi, F., its N-terminal and C-terminal fragments. Journal
Kocsis, B., Cassone, M., Wade, J.D., Nolte, O. of Peptide Science 14, 697707.
9 Structural Studies of Antimicrobial
Peptides Provide Insight into Their
Mechanisms of Action
Guangshun Wang
Abstract
This chapter reviews structural studies of antimicrobial peptides (AMPs), with a focus on human
defensins and cathelicidins. Also discussed are the major steps for structural determination of AMPs by
nuclear magnetic resonance spectroscopy, including bacterial expression and purification, sample
preparations, data collection, sequential signal assignments, structure calculations, validation and
coordinate deposition. A variety of three-dimensional structures (-helices, -strands, -fold and
non- structures) have been discovered for AMPs, which can induce similar biophysical consequences
in membranes such as positive curvature or lipid domain formation. Therefore, it is the amphipathic
surface, not polypeptide backbone scaolds, that is essential for the antimicrobial activity of AMPs. A
proper presentation of side chains (e.g. cationic and hydrophobic) on the surface of AMPs determines not
only membrane perturbation potential, but also other biological functions. Reduction in hydrophobicity
is a fundamental strategy to improve peptide selectivity. Structures of AMPs in complex with membranes
or non-membrane targets also form the foundation for engineering a new generation of antimicrobials
that will supplement or replace traditional resistant antibiotics.
human primates, but not in humans due to activity, human defensins possess other
the stop codon in the pseudogenes (Tran et functional roles in host defence such as anti-
al., 2002). Although the exact disulfide bond human inflammatory virus (HIV), antitoxin,
(SS) pattern varies, all defensins possess chemotaxis and immune-modulation proper-
multiple such bonds. In human -defensins ties (Lehrer et al., 2009).
(2933 residues), the three SS bonds are The precursor proteins of the cathelicidin
CysICysVI, CysIICysIV and CysIIICV; in family share a well-conserved N-terminal
human -defensins (3649 residues), they are cathelin domain while the sequences at the
CysICysV, CysIICysIV and CysIIICysVI. The C-terminus are highly variable, encoding a
SS bond connections in rhesus monkey plethora of AMPs (Zanetti, 2005). Several
-defensins are CysICysVI, CysIICysV and cathelicidins exist in animals such as sheep,
CysIIICysIV. The cysteines in these peptides cows and pigs. For example, PMAP-23,
are labelled from CysI to CysVI according to protegrin-1 and PR-39 are all pig cathelicidins
their order in the sequence. The SS bridges, that form -helix, -hairpin and extended
as well as the circular polypeptide chain in structures in membrane environments,
the case of -defensins, confer stability to the respectively. In contrast, only one cathelicidin
three-dimensional (3D) structure of defensins. gene has been found in humans. The
In addition to the known antibacterial precursor of human cathelicidin is an 18 kDa
Structural Studies of AMPs 143
human cationic antimicrobial protein defensins, LL-37 also possesses other bio-
(hCAP-18). Depending on the expression logical functions such as immune modulation,
sites, hCAP-18 can be cleaved into dierent angiogenesis, apoptosis and wound healing
forms of active peptides. In neutrophils, the (Nnik and Hancock, 2009).
peptide, released by proteinase 3, contains 37 To elucidate the functional roles of
residues and starts with two leucines, and is defensins and cathelicidins, 3D structures are
thereby referred to as LL-37 (Gudmundsson essential. While circular dichroism (CD) and
et al., 1995). In human production systems, Fourier transform infrared spectroscopy are
the released peptide is ALL-38. LL-37 and low-resolution techniques that give an
ALL-38 (Table 9.1) have similar antimicrobial estimation of secondary structures of proteins
activities (Srensen et al., 2003). In addition, in various states, X-ray crystallography and
LL-37 can be cleaved into short active nuclear magnetic resonance (NMR)
peptides in human skin or sweat, indicating a spectroscopy are able to determine the
regulatory role of proteases in host defence structure of proteins to the atomic resolution.
(Yamasaki et al., 2006). Of note, these LL-37 By February 2010, the Antimicrobial Peptide
fragments display greater antimicrobial Database (APD) (Wang et al., 2009) contained
activity in the presence of physiological salts 195 NMR structures and ten crystal
such as carbonate (Dorschner et al., 2006). structures, indicating that NMR is the major
Therefore, it is important to perform player (Wang, 2006; Haney et al., 2009). As a
antibacterial assays under physiological consequence, this chapter focuses on
conditions. Of these protease products of structural studies of AMPs by NMR. An
hCAP-18, LL-37 has been best studied. This outline of the procedure for NMR structural
molecule protects humans from infectious determination of AMPs is provided in Fig.
diseases (Nizet et al., 2001; Ptsep et al., 2002), 9.1. For structural analysis, highly purified
cystic fibrosis (Bucki et al., 2007) and sepsis by AMPs are required. Because cationic AMPs
neutralizing lipopolysaccharides (LPS or usually associate with anionic bacterial
endotoxin) (Cirioni et al., 2006). LL-37 and its membranes, NMR studies are often con-
fragments all inhibit HIV-1 infection ducted in membrane-mimetic systems. Also
(Bergman et al., 2007; Wang et al., 2008), but discussed are NMR methods, major structural
they may also perform dierent biological types (-helices, -sheets and extended
functions. While LL-37 promotes cancer structures), peptidemembrane interactions
metastasis, an N-terminal fragment (LL-25) and structure-based peptide design.
inhibits the process (Weber et al., 2009). In
addition, a C-terminal fragment has been
shown to have anticancer activity in vitro (Li
et al., 2006a). It is proposed that overexpressed 9.2 Bacterial Expression and
LL-37 attracts mesenchymal stem cells to Purification of Antimicrobial Peptides
tumour microenvironments by directly
interacting with formyl peptide receptor-like Most AMPs are relatively short and can
1 (FPRL-1). The chemotactic activity is readily be synthesized by the solid-phase
attributed to the N-terminal region of LL-37, method (Merrifield et al., 1995). In particular,
and the antibacterial region is mapped to the this method enables the incorporation of
C-terminus of the peptide (Bra et al., 2005). isotope-labelled amino acids at specific sites.
A recent review lists >100 LL-37 fragments Such peptides are important for solid-state
discovered by various laboratories (Burton NMR studies. Advances have also been made
and Steel, 2009). KR-12, the smallest anti- in the chemical synthesis of defensins (Raj et
microbial fragment with merely 12 residues al., 2000; Wu et al., 2004; Klver et al., 2006).
(Table 4.2, Chapter 4), has been mapped to For longer peptides or those with
residues 1829 of LL-37 (Wang, 2008c). multiple disulfide bonds, chemical synthesis
Evidently, polypeptide fragmentation is less ecient and bacterial expression shows
provides a useful approach for elucidating advantages. In addition, bacterial expression
the functional regions of AMPs. Like human enables uniform labelling of polypeptides
144 G. Wang
Table 9.3. Select enzymes and chemical agents and cleavage sequences.
Protease Cleavage sequence Chemical agent Cleavage site
Thrombin ArgGly, LysLeu or LysAla CNBr MetXxx
Enterokinase AspAspAspAspLys Formic acid AspPro
Factor Xa IleGluGlyArgXxx Hydroxylamine AspGly
SUMO protease 1 SUMOXxxa
TEV GluGlnLeuTyrPheGlnGly/Ser
a Xxx should not be proline.
Abbreviations: CNBr, cyanogen bromide; SUMO, small ubiquitin-related modifier; TEV, tobacco etch virus protease.
146 G. Wang
Fig. 9.3. Three membrane-mimetic models for NMR studies. (A) A lipid bilayer is composed of long-chain
lipids; (B) a bicelle comprises a mixture of short- and long-chain lipids; and (C) a micelle consists of
short-chain lipids (see the text for more details).
2008c). We also suggest the use of the NMR dimensional NMR methods are well suited
chemical shift standard externally, since for structural studies of AMPs because of
anionic compounds such as sodium their relatively small size (usually <50
2,2-dimethyl-silapentane-5-sulfonate (DSS) residues). The standard set of 2D (1H, 1H)
may interact with cationic AMPs (Wang et al., NMR experiments includes total correlation
2003). Note that DSS is unable to form a spectroscopy (TOCSY), nuclear Overhauser
micelle by itself based on translational eect spectroscopy (NOESY) and double-
diusion measurements by NMR (Keifer et quantum-filtered correlation spectroscopy
al., 2004). (DQF-COSY). These NMR experiments
allow for sequential signal assignments in
two steps (Wthrich, 1986). First, the amino
9.3.2 NMR methods
acid spin systems are identified in the
TOCSY spectrum and verified in DQF-
Two-dimensional NMR methods
COSY. Secondly, the relationships of these
NMR spectroscopy was established as a spin systems are established based on
structural tool by Kurt Wthrich (2002 NOESY spectra. In principle, nuclear
Nobel Laureate), whose group determined Overhauser eect (NOE) cross peaks can be
the first protein structure during 19841985. detected when two protons are within 5 .
The earlier 2D homonuclear NMR work of Signal assignments constitute a critical step
peptides and proteins is summarized in a towards structural determination of AMPs
classic book by Wthrich (1986). Two- (Fig. 9.1).
148 G. Wang
Natural abundance NMR spectroscopy and shifts can be employed to provide dynamic
its applications information. The basis of this application is
the observed correlation between 15N
The birth of cryogenic NMR probes increased
heteronuclear NOE values and secondary
the signal to noise ratio by several-fold, 13C shifts (Wang, 2010). A structured region
allowing the recording of 2D heteronuclear
is rigid with positive 15N NOE values and
correlated spectra at the natural abundance
large 13C secondary shifts. In contrast,
(0.36% for 15N and 1.11% for 13C). High
residues in a disordered region display
concentrations of peptides, if possible, are
negative 15N NOE and small 13C secondary
always recommended to further improve the
shifts. Therefore, a plot of the secondary 13C
signal to noise ratio. I routinely collect (1H,
15N) or (1H, 13C) heteronuclear single- shifts versus residue number of AMPs can be
used to locate rigid or flexible regions.
quantum coherence (HSQC) spectra for
Fourthly, the secondary 13C plot may also be
micelle-bound peptides. The heteronuclear
applied for structural validation. A poorly
chemical shifts can be assigned based on the
defined region may result from insucient
known proton resonances achieved from 2D
NMR restraints or peptide motion. The
NMR. The 15N and 13C chemical shifts have
poorly defined region may be regarded as
multiple applications. First, they can be
truly flexible if the 13C secondary shifts are
employed to verify 1H chemical shift
also small in the corresponding region. Lastly,
assignments (Wang, 2006). Secondly,
the plot of (1H, 13C) cross-peak intensity as a
heteronuclear chemical shifts also contain
function of residue number displays a wave
structural information. Statistical analysis of
pattern. Interestingly, residues with lower
protein NMR chemical shifts has revealed
peak intensities tend to be hydrophobic. This
that 13C are up- and downfield shifted in
finding may oer an approach for identifying
-sheet and -helical structures, respectively.
micelle-binding residues (Wang, 2010).
This means that the 13C secondary shifts
(dierences between the measured and
random shifts) are positive in helical regions
Heteronuclear 3D NMR studies of isotope-
and negative in -stranded regions (Wishart
labelled AMPs
and Sykes, 1994). Such empirical relation-
ships enable the identification of secondary For helical peptides or those with dicult
structures (-helices or -strands) in poly- sequences, 2D NMR may not provide
peptides (Fig. 9.1). Since 13C chemical shifts sucient spectral resolution (see Fig. 1 in
are related to protein dihedral angles, Ad Bax Wang, 2008c). Under these circumstances, 3D
and colleagues have established and heteronuclear NMR methods are needed.
improved the TALOS program, which The power of 3D NMR results from its high
predicts backbone dihedral angles of resolution by separating the signals in a
polypeptides based on 1H, 13C, 13C, 13C crowded 2D spectral region onto multiple 2D
carbonyl and 15N chemical shifts (Shen et al., planes along the third dimension, allowing
2009). For unlabelled AMPs, fewer than five structural studies of proteins in the 2030
chemical shifts have been found to be useful kDa range (Bax and Grzesiek, 1993). For
in predicting backbone angles (Wang, 2007). small AMPs, an 15N-labelled peptide might
Therefore, the measurement of natural be sucient to resolve the overlapped cross
abundance chemical shifts oers a practical peaks by recording 3D-edited TOCSY and
approach to obtaining dihedral angles, since NOESY (e.g. Park et al., 2007). In this case,
it is dicult to measure scalar couplings for sequential signal assignments can still be
micelle-bound peptides. The inclusion of achieved using the classic NOE-based
natural abundance chemical-shift-derived method (Wthrich, 1986). If spectral overlap
backbone angles into structural calculations persists, triple-resonance NMR experiments
has improved the structural quality of can be applied to a 15N,13C-labelled peptide.
micelle-bound peptides (Wang et al., 2005). In this method, sequential signal assignments
Thirdly, natural abundance 13C chemical are obtained by utilizing the simultaneous
Structural Studies of AMPs 149
linked to the APD and discussed herein. A pleurocidin (Syvitski et al., 2005), insect
few AMPs are helical even in aqueous spinigerin (Landon et al., 2006) and human
solutions, primarily due to the formation of LL-37 (Wang, 2008c) all contain a single
helix-bundle structures. Saposin-like proteins helical region with one or both ends
(approximately 80 residues) form pores in disordered. In the high-resolution structure
bacterial membranes. Such polypeptides of micelle-bound LL-37 determined by 3D
occur widely in nature, ranging from NMR, residues 231 form a continuous
protozoan parasites (e.g. Entamoeba histo- helical structure, whereas the C-terminal
lytica) to worms (e.g. Caenorhabditis elegans) residues 3237 are disordered (Fig. 9.4C).
and mammals (e.g. Sus scrofa), including Such a structure is fully consistent with
humans. The helix-bundle structure of these 15N heteronuclear NOE measurements, sup-
antimicrobial proteins is further stabilized by porting that only the C-terminus is flexible.
three disulfide bonds. In the structure of The high-resolution LL-37 structure deter-
caenopore-5 from C. elegans (Pro81 cis mined by 3D NMR methods is interesting in
conformer in Fig. 9.4A), the five helices are several aspects. First, it is the longest
located between residues 616, 2436, 4550, continuous helix (with 30 residues) found for
5866 and 7078 (Mysliwy et al., 2010). In AMPs to my knowledge. Secondly, the
addition, there are two SS bonds (Cys6- hydrophobic surface of the amphipathic
Cys80 and Cys9-Cys74) between helices I helix is punctuated by a hydrophilic residue
and V at the N- and C-termini of the protein; Ser9, leading to segregation of the hydro-
the third SS bond (Cys35-Cys49) is formed phobic surface into two regions. Thirdly,
between helices II and III. This AMP plays an there is a helical bend between residues
essential role in the survival of the worm by 1416, and all hydrophobic side chains are
eliminating any E. coli ingested. Upon located on the concave surface of the curved
association with bacterial membranes, the structure. Interestingly, the corresponding
helix bundle structure may open at a site that helical-bend residues in homologous primate
has several exposed hydrophobic side chains cathelicidins (Zelezetsky et al., 2006) are
(arrow in Fig. 9.4A). Despite a similar protein usually glycines (Wang, 2008c). These data
fold, the antibacterial activity of tick suggest that the LL-37 structure determined
microplusin is attributed to Cu2+ binding, in SDS micelles serves as a useful model
another antimicrobial mechanism dierent to understand the activity of homologous
from pore formation in bacterial membranes cathelicidins. Fourthly, the aromatic
(Silva et al., 2009). The structures of both aromatic packing at the N-terminal region of
caenopore-5 and microplusin were deter- LL-37 is proposed to play a role in peptide
mined by 3D heteronuclear NMR techniques. aggregation, as well as to confer chemotactic
Another unique AMP that forms a helix and cytotoxic properties to the peptide.
bundle structure in water (determined by 2D Some AMPs have been found to adopt a
solution NMR) is distinctin. This amphibian helix-hinge-helix motif (also referred to as a
peptide comprises two chains linked by one helix-break-helix or helix-turn-helix motif).
SS bond. This SS bond is critical for Examples are insect cecropin A (Holak et al.,
structure stability to proteases (Fig. 9.4B) 1988), fish pardaxin 4 (Porcelli et al., 2004),
rather than antimicrobial activity (Dalla Serra spider latarcin 2a (Dubovskii et al., 2006),
et al., 2008). Recent solid-state NMR studies amphibian gaegurin 4 (Chi et al., 2007) and
have revealed that both helices are located on dermaseptin B2 (Galanth et al., 2009). The
the membrane surface of lipid bilayers, linker region of these peptides was proposed
excluding the possibility of pore formation to be important for antimicrobial activity,
(Resende et al., 2009). allowing optimal binding of both helices to
In the membrane-bound state, a variety bacterial membranes (Park et al., 2007;
of helical structures have been determined Galanth et al., 2009). Normally, these AMPs
for linear AMPs with <40 residues. induce positive membrane curvature, leading
Amphibian magainin 2 (Gesell et al., 1997), to toroidal pore formation or micellization of
dermadistinctin K (Verly et al., 2009), fish membranes (Haney et al., 2010). However,
Structural Studies of AMPs 151
(A) (B)
81
(C) (D)
F2/F3
S9 F17
F15
F27
F5/F6
(E) (F)
F3 F13
F17
F27
Fig. 9.4. Three-dimensional structures of AMPs from the -helical family. Depicted are: (A) caenopore-5
from Caenorhabditis elegans (Protein Data Bank (PDB) ID: 2JS9) (arrow, exposed hydrophobic side
chains); (B) distinctin, a two-chain AMP from frogs (PDB ID: 1XKM); (C) LL-37, a cathelicidin from
humans (PDB ID: 2K6O); (D) pardaxin 4 from fish (PDB ID: 1XC0); (E) FK-13, an LL-37 antimicrobial
core peptide discovered by NMR (PDB ID: 2FBS); and (F) aurein 1.2 from Australian bell frogs (PDB ID:
1VM5). In the short peptides (panels CF), phenylalanine residues (labelled) are common and are
important for anchoring these AMPs into bacterial membrane. To clearly view the polypeptide fold, the
disulfide bonds in SS-containing peptides are not displayed here, but are described in the text.
pardaxin 4 was found to induce negative vesicles, but vesicle lysis in the presence of
curvature in lipid bilayers. The aromatic side anionic PG (Vad et al., 2010).
chains are not located on the same side in the In the helical family, AMPs with longer
3D structure (Fig. 9.4D). In fact, they might polypeptide chains tend to display toxicity to
not be needed or able to, because the mammalian cells (Ciornei et al., 2005;
N-terminal short helix is rather hydrophobic Dubovskii et al., 2006). This observation laid
and can insert into the membranes. The the foundation for truncating such AMPs to
C-terminal helix is amphipathic and its improve peptide selectivity. By using NMR,
membrane orientation and mode of action we have identified a 13-residue antibacterial
depend on lipid chain length and com- core peptide (FK-13) corresponding to
position (Porcelli et al., 2004). This peptide residues 1729 of human LL-37 (Li et al.,
induces pore formation in phosphocholine 2006a). FK-13 showed a similar antibacterial
152 G. Wang
activity to LL-37 and adopted a helical backbone dihedral angles of a d-amino acid.
structure upon association with micelles (Fig. Indeed, the protein fold was retained when
9.4E). Subsequently, Phe17 of FK-13 was Gly16 was changed to a d-amino acid
found to be critical for cytotoxicity to human (alanine, glutamic acid, phenylalanine, argin-
cells, because removal of this residue led to ine, threonine, valine or tyrosine), but the
KR-12 that displayed selective toxicity fold was disrupted when Gly16 was changed
against E. coli (Wang, 2008c). FK-13 is com- to l-alanine (Xie et al., 2005).
parable in size to aureins and temporins, the Likewise, structures of hBD-1, hBD-2
shortest helical peptides from amphibians in and hBD-3 have been determined by X-ray
the APD. For comparison, the high-quality diraction and NMR (Hoover et al., 2000,
structure of micelle-bound aurein 1.2 is 2001; Bauer et al., 2001; Sawai et al., 2001;
presented in Fig. 9.4F (Wang et al., 2005). Schibli et al., 2002). Human -defensins
Interestingly, we obtained a peptide that contain one N-terminal helix packed on the
shows sequence homology to aurein 1.2 after three -strands (Fig. 9.5, panels FH). Using
reversing the sequence of FK-13 (Li et al., hBD-1 as a model, Pazgier et al. (2007)
2006b). Retro-FK13 (sequence in Table 4.2) determined the structure of ten mutants by
showed a higher antibacterial activity than X-ray diraction. These mutants have a
aurein 1.2 owing to additional cationic protein fold that is identical to the wild type.
residues (five versus two). In contrast, aurein It is proposed that the charged residues
1.2 and the non-toxic bacterial membrane Arg29, Lys31, Lys33 and Lys36 are critical for
anchor both have two positively charged antibacterial activity, whereas the N-terminal
residues in their membrane-binding region. helical region (residues 18), including
The antibacterial activity of aurein 1.2 was adjacent residues such as Lys22, Arg29 and
attributed to a longer and broader hydro- Lys33, form the surface for chemotaxis to
phobic surface than that of the membrane CCR6-transfected HEK-293 cells. It is more
anchor. These structureactivity relationship convenient to map the binding sites by NMR.
studies have revealed and emphasize the This is because membrane binding to an
importance of both the hydrophobic surface isotope-labelled protein can cause selective
and cationic side chains of the amphipathic peak shifts of those residues that form the
helix in binding to bacterial membranes. The binding surface (this is also the principle for
combined consideration of charge and a screening potential drug molecules by NMR)
hydrophobic surface is important, because (Shuker et al., 1996). As an example, the
we have found it dicult to correlate a single NMR-identified membrane-binding sites on
structural parameter (e.g. helicity, net charge, the structure of plant defensin Psd1 are
hydrophobic surface area, transfer free indicated by an arrow in Fig. 9.5I (De
energy) with antibacterial activity in a series Medeiros et al., 2010).
of helical peptides (Wang et al., 2005). X-ray crystallography and NMR provide
complementary information under dierent
conditions. While the structural coordinates
AMPs with -sheet structures
determined in crystals are normally more
Human defensins have a folded structure in accurate, NMR can be applied to the study of
water, enabling crystallization and structural peptide dynamics in solution (Skalicky et al.,
determination by X-ray crystallography (Hill 1994), as well as the interaction with bacterial
et al., 1991; Szyk et al., 2006). Human membranes (see below). In addition, NMR
-defensins share a similar three antiparallel also provides insight into the oligomerization
-strand fold stabilized by three SS bonds of defensins in solution. While a dimer for
(Fig. 9.5, panels AE). The disulfide bonds, as hBD-1 and octamer for hBD-2 have been
well as salt bridges, are critical to maintaining found under crystal conditions (due to
the defensin fold. Using HNP-2 as a model, molecular packing), they are monomers in
the structural basis for the Gly-Xaa-Cys motif solution. However, hBD-3 is a dimer in
(Chapter 4) was elucidated. The conserved solution. The dimeric structure of hBD-3 is
glycine in the classic -bulge has the proposed to be important for its greater
Structural Studies of AMPs 153
indicated the existence of dierent oligomers adopted a non- structure with a distorted
for protegrin-1 in lipid bilayers (Mani et al., backbone. In these structures (Fig. 9.6), the
2006), molecular simulation led to an octamer six-membered aromatic rings of tryptophan
model as the active conformation in bacterial residues tend to point towards the
membranes (Langham et al., 2008). membrane. In the case of indolicidin and
lactoferrin, this is supported by fluorescence
spectroscopy as well as by 5-doxyl stearic
AMPs with non- structures
acid spin label experiments. The lactoferrin
Several tryptophan-rich peptides have been peptide could be further shortened to a
found to adopt non- structures (extended, hexamer fragment with a similar anti-
turns or loops). Rozek et al. (2000) found that microbial activity to the long fragment (Chan
the CD spectra of indolicidin in the presence et al., 2006). All of these NMR structures
of membrane-mimicking vesicles or micelles indicate a unique role for tryptophan in
did not resemble those of either -helical or micelle binding. Tryptophan residues have
-sheet proteins. In DPC micelles, NMR long been recognized as an important
structural determination revealed a unique membrane anchor, preferring the membrane
amphipathic structure, where the middle water interface (Wang et al., 1996; Yau et al.,
tryptophan-rich region plays the major role 1998; Schibli et al., 1999). Arginine residues
to anchor the peptide into the micelle, while provide positive charges for recognition of
the peptide termini are exposed and negatively charged bacterial membranes. The
sampling a wide conformational space (Fig. importance of these two residues is further
9.6A). In the structure, the aromatic rings of verified by the identification of hexapeptides
Trp6 and Trp9 pack against Pro7 and Pro10, based on combinatorial library screening
respectively. It is worth noting that the (Chapter 5). These hexamers are rich in both
structure of indolicidin in SDS micelles arginine and tryptophan residues and adopt
somewhat diers from that in DPC. Such a extended structures in micelles (Rezanso et
dierence could be real, as CD spectra of the al., 2005).
peptide in lipid vesicles or in micelles also Then what is the minimal length for a
dier (Rozek et al., 2000). Likewise, Vogel tryptophan/arginine-rich peptide to be active
and colleagues found that tritrpticin adopted against bacteria? Strm et al. (2003) answered
an amphipathic turn structure in SDS this question. While several hexapeptides
micelles (Fig. 9.6B), where the three with 50% of each residue were eective
tryptophan residues are clustered, but against both E. coli and S. aureus, only one
terminal cationic residues occupy a broader pentamer with three tryptophan residues
conformational space, implying motions (WRWRW-amidated) showed good activity
(Schibli et al., 1999). Tinoco et al. (2002) found against both bacteria. Synthetic tetramers
a WW+ motif in micelle-bound PW2 (Fig. were inactive. It appears that five residues are
9.6C), an AMP (sequence: HPLKQYWWRPSI) required for a tryptophan/arginine-rich
obtained from phage display libraries. The peptide to be active. (Coincidently, the
WW+ motif was also found in other peptides. shortest AMPs collected in the APD also have
It is remarkable that the side chain of Arg9 is five residues (sequences: ACSAG, AMVSS
well defined in this case and packs against and AMVGT). It is not clear whether these
the aromatic ring of Trp8, providing an earthworm peptides also act by binding to
example for the cation interaction. The bacterial membranes.) However, the inactive
aromatic ring has electron clouds on the tryptophan/arginine peptides are useful
surface, whereas the arginine side chain templates to design selective AMPs (for other
donates positive charge (Burghardt et al., strategies, see Chapter 4). By attaching three
2002; Chan et al., 2006). As a fourth example, hydrophobic tert-butyl moieties to the
the structure of a tryptophan-rich segment tryptophan ring, Haug et al. (2008) made an
corresponding to residues 414 of lactoferrin inactive tripeptide RWR bactericidal.
B2 is presented in Fig. 9.6D (Nguyen et al., Importantly, the tripeptide analogue only
2005). In SDS micelles, this peptide also associated with anionic membranes, but not
Structural Studies of AMPs 155
Fig. 9.6. Structural ensembles of AMPs from the non- family. Presented are: (A) 16 structures of
bovine indolicidin in dodecylphosphocholine micelles (Protein Data Bank (PDB) ID: 1G89) with the
backbone atoms of residues 611 superimposed; (B) 19 structures of tritrpticin in sodium dodecylsulfate
(SDS) micelles (PDB ID: 1D6X) with the backbone atoms of residues 411 superimposed; (C) 19
structures of PW2 in SDS micelles (PDB ID: 1M02) with the backbone atoms of residues 59
superimposed; and (D) 20 structures of lactoferrin B2(414) in SDS micelles (PDB ID: 1Y5C) with the
backbone atoms of residues 35 superimposed. Key residues are labelled. All structures were
determined by two-dimensional NMR.
one cross peak with a covalently bonded model, since it is a PG with shorter acyl
nitrogen nucleus. In water and at an acidic chains. The NMR T1/T2 ratio-derived
pH (Fig. 9.7A), the (1H, 15N) cross peaks of correlation times suggest that the LL-37
LL-37 are sharp and clustered in a narrow D8PG complex (6.9 ns at 50C) tumbles
spectral region of 8.08.6 ppm, indicating a slightly slower than the LL-37/SDS complex
randomly coiled structure as previously (7.0 ns at 25C). This may explain why the
suggested by CD (Johansson et al., 1998). At NMR spectra of LL-37 in D8PG were poor at
approximately pH 7, only a few cross peaks lower temperatures, but became well
remain (Fig. 9.7B), probably due to the dispersed at 50C (Fig. 9.7D). However, all
formation of elongated aggregates (Li et al., the NMR data collected for LL-37 in the two
2007) and perhaps structural heterogeneity. membrane models are remarkably similar
Since CD spectra indicate a helical structure and indicative of similar structures (Wang,
at this pH (Johansson et al., 1998) and size- 2008c). In addition, we found similar con-
exclusion chromatography suggests a formations for several other peptides in SDS
tetramer for synthetic LL-37 (Li et al., 2007), and D8PG micelles (Wang et al., 2003, 2004,
we propose that LL-37 adopts a four-helix 2005; Li et al., 2006a). Therefore, the LL-37
bundle structure at the physiological pH. As structure determined in SDS (Fig. 9.4C) can
only Ser37 is clearly visible, Fig. 9.7B also be applied to the PG case as well. In
implies that residues 236 of LL-37 par- summary, the high-quality structure of LL-37
ticipate in tetramer formation. Such a helix provides a basis for understanding its
bundle could dissociate into monomers interactions with bacterial outer and inner
when bound to anionic membranes (Oren et membranes. While nearly the entire LL-37 is
al., 1999). required for peptidepeptide interactions (i.e.
LPS is the major outer membrane oligomerization), only the long amphipathic
component of Gram-negative bacteria. For helix is involved in peptidemembrane
some AMPs, binding to LPS is the first step in interactions (Fig. 9.7).
interaction with such bacteria. Furthermore,
LPS can cause sepsis. In this sense, the
Atomic insight into peptidemembrane
binding of AMPs to LPS is a desired property.
interactions
In the presence of LPS (Fig. 9.7C), the cross
peaks for the N-terminal region of To provide additional insight into the
15N-labelled LL-37 disappeared, but the mechanism of action of LL-37, we also
signals for a few C-terminal residues were explored the possibility of detecting peptide
detected. This NMR spectrum implies that lipid interactions directly by NMR. We noted
the N-terminal portion of LL-37 is bound to that the amide proton signals of arginine side
LPS and forms large complexes, leading to chains of LL-37-derived fragments tend to
line broadening. The observation of intense overlap with aromatic protons of phenyl-
signals for C-terminal residues 3537 at alanine residues in SDS. In DPC micelles,
nearly the same positions in the spectrum as they can overlap with the backbone amide
observed for them in SDS or D8PG micelles protons. However, they are well resolved in
indicates that the tail of LL-37 is also flexible D8PG micelles (see Wang, 2008b). This
in complex with LPS. In the presence of lipid unique spectral window for arginine side
A (portion of LPS, see Fig. 7.1), CD spectra chains enabled the detection of argininePG
indicated a helical structure (Turner et al., interactions by solution NMR for the first
1998). Therefore, the ordereddisordered time (Wang, 2007). A useful strategy is to
structural model of LL-37 determined in SDS draw one-dimensional NMR slices at
micelles (Fig. 9.4C) can be applied to the LPS resolved lipid signals. The well-resolved
case. Consistent with this model, deletion of a D8PG protons at 2.34 ppm (C2-H) and 5.25
few N-terminal residues from LL-37 reduced ppm (H) (lipid head group) enabled
LPS binding activity (Kirikae et al., 1998). through space 1H1H dipolar interactions
Next, LL-37 moves towards the inner with the arginine side chain amide protons
membrane of bacteria. We utilized D8PG as a at 7.37.5 ppm (summarized in Fig. 9.8). As
Structural Studies of AMPs 157
Fig. 9.7. Heteronuclear single-quantum coherence spectra of 15N-labelled recombinant LL-37 in water at:
(A) pH 3.6 and (B) pH 6.9; (C) in the presence of lipopolysaccharide (LPS to peptide molar ratio 1:1, pH
5.4, 30C); and (D) in the presence of D8PG (peptide to D8PG ratio 1:30, pH 5.4, 50C). The arginine
side chain peaks are boxed and asparagine/glutamine side chain signals are connected by lines. Figure
adapted from Li et al. (2007) and Wang (2008c).
these intermolecular NOE cross peaks cationic AMPs can have a global impact on
showed normal build-up curves with the the bacterium, in particular those biological
increase in mixing time (Wang, 2007), they processes that require PGs. Examples are
resulted from direct dipolar interactions bacterial signal-transduction pathways (Wang
rather than spin diusions (Wthrich, 1986). et al., 2003) and voltage-dependent potassium
Thus, such NMR measurements provide channels (Schmidt et al., 2006). Therefore,
direct evidence for the association of cationic lipid domain formation will perturb the
peptides with anionic lipids. The argininePG bacterial membrane physiology, leading to
interactions observed herein provide the bacterial death (Wang, 2008c).
driving force for lipid domain formation in In addition, intermolecular NOE
lipid bilayers induced by cationic peptides. observations provide evidence for the
For KR-12, the smallest bactericidal fragment approximation of hydrophobic side chains of
of LL-37, the lipid domain formation is peptides to acyl chains of lipid micelles (Fig.
supported by solid-state NMR, dierential 9.8). For the bacterial membrane anchor,
scanning calorimetry and freeze-fracture aurein 1.2 and LL-37 fragments (e.g. KR-12
electron microscopy (Epand et al., 2009, and GI-20, sequences in Table 4.2),
2010). The clustering of anionic PGs around intermolecular NOEs were detected from the
158 G. Wang
peptide aromatic rings to the protons of the Interactions of AMPs with specific membrane
lipid head group as well as acyl chains (Wang components
et al., 2003, 2005; Wang, 2007). Similar results
were observed for the aromatic rings of Phe5, Bacterial AMPs also target bacterial mem-
Phe6, Phe17 and Phe27 on the hydrophobic branes. Hsu et al. (2002) have investigated the
surface of intact LL-37 in complex with D8PG interactions of nisin Z, a bacteriocin, with
micelles (Wang, 2008c). We conclude that cell-wall precursor lipid II by NMR. Using an
these peptides associated with the membrane 15N-labelled peptide, they found selective
surface. In the case of amphibian aurein 1.2 chemical shift perturbation at the N-terminal
and human LL-37, our conclusions are in line region of this lantibiotic, indicating that the
with solid-state NMR measurements in lipid N-terminal region, including rings A, B and
bilayers (Henzler Wildman et al., 2003; Balla et C, is responsible for nisin recognition. The
al., 2004). Solution NMR, however, provides C-terminal region (rings D and E) appeared
the long-desired evidence that these amphi- not to be involved in the recognition.
pathic AMPs indeed associate with bacterial However, the hinge between these two
membranes via both electrostatic and hydro- domains is critical for biological activity.
phobic interactions. These results for animal Such an interaction pattern is consistent with
AMPs indicate that solution and solid-state mutagenesis studies as well as other NMR
NMR studies provide complementary insight measurements such as temperature coef-
into peptidemembrane interactions in the ficients. The insertion of the C-terminus into
dierent membrane-mimetic systems depicted the membrane completes the pore formation
in Fig. 9.3. process.
Structural Studies of AMPs 159
design of non-peptidic compounds. By mim- have illustrated by NMR that the long
icking the 3D structure of cyclo(RRWWRF), amphipathic helix of LL-37 is responsible for
Appelt et al. (2007) successfully designed association with bacterial outer- and inner-
peptide analogues without using the peptide membrane components (LPS and PGs) (Fig.
backbone. Based on the novel amphipathic 9.7). In addition, we have observed direct
structure determined by Li et al. (2006a), phenylalaninePG and argininePG inter-
Gellman and colleagues succeeded in actions by solution NMR. These data depict
synthesizing random nylon co-polymers as a picture that the positively charged peptide
peptide mimetics (Mowery et al., 2009). These is indeed located on the negatively charged
studies, along with the synthesis of other membrane surface via both electrostatic and
antimicrobial peptidomimetics (Chapter 6), hydrophobic interactions. Such interactions
illustrates that the peptide backbone is not provide the driving forces for the formation
absolutely required. Rather, a proper of non-lamellar phases or lipid domains. It is
arrangement of functional groups into an useful to restate the membrane perturbation
amphipathic scaold is important. Structure- potential model (Wang et al., 2005). This
based engineering is not limited to membrane- model emphasizes the importance of a
bound AMPs. When a unique target molecule proper spatial presentation of cationic and
is verified within pathogenic microbes, novel hydrophobic side chains on AMP surfaces
peptides can be rationally designed based on that determine its ability to perturb the
the 3D structure of the AMPs in complex with bacterial membranes, as well as the outcome
non-membrane targets such as proteins or of peptidemembrane interactions that lead
DNA. Future structural determination of such to bacterial death. Our investigations of
complexes can borrow the NMR strategies peptidemembrane interactions have
already established and demonstrated for deepened and broadened our view on AMPs.
proteinprotein or proteinnucleic complexes In addition to lipid bilayers, AMPs also
(Wang et al., 2000; Marintchev et al., 2007). target specific molecules on the cell surface.
While lantibiotics such as nisins are known
to bind to cell-wall precursor lipid II, insect
9.5 Concluding Remarks and and plant defensins specifically recognize
Perspectives unique fungal sphingolipids. In other
situations, AMPs also recognize carbo-
AMPs can interact with membranes and non- hydrates. It is evident that NMR has played
membrane targets. Much research has been an important role in providing atomic
focused on the membrane-binding property insights into a variety of molecular inter-
of AMPs (Chapter 7). In the membrane- actions involving cationic AMPs.
bound state, they can adopt a variety of Not all AMPs target bacterial membranes
backbone scaolds (-helices, -strands and and interactions of AMPs with other targets
extended structures). Typical examples in the have emerged (for examples, see Chapter 8).
helical family are magainins, aureins, A hallmark for AMPs to act on non-chiral
dermaseptins, temporins and human bacterial membranes is that the d- and
cathelicidin LL-37. Human defensins are l-enantiomers with symmetric mirror images
typical members of the -sheet family. have essentially identical antimicrobial
Indolicidin and tritrpticin are representative activity (Wade et al., 1990; Wei et al., 2009).
members of the extended-structure (non-) This does not apply when AMPs target chiral
family. Although these polypeptide back- molecules such as proteins. The interactions
bones fold in dierent manners, their of AMPs with non-membrane targets are
surfaces share a common amphipathic nature important for many biological functions of
(i.e. a clear segregation of hydrophobic and human cathelicidin and defensins. Under
cationic side chains). Evidently, such a what conditions is there a correlation between
feature is essential for a positively charged peptide aggregation and biological activity?
amphipathic peptide to associate with How does an AMP traverse bacterial
negatively charged bacterial membranes. We membranes and bind to intracellular targets
Structural Studies of AMPs 161
such as DNA or proteins? What is the Appelt, C., Schrey, A.K., Sderhll, J.A. and
structural basis of human defensins HNP13 Schmieder, P. (2007) Design of antimicrobial
and HD-5, but not HNP-4, HD-6 or compounds based on peptide structures.
-defensins (Wei et al., 2009), in recognition of Bioorganic & Medicinal Chemistry Letters 17,
HIV envelope protein gp120 or protein toxins? 23342337.
Aumelas, A., Mangoni, M., Roumestand, C.,
How does LL-37 interact with membrane-
Chiche, L., Despaux, E., Grassy, G., Calas, B.
bound receptors to promote tumour
and Chavanieu, A. (1996) Synthesis and
metastasis, while a LL-37 fragment reverses solution structure of the antimicrobial peptide
the process? Considering that there are protegrin-1. European Journal of Biochemistry
thousands of such AMPs with various 237, 575583.
molecular targets, structural biology will Balla, M.S., Bowie, J.H. and Separovic, F. (2004)
continue to play an essential role in Solid-state NMR study of antimicrobial peptides
elucidating the basis of these ancient from Australian frogs in phospholipid
molecules in host defence and in regulating membranes. European Biophysics Journal 33,
immune responses. 109116.
Bang, S.K., Kang, C.S., Han, M.D. and Bang, I.S.
(2010) Expression of recombinant hybrid
peptide hinnavinII/-melanocyte-stimulating
Note Added in Proof
hormone in Escherichia coli: purification and
characterization. Journal of Microbiology 48,
During the production of this book, high- 2429.
level bacterial and yeast expression systems Bauer, F., Schweimer, K., Klver, E., Conejo-Garcia,
suitable for large-scale production of J.R., Forssmann, W.G., Rsch, P., Adermann,
recombinant LL-37 have also been reported K. and Sticht, H. (2001) Structure determination
(Krahulec et al., 2010; Liu et al., 2010). of human and murine -defensins reveals
structural conservation in the absence of
significant sequence similarity. Protein Science
Acknowledgement 10, 24702479.
Bax, A. and Grzesiek, S. (1993) Methodological
advances in protein NMR. Accounts of Chemical
The research was supported by the grant
Research 26, 131138.
award R56AI81975 from the National Bergman, P., Walter-Jallow, L., Broliden, K.,
Institute of Allergy and Infectious Diseases Agerberth, B. and Soderlund, J. (2007) The
(NIH, USA). antimicrobial peptide LL-37 inhibits HIV-1
replication. Current HIV Research 5, 410415.
Bhling, A., Hagge, S.O., Roes, S., Podschun, R.,
Sahly, H., Harder, J., Schrder, J.M., Grtzinger,
References J., Seydel, U. and Gutsmann, T. (2006) Lipid-
specific membrane activity of human
Aboitiz, N., Vila-Perell, M., Groves, P., Asensio, -defensin-3. Biochemistry 45, 56635670.
J.L., Andreu, D., Caada, F.J. and Jimnez- Boman, H.G. (2003) Antibacterial peptides: basic
Barbero, J. (2004) NMR and modeling studies facts and emerging concepts. Journal of Internal
of proteincarbohydrate interactions: synthesis, Medicine 254, 197215.
three-dimensional structure, and recognition Braff, M.H., Hawkins, M.A., Di Nardo, A., Lopez-
properties of a minimum hevein domain with Garcia, B., Howell, M.D., Wong, C., Lin, K.,
binding affinity for chitooligosaccharides. Streib, J.E., Dorschner, R., Leung, D.Y. and
Chembiochem: A European Journal of Chemical Gallo, R.L. (2005) Structurefunction relation-
Biology 5, 12451255. ships among human cathelicidin peptides:
Aerts, A.M., Franois, I.E., Meert, E.M., Li, Q.T., dissociation of antimicrobial properties from
Cammue, B.P. and Thevissen, K. (2007) The host immunostimulatory activities. Journal of
antifungal activity of RsAFP2, a plant defensin Immunology 174, 42714278.
from Raphanus sativus, involves the induction Bucki, R., Byfield, F.J. and Janmey, P.A. (2007)
of reactive oxygen species in Candida albicans. Release of the antimicrobial peptide LL-37 from
Journal of Molecular Microbiology and DNA/F-actin bundles in cystic fibrosis sputum.
Biotechnology 13, 243247. European Respiratory Journal 29, 624632.
162 G. Wang
Burghardt, T.P., Juranic, N., Macura, S. and Ajtai, K. and nucleic acid PDB entries. Journal of
(2002) Cation interaction in a folded Biomolecular NMR 45, 389396.
polypeptide. Biopolymers 63, 261272. Dorschner, R.A., Lopez-Garcia, B., Peschel, A.,
Burton, M.F. and Steel, P.G. (2009) The chemistry Kraus, D., Morikawa, K., Nizet, V. and Gallo, R.L.
and biology of LL-37. Natural Product Reports (2006) The mammalian ionic environment
26, 15721584. dictates microbial susceptibility to antimicrobial
Chan, D.I., Prenner, E.J. and Vogel, H.J. (2006) defense peptides. FASEB Journal 20, 3542.
Tryptophan- and arginine-rich antimicrobial Dubovskii, P.V., Volynsky, P.E., Polyansky, A.A.,
peptides: structures and mechanisms of action. Chupin, V.V., Efremov, R.G. and Arseniev, A.S.
Biochimica et Biophysica Acta 1758, (2006) Spatial structure and activity mechanism
11841202. of a novel spider antimicrobial peptide.
Chandrababu, K.B., Ho, B. and Yang, D. (2009) Biochemistry 45, 1075910767.
Structure, dynamics, and activity of an all- Epand, R.F., Wang, G., Berno, B. and Epand, R.M.
cysteine mutated human defensin-3 peptide (2009) Lipid segregation explains selective
analogue. Biochemistry 48, 60526061. toxicity of a series of fragments derived from the
Chen, H., Xu, Z., Peng, L., Fang, X., Yin, X., Xu, N. human cathelicidin LL-37. Antimicrobial Agents
and Cen, P. (2006) Recent advances in the and Chemotherapy 53, 37053714.
research and development of human defensins. Epand, R.M., Epand, R.F., Arnusch, C.J.,
Peptides 27, 931940. Papahadjopoulos-Sternberg, B., Wang, G. and
Chi, S.W., Kim, J.S., Kim, D.H., Lee, S.H., Park, Shai, Y. (2010) Lipid clustering by three
Y.H. and Han, K.H. (2007) Solution structure homologous arginine-rich antimicrobial peptides
and membrane interaction mode of an
is insensitive to amino acid arrangement and
antimicrobial peptide gaegurin 4. Biochemical
induced secondary structure. Biochimica et
and Biophysical Research Communications
Biophysica Acta 1798, 12721280.
352, 592597.
Galanth, C., Abbassi, F., Lequin, O., Ayala-
Ciornei, C.D., Sigurdardttir, T., Schmidtchen, A.
Sanmartin, J., Ladram, A., Nicolas, P. and
and Bodelsson, M. (2005) Antimicrobial and
Amiche, M. (2009) Mechanism of antibacterial
chemoattractant activity, lipopolysaccharide
action of dermaseptin B2: interplay between
neutralization, cytotoxicity, and inhibition by
helix-hinge-helix structure and membrane
serum of analogs of human cathelicidin LL-37.
curvature strain. Biochemistry 48, 313327.
Antimicrobial Agents and Chemotherapy 49,
Gesell, J., Zasloff, M. and Opella, S.J. (1997) Two-
28452850.
Cirioni, O., Giacometti, A., Ghiselli, R., Bergnach, dimensional 1H NMR experiments show that the
C., Orlando, F., Silvestri, C., Mocchegiani, F., 23-residue magainin antibiotic peptide is an
Licci, A., Skerlavaj, B., Rocchi, M., Saba, V., -helix in dodecylphosphocholine micelles,
Zanetti, M. and Scalise, G. (2006) LL-37 protects sodium dodecylsulfate micelles, and trifluoro-
rats against lethal sepsis caused by Gram- ethanol/water solution. Journal of Biomolecular
negative bacteria. Antimicrobial Agents and NMR 9, 127135.
Chemotherapy 50, 16721679. Gudmundsson, G.H., Magnusson, K.P., Chowdhary,
Dalla Serra, M., Cirioni, O., Vitale, R.M., Renzone, B.P., Johansson, M., Andersson, L. and Boman,
G., Coraiola, M., Giacometti, A., Potrich, C., H.G. (1995) Structure of the gene for porcine
Baroni, E., Guella, G., Sanseverino, M., De peptide antibiotic PR-39, a cathelin gene family
Luca, S., Scalise, G., Amodeo, P. and Scaloni, member: comparative mapping of the locus for
A. (2008) Structural features of distinctin the human peptide antibiotic FALL-39. Proceed-
affecting peptide biological and biochemical ings of the National Academy of Sciences of the
properties. Biochemistry 47, 78887899. USA 92, 70857089.
De Medeiros, L.N., Angeli, R., Sarzedas, C.G., Haney, E.F. and Vogel, H.J. (2009) NMR of
Barreto-Bergter, E., Valente, A.P., Kurtenbach, antimicrobial peptides. Annual Reports on NMR
E. and Almeida, F.C. (2010) Backbone dynamics Spectroscopy 65, 151.
of the antifungal Psd1 pea defensin and its Haney, E.F., Hunter, H.N., Matsuzaki, K. and Vogel,
correlation with membrane interaction by NMR H.J. (2009) Solution NMR studies of amphibian
spectroscopy. Biochimica et Biophysica Acta antimicrobial peptides: linking structure to
1798, 105113. function? Biochimica et Biophysica Acta 1788,
Doreleijers, J.F., Vranken, W.F., Schulte, C., Lin, J., 16391655.
Wedell, J.R., Penkett, C.J., Vuister, G.W., Vriend, Haney, E.F., Nathoo, S., Vogel, H.J. and Prenner,
G., Markley, J.L. and Ulrich, E.L. (2009) The E.J. (2010) Induction of non-lamellar lipid
NMR restraints grid at BMRB for 5,266 protein phases by antimicrobial peptides: a potential
Structural Studies of AMPs 163
link to mode of action. Chemistry and Physics of Jang, S.A., Sung, B.H., Cho, J.H. and Kim, S.C.
Lipids 163, 8293. (2009) Direct expression of antimicrobial
Haug, B.E., Stensen, W., Kalaaji, M., Rekdal, . and peptides in an intact form by a translationally
Svendsen, J.S. (2008) Synthetic antimicrobial coupled two-cistron expression system. Applied
peptidomimetics with therapeutic potential. and Environmental Microbiology 75,
Journal of Medicinal Chemistry 51, 43064314. 39803986.
Henzler Wildman, K.A., Lee, D.K. and Ramamoorthy, Johansson, J., Gudmundsson, G.H., Rottenberg,
A. (2003) Mechanism of lipid bilayer disruption M.E., Berndt, K.D. and Agerberth, B. (1998)
by the human antimicrobial peptide, LL-37. Conformation-dependent antibacterial activity
Biochemistry 42, 65456558. of the naturally occurring human peptide LL-37.
Hill, C.P., Yee, J., Selsted, M.E. and Eisenberg, D. Journal of Biological Chemistry 273,
(1991) Crystal structure of defensin HNP-3, an 37183724.
amphiphilic dimer: mechanisms of membrane Kapust, R.B., Tzsr, J., Fox, J.D., Anderson, D.E.,
permeabilization. Science 251, 14811485. Cherry, S., Copeland, T.D. and Waugh, D.S.
Holak, T.A., Engstrm, A., Kraulis, P.J., Lindeberg, (2001) Tobacco etch virus protease: mechanism
G., Bennich, H., Jones, T.A., Gronenborn, A.M. of autolysis and rational design of stable mutants
and Clore, G.M. (1988) The solution with wild-type catalytic proficiency. Protein
conformation of the antibacterial peptide Engineering 14, 9931000.
cecropin A: a nuclear magnetic resonance and Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone
dynamical simulated annealing study. dynamics of proteins as studied by 15N inverse
Biochemistry 27, 76207629. detected heteronuclear NMR spectroscopy:
Hoover, D.M., Rajashankar, K.R., Blumenthal, R.,
application to staphylococcal nuclease.
Puri, A., Oppenheim, J.J., Chertov, O. and
Biochemistry 28, 89728979.
Lubkowski, J. (2000) The structure of human
Keifer, P.A., Peterkofsky, A. and Wang, G. (2004)
-defensin-2 shows evidence of higher order
Effects of detergent alkyl chain length and
oligomerization. Journal of Biological Chemistry
chemical structure on the properties of a
275, 3291132918.
micelle-bound bacterial membrane targeting
Hoover, D.M., Chertov, O. and Lubkowski, J. (2001)
peptide. Analytical Biochemistry 331, 3339.
The structure of human -defensin-1: new
Kim, J.M., Jang, S.A., Yu, B.J., Sung, B.H., Cho,
insights into structural properties of -defensins.
J.H. and Kim, S.C. (2008) High-level expression
Journal of Biological Chemistry 276,
of an antimicrobial peptide histonin as a natural
3902139026.
Hoover, D.M., Wu, Z., Tucker, K., Lu, W. and form by multimerization and furin-mediated
Lubkowski, J. (2003) Antimicrobial character- cleavage. Applied Microbiology and Biotech-
ization of human -defensin 3 derivatives. nology 78, 123130.
Antimicrobial Agents and Chemotherapy 47, Kirikae, T., Hirata, M., Yamasu, H., Kirikae, F.,
28042809. Tamuta, H., Kayama, F., Nakatsuka, K., Yokochi,
Hsu, S.T., Breukink, E., de Kruijff, B., Kaptein, R., T. and Nakano, M. (1998) Protective effects of a
Bonvin, A.M. and van Nuland, N.A. (2002) human 18-kilodalton cationic antimicrobial
Mapping the targeted membrane pore formation protein (CAP18)-derived peptide against murine
mechanism by solution NMR: the nisin Z and endotoxemia. Infection and Immunity 66,
lipid II interaction in SDS micelles. Biochemistry 18611868.
41, 76707676. Klver, E., Adermann, K. and Schulz, A. (2006)
Hu, F., Ke, T., Li, X., Mao, P.H., Jin, X., Hui, F.L., Ma, Synthesis and structureactivity relationship of
X.D. and Ma, L.X. (2010) Expression and -defensins, multi-functional peptides of the
purification of an antimicrobial peptide by fusion immune system. Journal of Peptide Science 12,
with elastin-like polypeptides in Escherichia coli. 243257.
Applied Biochemistry and Biotechnology 160, Krahulec, J., Hyrsov, M., Pepeliaev, S., Jlkov, J.,
23772387. Cern, Z. and Machlkov, J. (2010) High level
Imamura, T., Yamamoto, N., Tamura, A., expression and purification of antimicrobial
Murabayashi, S., Hashimoto, S., Shimada, H. human cathelicidin LL-37 in Escherichia coli.
and Taguchi, S. (2008) NMR based structure Applied Microbiology Biotechnology 88,
activity relationship analysis of an antimicrobial 167175.
peptide, thanatin, engineered by site-specific Laederach, A., Andreotti, A.H. and Fulton, D.B.
chemical modification: activity improvement and (2002) Solution and micelle-bound structures of
spectrum alteration. Biochemical and Biophysical tachyplesin I and its active aromatic linear
Research Communications 369, 609615. derivatives. Biochemistry 41, 1235912368.
164 G. Wang
Landon, C., Meudal, H., Boulanger, N., Bulet, P. the activity of tobacco etch virus protease in
and Vovelle, F. (2006) Solution structures of detergent solutions. Analytical Biochemistry
stomoxyn and spinigerin, two insect antimicrobial 382, 6971.
peptides with an -helical conformation. Malakhov, M.P., Mattern, M.R., Malakhova, O.A.,
Biopolymers 81, 92103. Drinker, M., Weeks, S.D. and Butt, T.R. (2004)
Landon, C., Barbault, F., Legrain, M., Guenneugues, SUMO fusions and SUMO-specific protease for
M. and Vovelle, F. (2008) Rational design of efficient expression and purification of proteins.
peptides active against the Gram positive Journal of Structural and Functional Genomics
bacteria Staphylococcus aureus. Proteins 72, 5, 7586.
229239. Mandard, N., Sodano, P., Labbe, H., Bonmatin,
Langham, A.A., Ahmad, A.S. and Kaznessis, Y.N. J.M., Bulet, P., Hetru, C., Ptak, M. and Vovelle, F.
(2008) On the nature of antimicrobial activity: a (1998) Solution structure of thanatin, a potent
model for protegrin-1 pores. Journal of the bactericidal and fungicidal insect peptide,
American Chemical Society 130, 43384346. determined from proton two-dimensional
Lehrer, R.I., Jung, G., Ruchala, P., Wang, W., nuclear magnetic resonance data. European
Micewicz, E.D., Waring, A.J., Gillespie, E.J., Journal of Biochemistry 256, 404410.
Bradley, K.A., Ratner, A.J., Rest, R.F. and Lu, W. Mandard, N., Bulet, P., Caille, A., Daffre, S. and
(2009) Human -defensins inhibit hemolysis Vovelle, F. (2002) The solution structure of
mediated by cholesterol-dependent cytolysins. gomesin, an antimicrobial cysteine-rich peptide
Infection and Immunity 77, 40284040. from the spider. European Journal of
Li, X., Li, Y., Han, H., Miller, D.W. and Wang, G. Biochemistry 269, 11901198.
(2006a) Solution structures of human LL-37 Mani, R., Cady, S.D., Tang, M., Waring, A.J., Lehrer,
fragments and NMR-based identification of a R.I. and Hong, M. (2006) Membrane-dependent
minimal membrane-targeting antimicrobial and oligomeric structure and pore formation of a
anticancer region. Journal of the American -hairpin antimicrobial peptide in lipid bilayers
Chemical Society 128, 57765785. from solid-state NMR. Proceedings of the
Li, X., Li, Y., Peterkofsky, A. and Wang, G. (2006b) National Academy of Sciences of the USA 103,
NMR studies of aurein 1.2 analogs. Biochimica 1624216247.
et Biophysica Acta 1758, 12031214. Marintchev, A., Frueh, D. and Wagner, G. (2007)
Li, Y., Li, X., and Wang, G. (2006c) Cloning, NMR methods for studying proteinprotein
expression, isotope labeling, and purification of interactions involved in translation initiation.
human antimicrobial peptide LL-37 in Methods in Enzymology 430, 283331.
Escherichia coli for NMR studies. Protein Markley, J.L., Ulrich, E.L., Berman, H.M., Henrick,
Expression and Purification 47, 498505. K., Nakamura, H. and Akutsu, H. (2008)
Li, Y., Li, X., Li, H., Lockridge, O. and Wang, G. BioMagResBank (BMRB) as a partner in the
(2007) A novel method for purifying recombinant Worldwide Protein Data Bank (wwPDB): new
human host defense cathelicidin LL-37 by policies affecting biomolecular NMR depositions.
utilizing its inherent property of aggregation. Journal of Biomolecular NMR 40, 153155.
Protein Expression and Purification 54, Merrifield, E.L., Mitchell, S.A., Ubach, J., Boman,
157165. H.G., Andreu, D. and Merrifield, R.B. (1995)
Liu, D., He, J., Lin, Y. and Huang, Y. (2010) High D-Enantiomers of 15-residue cecropin Amelittin
expression of antimicrobial peptide LL-37 in hybrids. International Journal of Peptide and
yeast (SMD1168). Zhongguo Yufang Shouyi Protein Research 46, 214220.
Xuebao 32, 98101 (in Chinese). Moon, J.Y., Henzler-Wildman, K.A. and Ramamoothy,
Lobo, D.S., Pereira, I.B., Fragel-Madeira, L., A. (2006) Expression and purification of a
Medeiros, L.N., Cabral, L.M., Faria, J., Bellio, recombinant LL-37 in Escherichia coli. Biochimica
M., Campos, R.C., Linden, R. and Kurtenbach, et Biophysica Acta 1758, 13511358.
E. (2007) Antifungal Pisum sativum defensin 1 Mowery, B.P., Lindner, A.H., Weisblum, B., Stahl,
interacts with Neurospora crassa cyclin F S.S. and Gellman, S.H. (2009) Structureactivity
related to the cell cycle. Biochemistry 46, relationships among random nylon-3 copolymers
987996. that mimic antibacterial host-defense peptides.
Losonczi, J.A. and Prestegard, J.H. (1998) Improved Journal of the American Chemical Society 131,
dilute bicelle solutions for high-resolution NMR 97359745.
of biological macromolecules. Journal of Mysliwy, J., Dingley, A.J., Stanisak, M., Jung, S.,
Biomolecular NMR 12, 447451. Lorenzen, I., Roeder, T., Leippe, M. and
Lundbck, A.K., van den Berg, S., Hebert, H., Grtzinger, J. (2010) Caenopore-5: the three-
Berglund, H. and Eshaghi, S. (2008) Exploring dimensional structure of an antimicrobial protein
Structural Studies of AMPs 165
from Caenorhabditis elegans. Developmental Porcelli, F., Buck, B., Lee, D.K., Hallock, K.J.,
and Comparative Immunology 34, 323330. Ramamoorthy, A. and Veglia, G. (2004) Structure
Nguyen, L.T., Schibli, D.J. and Vogel, H.J. (2005) and orientation of pardaxin determined by NMR
Structural studies and model membrane experiments in model membranes. Journal of
interactions of two peptides derived from bovine Biological Chemistry 279, 4581545823.
lactoferricin. Journal of Peptide Science 11, Powers, J.P., Rozek, A. and Hancock, R.E. (2004)
379389. Structureactivity relationships for the -hairpin
Nijnik, A. and Hancock, R.E. (2009) The roles of cationic antimicrobial peptide polyphemusin I.
cathelicidin LL-37 in immune defences and Biochimica et Biophysica Acta 1698, 239250.
novel clinical applications. Current Opinion in Prosser, R.S., Evanics, F., Kitevski, J.L. and
Hematology 16, 4147. Al-Abdul-Wahid, M.S. (2006) Current
Nizet, V., Ohtake, T., Lauth, X., Trowbridge, J., applications of bicelles in NMR studies of
Rudisill, J., Dorschner, R.A., Pestonjamasp, V., membrane-associated amphiphiles and
Piraino, J., Huttner, K. and Gallo, R.L. (2001) proteins. Biochemistry 45, 84538465.
Innate antimicrobial peptide protects the skin Puigbo, P., Guzman, E., Romeu, A. and Garcia-
from invasive bacterial infection. Nature 414, Vallve, S. (2007) OPTIMIZER: a web server for
454457. optimizing the codon usage of DNA sequences.
Opella, S.J. and Marassi, F.M. (2004) Structure Nucleic Acids Research 35, W121W131.
determination of membrane proteins by NMR Ptsep, K., Carlsson, G., Boman, H.G. and
spectroscopy. Chemical Reviews 104, Andersson, M. (2002) Deficiency of antibacterial
35873606. peptides in patients with morbus Kostmann: an
Oren, Z., Lerman, J.C., Gudmundsson, G.H., observation study. Lancet 360, 11441149.
Agerberth, B. and Shai, Y. (1999) Structure and Raj, P.A., Antonyraj, K.J. and Karunakaran, T.
organization of the human antimicrobial peptide (2000) Large-scale synthesis and functional
LL-37 in phospholipid membranes: relevance to elements for the antimicrobial activity of
the molecular basis for its non-cell-selective defensins. Biochemical Journal 347, 633641.
activity. Biochemical Journal 341, 501513. Ramos, R., Domingues, L. and Gama, M. (2010)
Ovchinnikova, T.V., Shenkarev, Z.O., Nadezhdin, Escherichia coli expression and purification of
K.D., Balandin, S.V., Zhmak, M.N., Kudelina, LL37 fused to a family III carbohydrate-binding
I.A., Finkina, E.I., Kokryakov, V.N. and Arseniev, module from Clostridium thermocellum. Protein
A.S. (2007) Recombinant expression, synthesis, Expression and Purification 71, 17.
purification, and solution structure of arenicin. Resende, J.M., Moraes, C.M., Munhoz, V.H.,
Biochemical and Biophysical Research Aisenbrey, C., Verly, R.M., Bertani, P., Cesar, A.,
Communications 360, 156162. Pil-Veloso, D. and Bechinger, B. (2009)
Papo, N. and Shai, Y. (2004) Effect of drastic Membrane structure and conformational
sequence alteration and D-amino acid changes of the antibiotic heterodimeric peptide
incorporation on the membrane binding behavior distinctin by solid-state NMR spectroscopy.
of lytic peptides. Biochemistry 43, 63936403. Proceedings of the National Academy of
Park, S., Son, W.S., Kim, Y.J., Kwon, A.R. and Lee, Sciences of the USA 106, 1663916644.
B.J. (2007) NMR spectroscopic assessment of Rezansoff, A.J., Hunter, H.N., Jing, W., Park, I.Y.,
the structure and dynamic properties of an Kim, S.C. and Vogel, H.J. (2005) Interactions of
amphibian antimicrobial peptide (gaegurin 4) the antimicrobial peptide Ac-FRWWHR-NH2
bound to SDS micelles. Journal of Biochemistry with model membrane systems and bacterial
and Molecular Biology 40, 261269. cells. Journal of Peptide Research 65, 491501.
Park, T.J., Kim, J.S., Choi, S.S. and Kim, Y. (2009) Rodziewicz-Motowido, S., Mickiewicz, B., Greber,
Cloning, expression, isotope labeling, purifica- K., Sikorska, E., Szultka, L., Kamysz, E. and
tion, and characterization of bovine antimicrobial Kamysz, W. (2010) Antimicrobial and
peptide, lactophoricin in Escherichia coli. Protein conformational studies of the active and inactive
Expression and Purification 65, 2329. analogues of the protegrin-1 peptide. FEBS
Pazgier, M. and Lubkowski, J. (2006) Expression Journal 277, 10101022.
and purification of recombinant human Rozek, A., Friedrich, C.L. and Hancock, R.E. (2000)
-defensins in Escherichia coli. Protein Structure of the bovine antimicrobial peptide
Expression and Purification 49, 18. indolicidin bound to dodecylphosphocholine
Pazgier, M., Prahl, A., Hoover, D.M. and Lubkowski, and sodium dodecyl sulfate micelles.
J. (2007) Studies of the biological properties of Biochemistry 39, 1576515774.
human -defensin 1. Journal of Biological Rozek, A., Powers, J.P., Friedrich, C.L. and
Chemistry 282, 18191829. Hancock, R.E. (2003) Structure-based design
166 G. Wang
31P solid-state NMR spectroscopy. Biophysical Wang, G., Li, Y. and Li, X. (2005) Correlation of
Journal 96, 219421203. three-dimensional structures with the
Wade, D., Boman, A., Whlin, B., Drain, C.M., antibacterial activity of a group of peptides
Andreu, D., Boman, H.G. and Merrifield, R.B. designed based on a non-toxic bacterial
(1990) All-D amino acid-containing channel- membrane anchor. Journal of Biological
forming antibiotic peptides. Proceedings of the Chemistry 280, 58035811.
National Academy of Sciences of the USA 87, Wang, G., Watson, K.M. and Buckheit, R.W. Jr
47614765. (2008) Anti-human immunodeficiency virus type
Wang, G. (2006) Structural biology of antimicrobial 1 activities of antimicrobial peptides derived
peptides by NMR spectroscopy. Current Organic from human and bovine cathelicidins.
Chemistry 10, 569581. Antimicrobial Agents and Chemotherapy 52,
Wang, G. (2007) Determination of solution structure 34383440.
and lipid micelle location of an engineered Wang, G., Li, X. and Wang, Z. (2009) APD2: the
membrane peptide by using one NMR updated antimicrobial peptide database and its
experiment and one sample. Biochimica et application in peptide design. Nucleic Acids
Biophysica Acta 1768, 32713281. Research 37, D933D937.
Wang, G. (2008a) NMR of membrane-associated Weber, G., Chamorro, C.I., Granath, F., Liljegren,
peptides and proteins. Current Protein & Peptide A., Zreika, S., Saidak, Z., Sandstedt, B.,
Science 9, 5069. Rotstein, S., Mentaverri, R., Snchez, F.,
Wang, G. (2008b) NMR studies of a model Pivarcsi, A. and Sthle, M. (2009) Human
antimicrobial peptide in the micelles of SDS, antimicrobial protein hCAP18/LL-37 promotes a
dodecylphosphocholine, or dioctanoylphos-
metastatic phenotype in breast cancer. Breast
phatidylglycerol. Open Magnetic Resonance
Cancer Research 11, R6.
Journal 1, 915.
Wei, G., de Leeuw, E., Pazgier, M., Yuan, W., Zou,
Wang, G. (2008c) Structures of human host defense
G., Wang, J., Ericksen, B., Lu, W.Y., Lehrer, R.I.
cathelicidin LL-37 and its smallest antimicrobial
and Lu, W. (2009) Through the looking glass,
peptide KR-12 in lipid micelles. Journal of
mechanistic insights from enantiomeric human
Biological Chemistry 283, 3263732643.
defensins. Journal of Biological Chemistry 284,
Wang, G. (2010) Structure, dynamics, and mapping
2918029192.
of membrane-binding residues of micelle-bound
Welch, M., Govindarajan, S., Ness, J.E., Villalobos,
antimicrobial peptides by natural abundance
13C NMR spectroscopy. Biochimica et Biophysica A., Gurney, A., Minshull, J. and Gustafsson, C.
Acta 1798, 114121. (2009) Design parameters to control synthetic
Wang, G., Pierens, G.K., Treleaven, W.D., Sparrow, gene expression in Escherichia coli. PLoS One
J.T. and Cushley, R.J. (1996) Conformations of 4, e7002.
human apolipoprotein E(263286) and E(267 Wishart, D.S. and Sykes, B.D. (1994) The 13C
289) in aqueous solutions of sodium dodecyl chemical-shift index: a simple method for the
sulfate by CD and 1H-NMR. Biochemistry 35, identification of protein secondary structure
1035810366. using 13C chemical-shift data. Journal of
Wang, G., Louis, J.M., Sondej, M., Seok, Y.J., Biomolecular NMR 4, 171180.
Peterkofsky, A. and Clore, G.M. (2000) Solution Wu, H., Su, K., Guan, X., Sublette, M.E. and Stark,
structure of the phosphoryl transfer complex R.E. (2010) Assessing the size, stability, and
between the signal transducing proteins HPr and utility of isotropically tumbling bicelle systems
IIAGlucose of the Escherichia coli phospho- for structural biology. Biochimica et Biophysica
enolpyruvate:sugar phosphotransferase system. Acta 1798, 482488.
EMBO Journal 19, 56355649. Wu, Z., Ericksen, B., Tucker, K., Lubkowski, J. and
Wang, G., Keifer, P.A. and Peterkofsky, A. (2003) Lu, W. (2004) Synthesis and characterization of
Solution structure of the N-terminal amphitropic human -defensins 46. Journal of Peptide
domain of Escherichia coli glucose-specific Research 64, 118125.
enzyme IIA in membrane-mimetic micelles. Wthrich, K. (1986) NMR of Proteins and Nucleic
Protein Science 12, 10871096. Acids. Wiley, New York.
Wang, G., Keifer, P.A. and Peterkofsky, A. (2004) Xie, C., Prahl, A., Ericksen, B., Wu, Z., Zeng, P., Li,
Short-chain diacylphosphatidylglycerols: which X., Lu, W.Y., Lubkowski, J. and Lu, W. (2005)
one to choose for the NMR structural Reconstruction of the conserved -bulge in
determination of a membrane-associated mammalian defensins using D-amino acids.
peptide from Escherichia coil? Spectroscopy Journal of Biological Chemistry 280,
18, 257264. 3292132929.
168 G. Wang
Yamasaki, K., Schauber, J., Coda, A., Lin, H., Zanetti, M. (2005) The role of cathelicidins in the
Dorschner, R.A., Schechter, N.M., Bonnart C., innate host defenses of mammals. Current
Descargues, P., Hovnanian, A. and Gallo, R.L. Issues in Molecular Biology 7, 179196.
(2006) Kallikrein-mediated proteolysis regulates Zasloff, M. (2002) Antimicrobial peptides of
the antimicrobial effects of cathelicidins in skin. multicellular organisms. Nature 415, 389395.
FASEB Journal 20, 20682080. Zelezetsky, I., Pontillo, A., Puzzi, L., Antcheva, N.,
Yang, Y., Zheng, G., Li, G., Zhang, X., Cao, Z., Rao, Segat, L., Pacor, S., Crovella, S. and Tossi, A.
Q. and Wu, K. (2004) Expression of bioactive (2006) Evolution of the primate cathelicidin,
recombinant GSLL-39, a variant of human correlation between structural variations and
antimicrobial peptide LL-37, in Escherichia coli. antimicrobial activity. Journal of Biological
Protein Expression and Purification 37, Chemistry 281, 1986119871.
229235. Zhang, L., Falla, T., Wu, M., Fidai, S., Burian, J.,
Yau, W.M., Wimley, W.C., Gawrisch, K. and White, Kay, W. and Hancock, R.E. (1998) Determinants
S.H. (1998) The preference of tryptophan for of recombinant production of antimicrobial
membrane interfaces. Biochemistry 37, cationic peptides and creation of peptide
1471314718. variants in bacteria. Biochemical and Biophysical
Research Communications 247, 674680.
10
Lung Infection: Shifting the
Equilibrium Towards the Free and Active
Form of Human LL-37 and the Design of
Alternative Antimicrobial Agents
Abstract
Under disease conditions, human LL-37 and other cationic antimicrobial factors are expressed but
inactive due to their binding to anionic polyelectrolytes such as DNA, F-actin or acidic polysaccharides.
This chapter discusses the basis for this electrostatic interaction and alternative strategies that liberate
LL-37 and other amphipathic cations from the bound state so that they are available to fight invading
pathogenic bacteria. The potential of this approach for therapeutic use is discussed.
(F)-actin, glycosaminoglycans and mucins with the square of the distance between
produced by the host, as well as in anionic charges as: Epoint = q/4r2. Here, q is the
polyelectrolytes secreted by bacteria such as charge on the ion (in this case the cationic
Pseudomonas (Weiner et al., 2003; Baranska- antimicrobial peptide (AMP)), is the
Rybak et al., 2006; Bergsson et al., 2009). permittivity of water and r is the distance
between the two charges. In contrast, the
field from a line with linear charge density l
10.1.2 Interaction of polyvalent cations (such as F-actin, DNA or alginate) decays
with linear polyelectrolytes only linearly with r as: Eline = l/2r. The
field from an infinitely large charged surface
Charged lines and surfaces interact with (here, the anionic surface of the bacterium) is
soluble counterions dierently from the a constant with respect to r for distances that
interactions of two point charges, and when are small relative to the size of the cell. The
the linear charge density of a rod-like filament simplest consequence of these laws is that for
is suciently high, more multivalent than a given number of anionic charges, arranging
univalent counterions can condense on the them as distributed points, lines or surfaces
filament surface. If a sucient number of has a large eect on their attraction for
polyvalent counterions condense, the electro- counterions, with the greatest attraction for
static repulsion between like-charged linear the charged surface. Another result of
polyanions (DNA or F-actin in the case of CF) polyelectrolyte theory is that multivalent
is overcome by attractive interactions, and counterions are drawn to the filament much
multivalent cations, such as LL-37, induce the more avidly than ions of smaller valence,
formation of bundled filament arrays with largely because the entropy loss from
counterions trapped inside the bundle. sequestering a smaller number of high-
Experimental and theoretical work on valence ions to lower the surface charge of
polyelectrolyte condensation, developed the filaments is less than the same degree of
initially to model the condensation of DNA charge neutralization due to condensation of
(Perdue et al., 1976; Overman et al., 1998; a larger number of lower valence counterions.
Fuller et al., 2007; Devkota et al., 2009), shows In addition, because the fixed charges on
that multivalent counterions do not bind actin, DNA and other biopolymers are
tightly to the surface of the polyelectrolyte, univalent (phosphodiesters or carboxyls),
but maintain mobility along the filament sequestration of multivalent counterions
surface. When sucient counterions condense produces regions of positive charge that can
on the filament, the electrostatic repulsions lead to filamentfilament interactions due to
between filaments that keep them separated the formation of structures analogous to
become weaker than attractive interactions Wigner lattices (Lenac and Sunjic, 1991).
and the filaments collapse into bundles. These features of polyelectrolytes have
Previous studies of F-actin (Tang and Janmey, recently been shown to account for unusual
1996; Tang et al., 1997) and alginate (Bu et al., structures formed in rings of F-actin bundled
2004; Donati et al., 2006) confirm that the by divalent metal ions (Tang et al., 2001;
polyelectrolyte behaviour first observed with Cebers et al., 2006), and for the fact that
DNA also applies to these filaments, which filament bundles stabilized by counterions,
also have fixed charge spacing smaller than such as F-actin bundled by lysozyme
the Bjerrum length and are therefore classified (Guaqueta et al., 2006), cannot be dissociated
as strong polyelectrolytes. by increased monovalent ions.
The mechanism by which polyelectrolyte There are many assumptions including
filaments can trap multivalent cations such that the lines and surfaces are infinite and
as LL-37 depends on the strength of the uniform but at the crudest level, this
electric field attracting the counterion to the hierarchy of electrostatic attractions ration-
filament. The electric field, and therefore the alizes why cationic antimicrobial agents
force on an ion, emanating from a point selectively attack bacterial membranes
charge (such as a small soluble ion) decays without an apparent unique bacterial binding
Design of Alternative Antimicrobial Agents 171
partner, and why polyelectrolyte filaments monomeric actin, which can polymerize and
such as actin, alginate and DNA can inhibit form a mixed network of DNA and actin
their functions. Of course, AMPs are not filaments. F-actin bundles (Fig. 10.1) have
simply cations and hydrophobic moieties. been identified as an additional abnormal
The juxtaposition of cationic charges with biopolymer that can aect the viscous
spacing suitable for binding lipopoly- properties of lung airway fluid in respiratory
saccharide (LPS), lipoteichoic acid (LTA) or disease (Vasconcellos et al., 1994; Sheils et al.,
other anionic targets, and other modifications 1996).
that optimize lethal eects on bacteria, are Bacteria also produce anionic poly-
essential for the biological function of AMPs. saccharides, in part as extensions from the
lipid A moiety of LPS and often as much
longer polysaccharides extruded into the
10.2 Production of Anionic space surrounding the bacterium. During
Polyelectrolytes by Host and infection, bacteria can begin to produce
Microbial Sources exopolysaccharides alginate in the case of
Pseudomonas aeruginosa and form biofilms.
During physiological cell turnover, anionic These biofilms contain bacterial DNA
polyelectrolytes located inside the cell, such (Whitchurch et al., 2002) along with other
as DNA and F-actin, are not accumulated in polymers that create a hydrated and charged
extracellular compartments due to sucient environment around the bacterial surface,
phagocytic elimination of cell fragments preventing access by cationic antimicrobial
generated during apoptosis. Bacterial agents such as LL-37. Other polyanionic
infection rapidly leads to infiltration of white
blood cells, mainly neutrophils, which are (A)
highly enriched in cytoskeletal proteins.
During the course of necrosis, both the
F-actin
to attack bacteria. This hypothesis suggests combination of DNase I and gelsolin was
that strategies that destabilize polyelectrolyte more eective than either treatment alone.
bundles might release LL-37 and thereby Even high concentrations of LL-37 were not
enhance endogenous antibacterial function able by themselves to eradicate bacterial
even in the absence of increased LL-37 growth, but addition of gelsolin significantly
expression. Polyelectrolyte bundles can be increased the function of LL-37. However,
dissociated by several mechanisms. If the upon action of DNase and gelsolin, which
counterions are dynamic within the bundles, disaggregates CAPsDNAF-actin bundles,
LL-37 might be released by competitive the eciency of CAP release may still be
binding of a stronger polyvalent cation compromised if CAPs form precipitates with
(Purdy Drew et al., 2009). short DNA or F-actin fragments.
by DNA or F-actin have potential for lytic eect (a measure of peptide toxicity for
practical use. host cells) of CAPs has been based on rational
modifications of existing peptide sequences.
Since minor dierences in amino acid
10.5.1 Cationic steroids: minimizing and sequence can produce significant dierences
redistributing charge in antimicrobial activity (Raj et al., 2000), the
possibility of strategically varying key amino
AMP activity can be mimicked by cationic
acids has been explored by many
steroid antibiotics (CSAs), which were first
investigators (Travis et al., 2000; Hancock and
developed with the intent of reproducing or
Patrzykat, 2002; Jenssen et al., 2006; Saugar et
improving the antibacterial activities of
al., 2006; Zelezetsky and Tossi, 2006).
polymyxin B (Ding et al., 2004). These
Sequence modifications oer an enormous
antibacterial agents can have potency similar
number of combinatorial possibilities, includ-
to that of LL-37, but compared to natural
ing deleting, adding or replacing one or more
antibacterial peptides their net positive charge
residues, as well as truncating the peptide or
is usually lower and chemical synthesis
assembling chimeric peptides based on
provides the opportunity to control their
sequences from the same or dierent species.
charge distribution. One such synthetic com-
In addition, conjugating peptides with
pound, CSA-13, is significantly less sensitive
lipophilic acid (Avrahami and Shai, 2002) or
to inactivation by DNA or F-actin compared
rhodamine B (Bucki et al., 2004), incorporating
to LL-37 and shows better ecacy in CF
carbonate bond(s) (Lee and Oh, 2000) or
sputum (Bucki et al., 2007b). Synthetic CSAs
peptoid residues (Song et al., 2005) and
share some structural and functional
-amino acid epimerization (Mangoni et al.,
properties with squalamine, a membrane-
2006) have been found to change peptide
active CSA (Moore et al., 1993) that was first
physico-chemical properties and often
isolated from tissues of the dogfish shark.
translate to better biological activity. Enhanc-
Their bactericidal properties are due to
ing antibacterial potency by manipulating
membrane disruption and they display a
hydrophobicity proves the potential to reduce
moderate degree of selectivity for prokaryotic
or reorganize cationic moieties, thereby
over eukaryotic membranes (Salunke et al.,
weakening interactions with anionic poly-
2006). Several cholic acid derivates (Bellini et
electrolytes. However, modifications that
al., 1976) such as cholic acid with basic amino
increase hydrophobicity are also likely to
acids (Bellini et al., 1979; Li et al., 1999),
result in increasing peptide toxicity towards
guanidine (Savage and Li, 2000) and
host cells.
spermidine (Chen et al., 2006) have been
characterized as potent antimicrobial agents
that are eective against multidrug-resistant
bacteria (Schmidt et al., 2001) and fungi (Hazra 10.6 Possible Therapeutic Use
et al., 2004). In addition to these steroidal
conjugates (Salunke et al., 2006), a conjugate of 10.6.1 Selective use in some body fluids
spermine with two dexamethasone moieties and not others
displays strong antibacterial activity with low
Bacterial infection takes place in various
lytic eect on eukaryotic plasma membranes
areas of the body and CAP activity may vary
(Fein et al., 2009). This molecule also inhibits
depending on the local environment factors.
the inflammatory response induced by
Understanding the physiological homeostasis
bacterial wall products in vitro.
of CAPs in these settings is important to
design eective antimicrobial and anti-
10.5.2 Increasing the hydrophobicity of inflammatory therapies. Generally, CAPs,
antimicrobial agents including human cathelicidin LL-37, have
been shown to be active in body fluids with
The approach to enhancing the desired low protein concentrations such as airway
bactericidal activity and reducing the haemo- surface fluid (Bucki et al., 2007a), tears
176 P.A. Janmey and R. Bucki
(Huang et al., 2006), sweat (Rieg et al., 2006), phils (Barlow et al., 2006), the dierentiation
gastric juice (Leszczynska et al., 2009) and of dendritic cells that bridge innate with
saliva (Gutner et al., 2009). However, several adaptive immune responses (Kandler et al.,
body fluid components have been identified 2006; Skokos and Nussenzweig, 2007) and
as potent inhibitors of CAP functions. In the chemotactic activities of monocytes,
blood, most natural and synthetic cationic eosinophils and T cells (De et al., 2000;
antibacterial molecules lose their antibacterial Koczulla and Bals, 2003; Khine et al., 2006).
activity due to their insertion into plasma LL-37 directly activates endothelial cells,
lipoproteins and interaction with divalent which results in the increased proliferation
cations. Accordingly, LL-37 is inactive in the and formation of vessel-like structures in cell
presence of human serum, and its lack of culture. Decreased vascularization during
antibacterial activity cannot be explained by wound repair in mice deficient for CRAMP,
proteolytic degradation. LL-37 activity the murine homologue of LL-37/hCAP-18,
inhibition in human serum is mediated shows that cathelicidin-mediated angio-
mostly by binding to human apolipoprotein genesis is important for cutaneous wound
A-I (Wang et al., 1998, 2004). Interestingly, the neovascularization in vivo (Koczulla et al.,
de novo-engineered AMP WLBU2 resists the 2003). Additionally, LL-37 induces wound
inhibitory action of blood and physiological healing, proliferation and migration of
concentrations of Mg2+ and Ca2+ (Deslouches airway epithelial cells, suggesting that this
et al., 2005), suggesting that it is possible to peptide might be involved in the regulation
develop antibacterial peptides that will of tissue homeostasis in the airways
maintain activity in serum. Mucin is another (Shaykhiev et al., 2005). The regenerative
factor, widely present at the surfaces of activities of LL-37 support its therapeutic
airway, digestive or urogenital tracts, that potential to promote wound healing
can compromise the antibacterial function of
(Tokumaru et al., 2005; Carretero et al., 2008).
LL-37 (Bucki et al., 2008b). On the other hand,
interaction of CAPs with mucin can increase
the concentration of CAPs in the area most
subject to microorganism attack, and mucin 10.7 Conclusion
has been shown to be involved in protecting
peptides from proteolysis (Gutner et al., LL-37 and other cationic antimicrobial factors
2009). Antimicrobial activity at mucosal sites essential to the innate immune response
and ocular surfaces is aected by interactions appear to function largely by physico-
between dierent classes of antimicrobial chemical interactions with bacteria in which
factors (Nagaoka et al., 2000; McIntosh et al., electrostatic attraction is a fundamental
2005). feature. This mechanism also limits the
chemical environments in which these
cationic amphiphiles can perform as eective
10.6.2 Stimulation of host cells bactericidal agents. Release of polyanionic
filaments and other aggregates into the
Considering that bacterial killing by LL-37 extracellular space in which infection occurs
occurs primarily by depolarizing and can inactivate the antimicrobial eects of
permeabilizing bacterial membranes or by LL-37, even under conditions where it is
induced autolysis (Ginsburg, 2004), it has upregulated and protected against degrad-
been suggested that a similar mechanism of ation. Multiple strategies directed at
action can be used to target disruption of disrupting the complex of cationic AMPs
cancer cells by C-terminal LL-37 fragments with anionic DNA, F-actin, alginate or other
(Li et al., 2006). In addition to its bacteria- polyelectrolytes, or designing novel anti-
killing ability, LL-37 can also aect host microbial agents that have less anity for
leukocyte functions that are associated with polyelectrolytes, have the potential to
host immune defence. LL-37 aects the life improve antibacterial therapies in respiratory
span and inflammatory functions of neutro- infections and other diseases.
Design of Alternative Antimicrobial Agents 177
Chernick, W.S. and Barbero, G.J. (1959) Com- Guaqueta, C., Sanders, L.K., Wong, G.C. and
position of tracheobronchial secretions in cystic Luijten, E. (2006) The effect of salt on self-
fibrosis of the pancreas and bronchiectasis. assembled actinlysozyme complexes. Bio-
Pediatrics 24, 739745. physical Journal 90, 46304638.
De, Y., Chen, Q., Schmidt, A.P., Anderson, G.M., Gutner, M., Chaushu, S., Balter, D. and Bachrach,
Wang, J.M., Wooters, J., Oppenheim, J.J. and G. (2009) Saliva enables the antimicrobial
Chertov, O. (2000) LL-37, the neutrophil granule- activity of LL-37 in the presence of proteases of
and epithelial cell-derived cathelicidin, utilizes Porphyromonas gingivalis. Infection and
formyl peptide receptor-like 1 (FPRL1) as a Immunity 77, 55585563.
receptor to chemoattract human peripheral Gutsmann, T., Hagge, S.O., David, A., Roes, S.,
blood neutrophils, monocytes, and T cells. Bohling, A., Hammer, M.U. and Seydel, U.
Journal of Experimental Medicine 192, 1069 (2005) Lipid-mediated resistance of Gram-
1074. negative bacteria against various pore-forming
Deslouches, B., Islam, K., Craigo, J.K., Paranjape, antimicrobial peptides. Journal of Endotoxin
S.M., Montelaro, R.C. and Mietzner, T.A. (2005) Research 11, 167173.
Activity of the de novo engineered antimicrobial Hancock, R.E. and Patrzykat, A. (2002) Clinical
peptide WLBU2 against Pseudomonas aeru- development of cationic antimicrobial peptides:
ginosa in human serum and whole blood: from natural to novel antibiotics. Current Drug
implications for systemic applications. Anti- Targets: Infectious Disorders 2, 7983.
microbial Agents and Chemotherapy 49, 3208 Hatch, R.A. and Schiller, N.L. (1998) Alginate lyase
3216. promotes diffusion of aminoglycosides through
Devkota, B., Petrov, A.S., Lemieux, S., Boz, M.B., the extracellular polysaccharide of mucoid
Tang, L., Schneemann, A., Johnson, J.E. and Pseudomonas aeruginosa. Antimicrobial Agents
Harvey, S.C. (2009) Structural and electrostatic and Chemotherapy 42, 974977.
characterization of pariacoto virus: implications Hazra, B., Pore, V., Dey, S., Datta, S., Darokar, M.,
for viral assembly. Biopolymers 91, 530538. Saikia, D., Khanuja, S.P. and Thakur, A. (2004)
Ding, B., Yin, N., Liu, Y., Cardenas-Garcia, J., Bile acid amides derived from chiral amino
Evanson, R., Orsak, T., Fan, M., Turin, G. and alcohols: novel antimicrobials and antifungals.
Savage, P.B. (2004) Origins of cell selectivity of Bioorganic & Medicinal Chemistry Letters 14,
cationic steroid antibiotics. Journal of the 773777.
American Chemical Society 126, 13642 Huang, L.C., Petkova, T.D., Reins, R.Y., Proske,
13648. R.J. and McDermott, A.M. (2006) Multifunctional
Donati, I., Benegas, J.C., Cesaro, A. and Paoletti, roles of human cathelicidin (LL-37) at the ocular
S. (2006) Specific interactions versus counterion surface. Investigative Ophthalmology & Visual
condensation. 2. Theoretical treatment within Science 47, 23692380.
the counterion condensation theory. Biomacro- Jenssen, H., Hamill, P. and Hancock, R.E. (2006)
molecules 7, 15871596. Peptide antimicrobial agents. Clinical Micro-
Fein, D.E., Limberis, M.P., Maloney, S.F., Heath, biology Reviews 19, 491511.
J.M., Wilson, J.M. and Diamond, S.L. (2009) Kandler, K., Shaykhiev, R., Kleemann, P., Klescz,
Cationic lipid formulations alter the in vivo F., Lohoff, M., Vogelmeier, C. and Bals, R. (2006)
tropism of AAV2/9 vector in lung. Molecular The anti-microbial peptide LL-37 inhibits the
Therapy 17, 207887 activation of dendritic cells by TLR ligands.
Felgentreff, K., Beisswenger, C., Griese, M., Gulder, International Immunology 18, 17291736.
T., Bringmann, G. and Bals, R. (2006) The Khine, A.A., Del Sorbo, L., Vaschetto, R., Voglis, S.,
antimicrobial peptide cathelicidin interacts with Tullis, E., Slutsky, A.S., Downey, G.P. and Zhang,
airway mucus. Peptides 27, 31003106. H. (2006) Human neutrophil peptides induce
Fuller, D.N., Rickgauer, J.P., Jardine, P.J., Grimes, interleukin-8 production through the P2Y6
S., Anderson, D.L. and Smith, D.E. (2007) Ionic signaling pathway. Blood 107, 29362942.
effects on viral DNA packaging and portal motor Koczulla, A.R. and Bals, R. (2003) Antimicrobial
function in bacteriophage 29. Proceedings of peptides: current status and therapeutic
the National Academy of Sciences of the USA potential. Drugs 63, 389406.
104, 1124511250. Koczulla, R., von Degenfeld, G., Kupatt, C., Krotz,
Ginsburg, I. (2004) Bactericidal cationic peptides F., Zahler, S., Gloe, T., Issbrucker, K.,
can also function as bacteriolysis-inducing Unterberger, P., Zaiou, M., Lebherz, C., Karl, A.,
agents mimicking beta-lactam antibiotics; it is Raake, P., Pfosser, A., Boekstegers, P., Welsch,
enigmatic why this concept is consistently U., Hiemstra, P.S., Vogelmeier, C., Gallo, R.L.,
disregarded. Medical Hypotheses 62, 367374. Clauss, M. and Bals, R. (2003) An angiogenic
Design of Alternative Antimicrobial Agents 179
role for the human peptide antibiotic LL-37/ expression of TNF- by blocking the binding
hCAP-18. Journal of Clinical Investigation 111, of LPS to CD14+ cells. Journal of Immunology
16651672. 167, 33293338.
Lee, K.H. and Oh, J.E. (2000) Design and synthesis Overman, S.A., Aubrey, K.L., Reilly, K.E., Osman,
of novel antimicrobial pseudopeptides with O., Hayes, S.J., Serwer, P. and Thomas, G.J. Jr
selective membrane-perturbation activity. Bio- (1998) Conformation and interactions of the
organic & Medicinal Chemistry 8, 833839. packaged double-stranded DNA genome of
Lenac, Z. and Sunjic, M. (1991) Dynamical bacteriophage T7. Biospectroscopy 4, S4756.
properties and Wigner transitions of two- Parks, Q.M., Young, R.L., Poch, K.R., Malcolm,
dimensional electron lattices on dielectric K.C., Vasil, M.L. and Nick, J.A. (2009) Neutrophil
substrates. Physical Review: B, Condensed enhancement of Pseudomonas aeruginosa
Matter 44, 1146511471. biofilm development: human F-actin and DNA
Leszczynska, K., Namiot, A., Fein, D.E., Wen, Q., as targets for therapy. Journal of Medical
Namiot, Z., Savage, P.B., Diamond, S., Janmey, Microbiology 58, 492502.
P.A. and Bucki, R. (2009) Bactericidal activities Perdue, M.L., Cohen, J.C., Randall, C.C. and
of the cationic steroid CSA-13 and the OCallaghan, D.J. (1976) Biochemical studies of
cathelicidin peptide LL-37 against Helicobacter the maturation of herpesvirus nucleocapsid
pylori in simulated gastric juice. BMC species. Virology 74, 194208.
Microbiology 9, 187. Purdy Drew, K.R., Sanders, L.K., Culumber, Z.W.,
Li, C., Lewis, M.R., Gilbert, A.B., Noel, M.D., Zribi, O. and Wong, G.C. (2009) Cationic
Scoville, D.H., Allman, G.W. and Savage, P.B. amphiphiles increase activity of aminoglycoside
(1999) Antimicrobial activities of amine- and
antibiotic tobramycin in the presence of airway
guanidine-functionalized cholic acid derivatives.
polyelectrolytes. Journal of the American
Antimicrobial Agents and Chemotherapy 43,
Chemical Society 131, 486493.
13471349.
Raj, P.A., Antonyraj, K.J. and Karunakaran, T.
Li, X., Li, Y., Han, H., Miller, D.W. and Wang, G.
(2000) Large-scale synthesis and functional
(2006) Solution structures of human LL-37
elements for the antimicrobial activity of
fragments and NMR-based identification of a
defensins. Biochemical Journal 347, 633641.
minimal membrane-targeting antimicrobial and
Rieg, S., Seeber, S., Steffen, H., Humeny, A.,
anticancer region. Journal of the American
Kalbacher, H., Stevanovic, S., Kimura, A.,
Chemical Society 128, 57765785.
Garbe, C. and Schittek, B. (2006) Generation of
Mangoni, M.L., Papo, N., Saugar, J.M., Barra, D.,
Shai, Y., Simmaco, M. and Rivas, L. (2006) multiple stable dermcidin-derived antimicrobial
Effect of natural L- to D-amino acid conversion peptides in sweat of different body sites. Journal
on the organization, membrane binding, and of Investigative Dermatology 126, 354365.
biological function of the antimicrobial peptides Salunke, D.B., Hazra, B.G. and Pore, V.S. (2006)
bombinins H. Biochemistry 45, 42664276. Steroidal conjugates and their pharmacological
McIntosh, R.S., Cade, J.E., Al-Abed, M., applications. Current Medicinal Chemistry 13,
Shanmuganathan, V., Gupta, R., Bhan, A., 813847.
Tighe, P.J. and Dua, H.S. (2005) The spectrum Saugar, J.M., Rodriguez-Hernandez, M.J., de la
of antimicrobial peptide expression at the ocular Torre, B.G., Pachon-Ibanez, M.E., Fernandez-
surface. Investigative Ophthalmology & Visual Reyes, M., Andreu, D., Pachon, J. and Rivas, L.
Science 46, 13791385. (2006) Activity of cecropin Amelittin hybrid
Moore, K.S., Wehrli, S., Roder, H., Rogers, M., peptides against colistin-resistant clinical strains
Forrest, J.N. Jr, McCrimmon, D. and Zasloff, M. of Acinetobacter baumannii: molecular basis for
(1993) Squalamine: an aminosterol antibiotic the differential mechanisms of action. Anti-
from the shark. Proceedings of the National microbial Agents and Chemotherapy 50,
Academy of Sciences of the USA 90, 1354 12511256.
1358. Savage, P.B. and Li, C. (2000) Cholic acid
Nagaoka, I., Hirota, S., Yomogida, S., Ohwada, A. derivatives: novel antimicrobials. Expert Opinion
and Hirata, M. (2000) Synergistic actions of on Investigational Drugs 9, 263272.
antibacterial neutrophil defensins and cath- Schmidt, E.J., Boswell, J.S., Walsh, J.P.,
elicidins. Inflammation Research 49, 7379. Schellenberg, M.M., Winter, T.W., Li, C., Allman,
Nagaoka, I., Hirota, S., Niyonsaba, F., Hirata, M., G.W. and Savage, P.B. (2001) Activities of cholic
Adachi, Y., Tamura, H. and Heumann, D. acid-derived antimicrobial agents against
(2001) Cathelicidin family of antibacterial multidrug-resistant bacteria. Journal of Anti-
peptides CAP18 and CAP11 inhibit the microbial Chemotherapy 47, 671674.
180 P.A. Janmey and R. Bucki
Shak, S., Capon, D.J., Hellmiss, R., Marsters, S.A. antimicrobial peptide LL-37. Journal of
and Baker, C.L. (1990) Recombinant human Immunology 175, 46624668.
DNase I reduces the viscosity of cystic fibrosis Tolker-Nielsen, T. and Hoiby, N. (2009) Extracellular
sputum. Proceedings of the National Academy DNA and F-actin as targets in antibiofilm cystic
of Sciences of the USA 87, 91889192. fibrosis therapy. Future Microbiology 4, 645
Shaykhiev, R., Beisswenger, C., Kandler, K., 647.
Senske, J., Puchner, A., Damm, T., Behr, J. and Travis, S.M., Anderson, N.N., Forsyth, W.R.,
Bals, R. (2005) Human endogenous antibiotic Espiritu, C., Conway, B.D., Greenberg, E.P.,
LL-37 stimulates airway epithelial cell McCray, P.B. Jr, Lehrer, R.I., Welsh, M.J. and
proliferation and wound closure. American Tack, B.F. (2000) Bactericidal activity of
Journal of Physiology. Lung Cellular and mammalian cathelicidin-derived peptides.
Molecular Physiology 289, L842L848. Infection and Immunity 68, 27482755.
Sheils, C.A., Kas, J., Travassos, W., Allen, P.G., Vasconcellos, C.A., Allen, P.G., Wohl, M.E., Drazen,
Janmey, P.A., Wohl, M.E. and Stossel, T.P. J.M., Janmey, P.A. and Stossel, T.P. (1994)
(1996) Actin filaments mediate DNA fiber Reduction in viscosity of cystic fibrosis sputum
formation in chronic inflammatory airway in vitro by gelsolin. Science 263, 969971.
disease. American Journal of Pathology 148, Walker, T.S., Tomlin, K.L., Worthen, G.S., Poch,
919927. K.R., Lieber, J.G., Saavedra, M.T., Fessler,
Skokos, D. and Nussenzweig, M.C. (2007) CD8 M.B., Malcolm, K.C., Vasil, M.L. and Nick, J.A.
DCs induce IL-12-independent Th1 (2005) Enhanced Pseudomonas aeruginosa
differentiation through Delta 4 Notch-like ligand biofilm development mediated by human
in response to bacterial LPS. Journal of neutrophils. Infection and Immunity 73, 3693
Experimental Medicine 204, 15251531. 3701.
Song, Y.M., Park, Y., Lim, S.S., Yang, S.T., Woo, Wang, Y., Agerberth, B., Lothgren, A., Almstedt, A.
E.R., Park, I.S., Lee, J.S., Kim, J.I., Hahm, K.S., and Johansson, J. (1998) Apolipoprotein A-I
Kim, Y. and Shin, S.Y. (2005) Cell selectivity and binds and inhibits the human antibacterial/
mechanism of action of antimicrobial model cytotoxic peptide LL-37. Journal of Biological
peptides containing peptoid residues. Chemistry 273, 3311533118.
Biochemistry 44, 1209412106. Wang, Y., Johansson, J., Agerberth, B., Jornvall, H.
Tang, J. and Janmey, P.A. (1996) The polyelectrolyte and Griffiths, W.J. (2004) The antimicrobial
nature of F-actin and the mechanism of actin peptide LL-37 binds to the human plasma
bundle formation. Journal of Biological protein apolipoprotein A-I. Rapid
Chemistry 271, 85568563. Communications in Mass Spectrometry 18,
Tang, J., Wong, S., Tran, P. and Janmey, P.A. (1996) 588589.
Counterion induced bundle formation of rodlike Weiner, D.J., Bucki, R. and Janmey, P.A. (2003) The
polyelectrolytes.Berichte der Bunsengesellschaft antimicrobial activity of the cathelicidin LL37 is
fr physikalische Chemie 100, 796806. inhibited by F-actin bundles and restored by
Tang, J., Ito, T., Tao, T., Traub, P. and Janmey, P.A. gelsolin. American Journal of Respiratory Cell
(1997) Opposite effects of electrostatics and and Molecular Biology 28, 738745.
steric exclusion on bundle formation by F-actin Whitchurch, C.B., Tolker-Nielsen, T., Ragas, P.C.
and other filamentous polyelectrolytes. Bio- and Mattick, J.S. (2002) Extracellular DNA
chemistry 36, 1260012607. required for bacterial biofilm formation. Science
Tang, J., Kas, J.A., Shah, J.V. and Janmey, P.A. 295, 1487.
(2001) Counterion-induced actin ring formation. Yang, L., Gordon, V.D., Mishra, A., Som, A., Purdy,
European Biophysics Journal 30, 477484. K.R., Davis, M.A., Tew, G.N. and Wong, G.C.
Tang, J., Wen, Q., Bennett, A., Kim, B., Sheils, C.A., (2007) Synthetic antimicrobial oligomers induce
Bucki, R. and Janmey, P.A. (2005) Anionic a composition-dependent topological transition
poly(amino acid)s dissolve F-actin and DNA in membranes. Journal of the American
bundles, enhance DNase activity, and reduce Chemical Society 129, 1214112147.
the viscosity of cystic fibrosis sputum. American Yin, H.L., Kwiatkowski, D.J., Mole, J.E. and Cole,
Journal of Physiology: Lung Cellular and F.S. (1984) Structure and biosynthesis of
Molecular Physiology 289, L599L605. cytoplasmic and secreted variants of gelsolin.
Tokumaru, S., Sayama, K., Shirakata, Y., Journal of Biological Chemistry 259, 5271
Komatsuzawa, H., Ouhara, K., Hanakawa, Y., 5276.
Yahata, Y., Dai, X., Tohyama, M., Nagai, H., Zelezetsky, I. and Tossi, A. (2006) Alpha-helical
Yang, L., Higashiyama, S., Yoshimura, A., Sugai, antimicrobial peptides using a sequence
M. and Hashimoto, K. (2005) Induction of template to guide structureactivity relationship
keratinocyte migration via transactivation of the studies. Biochimica et Biophysica Acta 1758,
epidermal growth factor receptor by the 14361449.
11
Role of Vitamin D in the
Enhancement of Antimicrobial Peptide
Gene Expression
Abstract
1,25-Dihydroxyvitamin D (1,25D), the hormonally active form of vitamin D, was initially identified as a
key regulator of calcium homeostasis. 1,25D signals through the nuclear vitamin D receptor, a ligand-
regulated transcription factor. More recent studies have revealed that 1,25D regulates a number of
physiological processes, including innate immunity. The innate immune system is responsible for rapid,
non-specific host responses to pathogenic infection. Unlike adaptive immunity, innate immune responses
do not confer lasting protection on the host. 1,25D induces the expression of LL-37 and hBD-2,
antimicrobial peptides that serve as vanguards of innate immune responses. Antimicrobial peptides are
small proteins with antibiotic properties that can also acts as signalling molecules to modulate specific
responses such as wound healing and cytokine induction. This chapter focuses on the gene regulatory
activity of 1,25D, with an emphasis on its role in the potentiation of innate immune responses and the
promotion of antimicrobial peptide expression.
* Corresponding author.
use. Cod-liver oil was first described as a temperate climates, this phenomenon is
medicinal agent for the treatment of chronic commonly referred to as vitamin D winter.
rheumatism in 1789. Throughout the next For example, one survey found widespread
century, medical literature documented its vitamin D deficiency among female
eectiveness for treating a number of populations across Northern Europe
prevalent conditions such as gout and (Andersen et al., 2005). Another study found
scrofula, a form of TB that infects the lymph that a significant portion of African-American
nodes (Guy, 1923). Beginning in the 1820s, women living in the USA are seriously
studies showed that administering doses of vitamin D deficient (Chapuy et al., 1997).
cod-liver oil to aicted children could cure While correlations between vitamin D
rickets (Guy, 1923), a nutritional disease deficiency and disease go back for well over
characterized by a lack of vitamin D or a century, more recent studies have estab-
calcium leading to bone softening and lished a direct connection between the two
deformity. However, it was not until several conditions. Since the association between TB
decades later that the active compound in and vitamin D deficiency was described over
cod-liver oil was identified as vitamin D. By 20 years ago (Davies et al., 1985), numerous
1849, the list of conditions treatable with cod- epidemiological studies have linked vitamin
liver oil had grown to include TB (Grad, D deficiency to increased rates of cancer,
2004; Martineau et al., 2007). multiple sclerosis, type I diabetes, Crohns
Several independent observations in the disease and infection (Schwalfenberg, 2007;
19th and early 20th centuries fostered further White, 2008).
links between sunlight and cutaneous
vitamin D synthesis. This was highlighted by
reports such as the following (Guy, 1923; 11.4 Vitamin D Signalling and
Crowle et al., 1987; Grad, 2004; Lin and Mechanisms of Action
White, 2004; Martineau et al., 2007):
A relationship was found between the Vitamin D is generated primarily by the
prevalence of rickets and lack of exposure photochemical action of UVB radiation (295
to sunlight. 320 nm) in the skin (Fig. 11.1), but is also
UV light therapy was found to cure obtained from limited dietary sources such
rickets. as fish oils and fortified dairy products.
Feeding UV-irradiated skin to rats was Vitamin D refers collectively to closely
found to have the same protective eects related molecules (Fig. 11.2) known as
against rickets as did cod-liver oil. vitamin D2 (ergocalciferol) and vitamin D3
UV light was also found to be useful in (cholecalciferol) (DAldebert et al., 2009).
treating cutaneous TB (lupus vulgaris). Dietary vitamin D2 is derived from the
fungal steroid ergosterol. Vitamin D3 is
These studies support associations estab- found in animal sources such as cod-liver oil
lished between vitamin D deficiency and the and is generated by cutaneous photochemical
prevalence of certain diseases. conversion of the cholesterol metabolite
7-dehydrocholesterol.
Vitamin D undergoes a series of
11.3 Vitamin D Deficiency hydroxylation reactions, which are required
for both biological activation and deactiva-
Today, vitamin D deficiency is regarded as a tion of the molecule. First, 25-hydroxylation
widespread and completely preventable is catalysed by the enzymes cytochrome
health concern. Although there is no strict P (CYP)2R1 or CYP27A1, producing
definition, vitamin D deficiency is char- 25-hydroxyvitamin D (25D), the major
acterized by low circulating vitamin D circulating vitamin D metabolite (Jones et al.,
metabolite levels, caused by a combination of 1998; Cheng et al., 2004; Prosser and Jones,
seasonal variations in sunlight and 2004; Shinkyo et al., 2004; Holick, 2007). This
inadequate dietary consumption. In more occurs primarily in the liver, but also in the
Vitamin D and AMP Gene Expression 183
Vitamin D3
Skin
Provitamin D3
Liver (skin)
25-OHase
(CYP27A1
and others)
Kidney and
peripheral tissues
(incl. skin)
1-OHase
(CYP27B1)
1,25-Dihydroxy- 25-Hydroxy-
vitamin D3 vitamin D3
Fig. 11.1. Biosynthesis of 1,25-dihydroxyvitamin D3. CYP, cytochrome P; VDR, vitamin D receptor.
(VDREs), located in the regulatory regions of gene encoding RANK ligand, a tumour
specific target genes. VDREs are direct repeats necrosis factor-like ligand secreted by
(DR) or everted (inverted) repeats (ER) of mesenchymal cells (Kim et al., 2006).
hexameric nucleotide motifs composed of
PuG(G/T)TCA consensus sequences,
characterized by their spacing of either three 11.5 Overview of Vitamin D and
(DR3), six (ER6) or eight (ER8) nucleotides Immune System Regulation
(Lin and White, 2004; Tavera-Mendoza et al.,
2006). Two copies of cognate DNA sequence A substantial body of evidence exists linking
motifs reflect the binding of heterodimeric vitamin D signalling with regulation of the
VDRRXRs in heel-to-toe or toes out innate and adaptive arms of the immune
orientations (Fig. 11.3) (Rochel et al., 2000; system. The VDR is present in most cells of
Chawla et al., 2001; Holick, 2007). the immune system, including T lympho-
After DNA binding, VDRRXRs recruit cytes, neutrophils and antigen-presenting
a series of co-regulatory proteins required for cells such as macrophages and dendritic cells
the regulation of transcription of adjacent (DCs) (Provvedini et al., 1983; Bhalla et al.,
target genes. Regulation of transcription of 1984; Brennan et al., 1987; Adorini, 2005;
protein-coding genes in eukaryotes is a bio- Norman, 2006). 1,25D polarizes T-helper (Th)
chemically complex process that culminates responses towards a more regulatory Th2
in recruitment of RNA polymerase II and phenotype, which is considered to be a key
initiation of transcription. For further component in its capacity to suppress Th1-
information, readers are referred to a series driven autoimmune responses. It inhibits DC
of excellent reviews on the subject (Glass and maturation and T-cell proliferation (van Etten
Rosenfeld, 2000; Dilworth and Chambon, and Mathieu, 2005). Gene expression studies
2001; McKenna and OMalley, 2002). Recent have shown that 1,25D represses the
studies suggest that the VDR can regulate transcription of genes encoding key Th1
target gene transcription from a considerable cytokines, including interferon- and inter-
distance. For example, work has suggested leukin (IL)-2 (Alroy et al., 1995; van Etten and
that a VDRE located as far as 75 kb from the Mathieu, 2005). The net eect of 1,25D
start site can regulate transcription of the signalling leads to a suppression of antigen
PuG(G/T)TCAN3PuG(G/T)TCA TGA(A/C)CPyN6PuG(G/T)TCA
DR3 ER6
Fig. 11.3. Vitamin D receptor (VDR)retinoid X receptor (RXR) dimeric binding to vitamin D response
elements of the direct repeat (DR; left) or everted repeat (ER; right) type. DBD, DNA-binding domain;
LBD, ligand-binding domain.
Vitamin D and AMP Gene Expression 185
presentation and activation and recruitment regulation of DEFB2 transcription was found
of Th1 cells. to be modest (Wang et al., 2004). However, it
Innate immune responses are found in a is now clear that 1,25D can enhance DEFB2
wide variety of plant and animal life and expression with stimulation by other
represent the front-line defences to patho- regulators (see below). Remarkably, several
genic challenge. The innate immune system studies showed that 1,25D strongly stimu-
defends the host from infection in a non- lated LL-37 expression in a number of human
specific manner but, unlike adaptive cells and tissues (Wang et al., 2004; Gombart
immunity, does not confer long-lasting or et al., 2005). These findings are highly
protective immunity to the host. Molecular significant because AMPs represent the
evidence for the regulation of innate immune vanguards of innate immune responses to
responses by 1,25D has been accumulating infection.
for some time. However, it is only in the last
5 years or so that we have begun to fill in
many of the details of the central role of 11.7 Antimicrobial Peptides
vitamin D signalling in innate immune
responses in humans. AMPs are a group of small proteins with
intrinsic antibiotic properties that are
synthesized and secreted in cells and tissues
11.6 Vitamin D as a Regulator of exposed to environmental pathogens. Much
Antimicrobial Peptide Gene of the eectiveness of AMPs stems from their
Expression ability to eradicate a broad spectrum of
pathogens, including bacteria, fungi and
Modern genomics techniques have shifted viruses, without triggering antibiotic resist-
the focus of vitamin D into fields other than ance (Patil et al., 2004; Hancock and Sahl,
regulation of calcium homeostasis, and have 2006; Jenssen et al., 2006). Additionally, being
begun to reveal the molecular mechanisms a natural constituent of host immune
underlying the ancient use of heliotherapy to systems, AMPs have been discovered to have
treat infectious diseases. This new focus has additional immunoregulatory properties,
its roots in studies of how 1,25D modulates leading to the term immune effector mol-
gene expression. As detailed above, the VDR ecules. These regulatory properties include
is a direct regulator of gene transcription alteration of gene expression, cytokine
with a well-defined binding site (Lin and induction and modulation of DC responses
White, 2004). Therefore, initial large-scale (Patil et al., 2004; Hancock and Sahl, 2006;
identification of 1,25D target genes began by Jenssen et al., 2006; Giuliani et al., 2007). As
utilizing a combination of microarrays and pathogens continue evolving into increas-
genome-wide screens for VDREs, yielding ingly antibiotic-resistant strains, AMPs hold
significant results (Akutsu et al., 2001; Lin et promise as a potent method in the control of
al., 2002; White, 2004; Wang et al., 2005). Of microbial infection.
particular interest, these screens identified Cathelicidins are a family of anti-
consensus DR3 VDREs located in the microbial polypeptides, the precursors of
promoter region of two genes encoding the which are characterized by a highly con-
antimicrobial peptides (AMPs) defensin 2 served N-terminal cathelin region with a
(DEFB2/hBD-2) and LL-37 (Wang et al., 2004). variable C-terminal antimicrobial domain. As
The 1,25D-dependent binding of the VDR to a subset of a larger group, the cathelin
these elements was confirmed by chromatin domain shares sequence homology and
immunoprecipitation assays. These assays of function with the cystatin family of cysteine
VDR binding entail immunoprecipitation of proteinase inhibitors (Zanetti, 2004). Cath-
chemically cross-linked complexes of the elicidins serve as a critical component of the
VDR bound to target DNA sequences, innate immune response, providing rapid
followed by detection of specific DNA defence against infection. While genes
sequences by PCR amplification. 1,25D encoding for cathelicidins are widely
186 J.H. White and A.J. Bitton
conserved among vertebrates (Zanetti, 2004; immune function (Lehrer, 2004; Klotman and
Chang et al., 2005; van Dk et al., 2005), the Chang, 2006). Structurally, defensins are
human gene remains the subject of focus. characterized by a cysteine-rich backbone
In humans, a single gene encodes the forming -pleated sheets stabilized by
18-kDa cathelicidin precursor protein (hCAP- disulfide bonds (Klotman and Chang, 2006).
18), widely expressed throughout cells of the Classified by their orientation of disulfide
immune system. Proteolytic cleavage of the bonds to cysteine residues, two subfamilies
proprotein releases LL-37, also known as of defensins exist in humans: -defensins,
human cathelicidin AMP (CAMP) (Fig. 11.4). which are commonly produced by
LL-37 is a linear-chain polypeptide with a net neutrophils and Paneth cells, and
positive charge of +6 due to an excess of basic -defensins, which are secreted by epithelial
amino acids (Hancock and Diamond, 2000). tissues (for primary and tertiary structures,
The basis of cathelicidins antimicrobial see Table 9.1 and Fig. 9.5, respectively).
activity is due to the cationic nature of the
peptide. Bacterial cell membranes contain
high lipid compositions with a negatively 11.8 Regulation of DEFB2/hBD-2
charged surface, thereby creating a selective Expression by 1,25D
preference for the positively charged AMP.
Upon binding to bacterial cell surfaces, 1,25D induction of DEFB2 has been linked
human cathelicidin LL-37 folds into an with cytokine production during the immune
amphipathic -helical structure (see Fig. response. As mentioned above, Wang et al.
9.4C, Chapter 9), leading to insertion of the (2004) found the induction of DEFB2
protein and interference with cell membrane expression by 1,25D alone to be relatively
integrity. Increasingly, in vivo studies have modest (maximum twofold). However, this
documented a more complete role for human initial study also showed that fold expression
cathelicidin beyond the direct eradication of of DEFB2 could be elevated by the addition
microbes. Cathelicidins have been shown to of IL-1 to the incubation media. IL-1 is a
mediate a number of immune system cytokine and key regulator of inflammatory
responses, including chemotaxis, altering immune responses. The combined eects of
transcription, directing the inflammatory 1,25D and IL-1 on DEFB2 transcription
response and promoting wound healing hinted at crosstalk between the two
(Hancock and Diamond, 2000; Zanetti, 2004; signalling pathways. Expanding upon these
Chang et al., 2005; van Dk et al., 2005). results, Liu et al. (2009b) demonstrated the
Defensins represent another family of synergistic activity of IL-1 with 1,25D in the
1,25D-induced AMPs that contribute to potentiation of antimicrobial responses
innate immune defence. Similar to cath- induced by Toll-like receptors (TLRs). TLRs
elicidins, defensins are small polypeptides are pattern-recognition receptors that stimu-
with cationic properties that are integral to late innate immune responses upon
their antimicrobial activity. Found in both recognition of molecular motifs characteristic
vertebrates and invertebrates, defensins are of pathogens. More recent research has
also well recognized for their dual capacity found that 1,25D stimulates the expression of
to both eradicate microbes and modulate another pattern-recognition receptor called
Fig. 11.4. A schematic representation of the precursor protein hCAP-18 and the cleavage product LL-37.
Basic amino acids in the LL-37 peptide are noted in bold.
Vitamin D and AMP Gene Expression 187
NOD2/CARD15 (Wang et al., 2010). NOD2/ a number of findings: (i) the VDR is found in
CARD15 is an intracellular receptor that most immune cells, including T lymphocytes,
recognizes lysosomal breakdown products of neutrophils, DCs and macrophages (Prov-
bacterial peptidoglycan in the form of vedini et al., 1983; Bhalla et al., 1984; Brennan
muramyl dipeptide (MDP). Notably, others et al., 1987; Adorini, 2005; Norman, 2006); (ii)
have shown that signalling by MDP through studies have linked VDR induction of AMP
NOD2/CARD15 induces expression of DEFB4 production with TLR signalling in innate
(formerly DEFB2/hBD-2) (Liu, P.T. et al., immune responses (Stead et al., 1990;
2009b). The combination of MDP and 1,25D Brightbill et al., 1999; Thoma-Uszynski et al.,
synergistically induced DEFB4 expression. 2001; Liu et al., 2006, 2007); and (iii) VDR
These results are of clinical significance signalling is important for both the process
because attenuated or defective expression of of pathogen recognition and antimicrobial
NOD2/CARD15 or DEFB2/hBD-2 has been response during tissue injury (Schauber et al.,
associated with the pathogenesis of Crohns 2007).
disease (Wang et al., 2010). Crohns disease is TLR pattern-recognition receptors are a
a chronic inflammatory bowel disease that family of cell-surface and intracellular recep-
arises from a defect in intestinal innate tors. TLRs have long been studied for their
immune responses to bacterial flora. roles in responses to microbial infection, and
have recently been linked to VDR
immunoregulation. TLR2 receptors are cell-
11.9 1,25D Regulation of LL-37 is surface proteins that recognize bacterial
Human and Primate Specific lipopeptides. By 2006, it was established that
TLR2 activation of human or murine
The induction of antimicrobial gene macrophages directly stimulates anti-
expression by 1,25D does not appear to be microbial activity against TB infection (Liu et
widely conserved among mammals. The al., 2006). However, the intermediate
VDRE in the human hBD-2/DEFB2 gene is signalling pathways leading to AMP
not conserved in the mouse, for example. production are believed to be dierent
Moreover, work by Gombart et al. (2005) between the two species. In mice, studies
showed that the regulation of LL-37 showed that antimicrobial activity is
expression seen in humans is not conserved dependent upon the induction of nitric oxide
in mice. Indeed, a comparison of several synthase (iNOS) and the production of nitric
mammalian genomes showed that the oxide (NO) in infected tissue (Liu et al., 2006,
mechanism in 1,25D induction of LL-37 is 2007). Further work showed that NO-induced
conserved specifically in humans and non- antimicrobial activity is mediated through
human primates. Conservation of the VDRE TLR signalling in mice, and that iNOS
in the LL-37 promoter can be attributed to the inhibitors are able to block this induction of
presence of the element within a short antimicrobial activity (Liu et al., 2006).
interspersed element of the Alu subfamily. Additionally, these studies demonstrated
Alu repeats are a family of transposable that human macrophages do not depend
DNA elements that are primate and human upon the production of NO for their anti-
specific. This specific conservation of the Alu microbial activity. However, one intriguing
repeat containing the LL-37 VDRE can be study has cast doubts on these initial results
traced to an insertion event dating back to and shown that iNOS activity does indeed
5560 million years ago (Gombart et al., play a role in host defences to TB infection in
2009), prior to the divergence of the primate human macrophages (Lee et al., 2009).
lineage leading to humans, apes and Old and Research has confirmed the induction of
New World monkeys. AMPs as the basis for TLR-induced
Regulation of the LL-37 gene by 1,25D is antimicrobial activity in human macrophages
believed to hold a selective advantage given and linked VDR signalling to AMP
its functional significance in innate immune production (Liu et al., 2007). Using micro-
responses. This significance is highlighted by arrays, studies found that TLR stimulation
188 J.H. White and A.J. Bitton
by blocking CYP450-driven vitamin D meta- al., 2009, Peric et al., 2009). Furthermore, the
bolism (Yamasaki et al., 2007). synergistic enhancement of LL-37 expression
Vitamin D analogues are widely used to is induced by the combination of vitamin D
treat psoriasis, an autoimmune inflammatory and bile salts, suggesting a novel therapeutic
skin condition. It is thus paradoxical that approach to treating inflammatory biliary
LL-37 is overexpressed in psoriasis and may disease (DAldebert et al., 2009).
contribute to its pathogenesis. Cathelicidin Similarly, VDR regulation of the innate
expression is limited to keratinocytes immune response is essential for maintaining
exposed to injury, stimulating immune lung homeostasis. The epithelial lining of the
responses and promoting wound healing lungs is constantly exposed to a variety of
(Dorschner et al., 2001; Heilborn et al., 2003). environmental pathogens, and induction of
During psoriasis, this tightly regulated antimicrobial gene expression is vital in
process is altered, leading to overexpression providing a front line of defence against
of the LL-37 peptide and triggering immune inhaled microbes. 1,25D has been shown to
responses by activating plasmacytoid DCs strongly induce LL-37 expression in Calu-3
(Lande et al., 2007). Plasmacytoid DCs are cells, a human epithelial airway cell line
specialized skin cells that sense viral and (Wang et al., 2004). Moreover, treatment with
bacterial DNA through TLR signalling. In 1,25D yielded a significant reduction in the
particular cases of autoimmune disease, a microbial activity of either Escherichia coli or
breakdown in the sensory apparatus occurs Pseudomonas aeruginosa in infected Calu-3
allowing for self-DNA to trigger an immune cells. P. aeruginosa is an opportunistic patho-
response. In psoriatic skin, Lande et al. (2007) gen associated with the vast majority of
established that LL-37 is the mediating factor respiratory infections in patients aicted
triggering plasmacytoid DC activation. with cystic fibrosis (Speert et al., 2002).
Lending to the cationic, amphipathic nature Notably, patients with cystic fibrosis have
of protein, LL-37 directly binds to DNA in also been observed to have low circulating
plasmacytoid DCs, forming aggregated and serum 25D levels (Hecker and Aris, 2004).
condensed structures that are delivered to Expanding upon these results, Yim et al.
TLR-9 receptors, thereby activating immune (2007) documented the presence of the VDR,
response mechanisms leading to chronic skin and robust induction of cathelicidin, in
inflammation. normal human bronchial epithelial cells.
VDR-regulated signalling is not limited Additionally, this particular study demon-
to dermal epithelial cell types. One recent strated the direct contribution of cathelicidin
study showed that both LL-37 and the VDR to antimicrobial activity through preincub-
are expressed in hepatic biliary epithelial ation of infected normal human bronchial
cells (DAldebert et al., 2009). Under normal epithelial cells with an anti-LL-37 antibody.
physiological conditions, the biliary tract Active vitamin D metabolism has also
maintains a number of defence mechanisms, been established in the endometrium and
preserving a microbe-free environment. The placenta during gestation. Beginning in the
authors found that antibacterial activity in 1980s, studies conducted with rats connected
the biliary tract is maintained by signalling vitamin D deficiency with significantly
actions of bile salts through the VDR and a diminished rates of reproduction (Halloran
related farnesoid X receptor (FXR). FXR is a and DeLuca, 1980; Hickie et al., 1983;
member of the nuclear receptor family Kwiecinksi et al., 1989). Recently, one
activated by bile salts. Remarkably, the VDR intriguing study linked high levels of
has also been established as a functional bile placental 1-hydroxylase expression during
acid receptor (Makishima et al., 2002). early gestation with the pleiotropic actions of
Activation of either the FXR or VDR by bile vitamin D signalling beyond fetal musculo-
acids has been found to mediate innate skeletal development (Zehnder et al., 2002).
immune function through cathelicidin Expanding upon these results, another study
induction in both hepatic epithelial cells and correlated vitamin D signalling with
normal human keratinocytes (DAldebert et attenuated immune function during the early
190 J.H. White and A.J. Bitton
Bhalla, A.K., Amento, E.P., Serog, B. and Glimcher, cells in vivo and in vitro. British Journal of
L.H. (1984) 1,25-Dihydroxyvitamin D3 inhibits Cancer 76, 10171020.
antigen-induced T cell activation. Journal of Coulombe, F., Divangahi, M., Veyrier, F., De
Immunology 133, 17481754. Leseleuc, L., Gleason, J.L., Yang, Y., Kelliher,
Brennan, A., Katz, D.R., Nunn, J.D., Barker, S., M.A., Pandey, A.K., Sassetti, C.M., Reed, M.B.
Hewison, M., Fraher, L.J. and ORiordan, J.L. and Behr, M.A. (2009) Increased NOD2-
(1987) Dendritic cells from human tissues mediated recognition of N-glycolyl muramyl
express receptors for the immunoregulatory dipeptide. Journal of Experimental Medicine
vitamin D3 metabolite, dihydroxycholecalciferol. 206, 17091716.
Immunology 61, 457461. Crowle, A.J., Ross, E.J. and May, M.H. (1987)
Brightbill, H.D., Libraty, D.H., Krutzik, S.R., Yang, Inhibition by 1,25(OH)2-vitamin D3 of the
R.B., Belisle, J.T., Bleharski, J.R., Maitland, M., multiplication of virulent tubercle bacilli in
Norgard, M.V., Plevy, S.E., Smale, S.T., cultured human macrophages. Infection and
Brennan, P.J., Bloom, B.R., Godowski, P.J. and Immunity 55, 29452950.
Modlin, R.L. (1999) Host defense mechanisms DAldebert, E., Biyeyeme Bi Mve, M.J., Mergey, M.,
triggered by microbial lipoproteins through Toll- Wendum, D., Firrincieli, D., Coilly, A., Fouassier,
like receptors. Science 285, 732736. L., Corpechot, C., Poupon, R., Housset, C. and
Brown, A.J. and Slatopolsky, E. (2008) Vitamin D Chignard, N. (2009) Bile salts control the
analogs: therapeutic applications and antimicrobial peptide cathelicidin through
mechanisms for selectivity. Molecular Aspects nuclear receptors in the human biliary
of Medicine 29, 433452. epithelium. Gastroenterology 136, 14351443.
Carretero, M., Escamez, M.J., Garcia, M., Duarte, Davies, P.D., Brown, R.C. and Woodhead, J.S.
B., Holguin, A., Retamosa, L., Jorcano, J.L.,
(1985) Serum concentrations of vitamin D
Rio, M.D. and Larcher, F. (2008) In vitro and in
metabolites in untreated tuberculosis. Thorax
vivo wound healing-promoting activities of
40, 187190.
human cathelicidin LL-37. Journal of
Deretic, V. (2009) Multiple regulatory and effector
Investigative Dermatology 128, 223236.
roles of autophagy in immunity. Current Opinion
Chang, C.I., Pleguezuelos, O., Zhang, Y.A., Zou, J.
in Immunology 21, 5362.
and Secombes, C.J. (2005) Identification of a
Deretic, V. (2010) Autophagy in infection. Current
novel cathelicidin gene in the rainbow trout,
Opinion in Cell Biology 22, 252262.
Oncorhynchus mykiss. Infection and Immunity
Dilworth, F.J. and Chambon, P. (2001) Nuclear
73, 50535064.
receptors coordinate the activities of chromatin
Chapuy, M.C., Preziosi, P., Maamer, M., Arnaud,
remodeling complexes and coactivators to
S., Galan, P., Hercberg, S. and Meunier, P.J.
(1997) Prevalence of vitamin D insufficiency in facilitate initiation of transcription. Oncogene 20,
an adult normal population. Osteoporosis 30473054.
International 7, 439443. Dorschner, R.A., Pestonjamasp, V.K., Tamakuwala,
Chawla, A., Repa, J.J., Evans, R.M. and S., Ohtake, T., Rudisill, J., Nizet, V., Agerberth,
Mangelsdorf, D.J. (2001) Nuclear receptors and B., Gudmundsson, G.H. and Gallo, R.L. (2001)
lipid physiology: opening the X-files. Science Cutaneous injury induces the release of
294, 18661870. cathelicidin anti-microbial peptides active
Cheng, J.B., Levine, M.A., Bell, N.H., Mangelsdorf, against group A Streptococcus. Journal of
D.J. and Russell, D.W. (2004) Genetic evidence Investigative Dermatology 117, 9197.
that the human CYP2R1 enzyme is a key Evans, K.N., Nguyen, L., Chan, J., Innes, B.A.,
vitamin D 25-hydroxylase. Proceedings of the Bulmer, J.N., Kilby, M.D. and Hewison, M. (2006)
National Academy of Sciences of the USA 101, Effects of 25-hydroxyvitamin D3 and
77117715. 1,25-dihydroxyvitamin D3 on cytokine production
Colston, K.W., Mackay, A.G., James, S.Y., Binderup, by human decidual cells. Biology of Reproduction
L., Chander, S. and Coombes, R.C. (1992) 75, 816822.
EB1089: a new vitamin D analogue that inhibits Giuliani, A., Pirri, G. and Nicoletto, S. (2007)
the growth of breast cancer cells in vivo and in Antimicrobial peptides: an overview of a
vitro. Biochemical Pharmacology 44, 2273 promising class of therapeutics. Central
2280. European Journal of Biology 2, 133.
Colston, K.W., James, S.Y., Ofori-Kuragu, E.A., Glass, C.K. and Rosenfeld, M.G. (2000) The
Binderup, L. and Grant, A.G. (1997) Vitamin D coregulator exchange in transcriptional functions
receptors and anti-proliferative effects of vitamin of nuclear receptors. Genes & Development 14,
D derivatives in human pancreatic carcinoma 121141.
192 J.H. White and A.J. Bitton
Gombart, A.F., Borregaard, N. and Koeffler, H.P. multiple long-range enhancers. Molecular and
(2005) Human cathelicidin antimicrobial peptide Cellular Biology 26, 64696486.
(CAMP) gene is a direct target of the vitamin D Klotman, M.E. and Chang, T.L. (2006) Defensins in
receptor and is strongly up-regulated in myeloid innate antiviral immunity. Nature Reviews
cells by 1,25-dihydroxyvitamin D3. FASEB Immunology 6, 447456.
Journal 19, 10671077. Kwiecinksi, G.G., Petrie, G.I. and Deluca, H.F.
Gombart, A.F., Saito, T. and Koeffler, H.P. (2009) (1989) 1,25-Dihydroxyvitamin D3 restores
Exaptation of an ancient Alu short interspersed fertility of vitamin D-deficient female rats.
element provides a highly conserved vitamin American Journal of Physiology 256,
D-mediated innate immune response in humans E483E487.
and primates. BMC Genomics 10, 321. Lande, R., Gregorio, J., Facchinetti, V., Chatterjee,
Grad, R. (2004) Cod and the consumptive: a brief B., Wang, Y.H., Homey, B., Cao, W., Su, B.,
history of cod-liver oil in the treatment of Nestle, F.O., Zal, T., Mellman, I., Schroder, J.M.,
pulmonary tuberculosis. Pharmacy in History Liu, Y.J. and Gilliet, M. (2007) Plasmacytoid
46, 106120. dendritic cells sense self-DNA coupled with
Guy, R.A. (1923) The history of cod liver oil as a antimicrobial peptide. Nature 449, 564569.
remedy. American Journal of Diseases of Larsson, P., Mattsson, L., Klareskog, L. and
Children 26, 112116. Johnsson, C. (1998) A vitamin D analogue (MC
Halloran, B.P. and DeLuca, H.F. (1980) Effect of 1288) has immunomodulatory properties and
vitamin D deficiency on fertility and reproductive suppresses collagen-induced arthritis (CIA)
capacity in the female rat. Journal of Nutrition without causing hypercalcaemia. Clinical and
Experimental Immunology 114, 277283.
110, 15731580.
Lee, J.S., Yang, C.S., Shin, D.M., Yuk, J.M., Son,
Hancock, R.E. and Diamond, G. (2000) The role of
J.W. and Jo, E.K. (2009) Nitric oxide synthesis is
cationic antimicrobial peptides in innate host
modulated by 1,25-dihydroxyvitamin D3 and
defences. Trends in Microbiology 8, 402410.
interferon- in human macrophages after
Hancock, R.E. and Sahl, H.G. (2006) Antimicrobial
mycobacterial infection. Immune Network 9,
and host-defense peptides as new anti-infective
192202.
therapeutic strategies. Nature Biotechnology
Lehrer, R.I. (2004) Primate defensins. Nature
24, 15511557.
Reviews Microbiology 2, 727738.
Hecker, T.M. and Aris, R.M. (2004) Management of
Lin, R. and White, J.H. (2004) The pleiotropic
osteoporosis in adults with cystic fibrosis. Drugs
actions of vitamin D. Bioessays 26, 2128.
64, 133147.
Lin, R., Nagai, Y., Sladek, R., Bastien, Y., Ho, J.,
Heilborn, J.D., Nilsson, M.F., Kratz, G., Weber, G., Petrecca, K., Sotiropoulou, G., Diamandis, E.P.,
Sorensen, O., Borregaard, N. and Stahle- Hudson, T.J. and White, J.H. (2002) Expression
Backdahl, M. (2003) The cathelicidin anti- profiling in squamous carcinoma cells reveals
microbial peptide LL-37 is involved in pleiotropic effects of vitamin D3 analog EB1089
re-epithelialization of human skin wounds and is signaling on cell proliferation, differentiation,
lacking in chronic ulcer epithelium. Journal of and immune system regulation. Molecular
Investigative Dermatology 120, 379389. Endocrinology 16, 12431256.
Hickie, J.P., Lavigne, D.M. and Woodward, W.D. Liu, N., Kaplan, A.T., Low, J., Nguyen, L., Liu, G.Y.,
(1983) Reduced fecundity of vitamin D deficient Equils, O. and Hewison, M. (2009) Vitamin D
rats. Comparative Biochemistry and Physiology induces innate antibacterial responses in human
A: Comparative Physiology 74, 923925. trophoblasts via an intracrine pathway. Biology
Holick, M.F. (2007) Vitamin D deficiency. New of Reproduction 80, 398406.
England Journal of Medicine 357, 266281. Liu, P.T., Stenger, S., Li, H., Wenzel, L., Tan, B.H.,
Jenssen, H., Hamill, P. and Hancock, R.E. (2006) Krutzik, S.R., Ochoa, M.T., Schauber, J., Wu,
Peptide antimicrobial agents. Clinical Micro- K., Meinken, C., Kamen, D.L., Wagner, M., Bals,
biology Reviews 19, 491511. R., Steinmeyer, A., Zugel, U., Gallo, R.L.,
Jones, G., Strugnell, S.A. and Deluca, H.F. (1998) Eisenberg, D., Hewison, M., Hollis, B. W.,
Current understanding of the molecular actions Adams, J.S., Bloom, B.R. and Modlin, R.L.
of vitamin D. Physiological Reviews 78, (2006) Toll-like receptor triggering of a vitamin
11931231. D-mediated human antimicrobial response.
Kim, S., Yamazaki, M., Zella, L.A., Shevde, N.K. Science 311, 17701773.
and Pike, J.W. (2006) Activation of receptor Liu, P.T., Stenger, S., Tang, D.H. and Modlin, R.L.
activator of NF-B ligand gene expression by (2007) Cutting edge: vitamin D-mediated human
1,25-dihydroxyvitamin D3 is mediated through antimicrobial activity against Mycobacterium
Vitamin D and AMP Gene Expression 193
tuberculosis is dependent on the induction of Provvedini, D.M., Tsoukas, C.D., Deftos, L.J. and
cathelicidin. Journal of Immunology 179, Manolagas, S.C. (1983) 1,25-Dihydroxyvitamin
20602063. D3 receptors in human leukocytes. Science 221,
Liu, P.T., Schenk, M., Walker, V.P., Dempsey, P.W., 11811183.
Kanchanapoomi, M., Wheelwright, M., Vazirnia, Rochel, N., Wurtz, J.M., Mitschler, A., Klaholz, B.
A., Zhang, X., Steinmeyer, A., Zgel, U., Hollis, and Moras, D. (2000) The crystal structure of
B.W., Cheng, G. and Modlin, R.L. (2009) the nuclear receptor for vitamin D bound to its
Convergence of IL-1B and VDR activation natural ligand. Molecular Cell 5, 173179.
pathways in human TLR2/1-induced anti- Schauber, J., Dorschner, R.A., Coda, A.B., Buchau,
microbial responses. PLoS ONE 4, e5810. A.S., Liu, P.T., Kiken, D., Helfrich, Y.R., Kang, S.,
Makishima, M., Lu, T.T., Xie, W., Whitfield, G.K., Elalieh, H.Z., Steinmeyer, A., Zugel, U., Bikle,
Domoto, H., Evans, R.M., Haussler, M.R. and D.D., Modlin, R.L. and Gallo, R.L. (2007) Injury
Mangelsdorf, D.J. (2002) Vitamin D receptor as enhances TLR2 function and antimicrobial
an intestinal bile acid sensor. Science 296,
peptide expression through a vitamin
13131316.
D-dependent mechanism. Journal of Clinical
Martineau, A.R., Honecker, F.U., Wilkinson, R.J.
Investigation 117, 803811.
and Griffiths, C.J. (2007) Vitamin D in the
Schwalfenberg, G. (2007) Not enough vitamin D:
treatment of pulmonary tuberculosis. Journal of
health consequences for Canadians. Canadian
Steroid Biochemistry and Molecular Biology
103, 793798. Family Physician 53, 841854.
Masten, A.R. (1935) Sunlight in tuberculosis. Chest Schwartz, G., Eads, D., Naczki, C., Northrup, S.,
1, 823. Chen, T. and Koumenis, C. (2008) 19-nor-1
McKenna, N.J. and OMalley, B.W. (2002) ,25-Dihydroxyvitamin D2 (paricalcitol) inhibits
Combinatorial control of gene expression by the proliferation of human pancreatic cancer
nuclear receptors and coregulators. Cell 108, cells in vitro and in vivo. Cancer Biology &
465474. Therapy 7, 430436.
Nesby-ODell, S., Scanlon, K.S., Cogswell, M.E., Segaert, S. and Simonart, T. (2008) The epidermal
Gillespie, C., Hollis, B.W., Looker, A.C., Allen, vitamin D system and innate immunity: some
C., Doughertly, C., Gunter, E.W. and Bowman, more light shed on this unique photoendocrine
B.A. (2002) Hypovitaminosis D prevalence and system? Dermatology 217, 711.
determinants among African American and Shinkyo, R., Sakaki, T., Kamakura, M., Ohta, M.
white women of reproductive age: third National and Inouye, K. (2004) Metabolism of vitamin D
Health and Nutrition Examination Survey, by human microsomal CYP2R1. Biochemical
19881994. American Journal of Clinical and Biophysical Research Communications
Nutrition 76, 187192. 324, 451457.
Norman, A.W. (1998) Sunlight, season, skin Speert, D.P., Campbell, M.E., Henry, D.A., Milner,
pigmentation, vitamin D, and 25-hydroxyvitamin R., Taha, F., Gravelle, A., Davidson, A.G., Wong,
D: integral components of the vitamin D L.T. and Mahenthiralingam, E. (2002)
endocrine system. American Journal of Clinical Epidemiology of Pseudomonas aeruginosa in
Nutrition 67, 11081110. cystic fibrosis in British Columbia, Canada.
Norman, A.W. (2006) Minireview: vitamin D American Journal of Respiratory and Critical
receptor: new assignments for an already busy
Care Medicine 166, 988993.
receptor. Endocrinology 147, 55425548.
Stead, W.W., Senner, J.W., Reddick, W.T. and
Patil, A., Hughes, A.L. and Zhang, G. (2004) Rapid
Lofgren, J.P. (1990) Racial differences in
evolution and diversification of mammalian
susceptibility to infection by Mycobacterium
-defensins as revealed by comparative analysis
tuberculosis. New England Journal of Medicine
of rodent and primate genes. Physiological
Genomics 20, 111. 322, 422427.
Peric, M., Koglin, S., Dombrowski, Y., Gross, K., Tavera-Mendoza, L., Wang, T.T., Lallemant, B.,
Bradac, E., Ruzicka, T. and Schauber, J. (2009) Zhang, R., Nagai, Y., Bourdeau, V., Ramirez-
VDR and MEK-ERK dependent induction of the Calderon, M., Desbarats, J., Mader, S. and
antimicrobial peptide cathelicidin in keratinocytes White, J.H. (2006) Convergence of vitamin D
by lithocholic acid. Molecular Immunology 46, and retinoic acid signalling at a common
31833187. hormone response element. EMBO Reports 7,
Prosser, D.E. and Jones, G. (2004) Enzymes 180185.
involved in the activation and inactivation of Thoma-Uszynski, S., Stenger, S., Takeuchi, O.,
vitamin D. Trends in Biochemical Sciences 29, Ochoa, M.T., Engele, M., Sieling, P.A., Barnes,
664673. P.F., Rollinghoff, M., Bolcskei, P.L., Wagner, M.,
194 J.H. White and A.J. Bitton
Akira, S., Norgard, M.V., Belisle, J.T., Godowski, Mader, S., Behr, M.A. and White, J.H. (2010)
P.J., Bloom, B.R. and Modlin, R.L. (2001) Direct and indirect induction by 1,25-dihydroxy-
Induction of direct antimicrobial activity through vitamin D3 of the NOD2/CARD15-defensin 2
mammalian Toll-like receptors. Science 291, innate immune pathway defective in Crohn
15441547. disease. Journal of Biological Chemistry 285,
van Dijk, A., Veldhuizen, E.J., Van Asten, A.J. and 22272231.
Haagsman, H.P. (2005) CMAP27, a novel White, J.H. (2004) Profiling 1,25-dihydroxyvitamin
chicken cathelicidin-like antimicrobial protein. D3-regulated gene expression by microarray
Veterinary Immunology and Immunopathology analysis. Journal of Steroid Biochemistry and
106, 321327. Molecular Biology 8990, 239244.
van Etten, E. and Mathieu, C. (2005) Immuno- White, J.H. (2008) Vitamin D signaling, infectious
regulation by 1,25-dihydroxyvitamin D3: basic diseases, and regulation of innate immunity.
concepts. Journal of Steroid Biochemistry and Infection and Immunity 76, 38373843.
Molecular Biology 97, 93101. Yamasaki, K., Di Nardo, A., Bardan, A., Murakami,
van Etten, E., Decallonne, B., Verlinden, L., Verstuyf, M., Ohtake, T., Coda, A., Dorschner, R.A.,
A., Bouillon, R. and Mathieu, C. (2003) Analogs Bonnart, C., Descargues, P., Hovnanian, A.,
of 1,25-dihydroxyvitamin D3 as pluripotent Morhenn, V.B. and Gallo, R.L. (2007) Increased
immunomodulators. Journal of Cellular serine protease activity and cathelicidin
Biochemistry 88, 223226. promotes skin inflammation in rosacea. Nature
Wali, R.K., Bissonnette, M., Khare, S., Hart, J., Medicine 13, 975980.
Sitrin, M.D. and Brasitus, T.A. (1995) 1,25- Yim, S., Dhawan, P., Ragunath, C., Christakos, S.
Dihydroxy-16-ene-23-yne-26,27-hexafluoro- and Diamond, G. (2007) Induction of cathelicidin
cholecalciferol, a noncalcemic analogue of in normal and CF bronchial epithelial cells by
1,25-dihydroxyvitamin D3, inhibits azoxy- 1,25-dihydroxyvitamin D3. Journal of Cystic
methane-induced colonic tumorigenesis. Cancer Fibrosis 6, 403410.
Research 55, 30503054. Yuk, J.M., Shin, D.M., Lee, H.M., Yang, C.S., Jin,
Wang, T.T., Nestel, F.P., Bourdeau, V., Nagai, Y., H.S., Kim, K.K., Lee, Z.W., Lee, S.H., Kim, J.M.
Wang, Q., Liao, J., Tavera-Mendoza, L., Lin, R., and Jo, E.K. (2009) Vitamin D3 induces
Hanrahan, J.W., Mader, S. and White, J.H. autophagy in human monocytes/macrophages
(2004) Cutting edge: 1,25-dihydroxyvitamin D3 via cathelicidin. Cell Host & Microbe 6, 231
is a direct inducer of antimicrobial peptide gene 243.
expression. Journal of Immunology 173, 2909 Zanetti, M. (2004) Cathelicidins, multifunctional
2912. peptides of the innate immunity. Journal of
Wang, T.T., Tavera-Mendoza, L.E., Laperriere, D., Leukocyte Biology 75, 3948.
Libby, E., Macleod, N.B., Nagai, Y., Bourdeau, Zehnder, D., Evans, K.N., Kilby, M.D., Bulmer, J.N.,
V., Konstorum, A., Lallemant, B., Zhang, R., Innes, B.A., Stewart, P.M. and Hewison, M.
Mader, S. and White, J.H. (2005) Large-scale in (2002) The ontogeny of 25-hydroxyvitamin D3
silico and microarray-based identification of 1-hydroxylase expression in human placenta
direct 1,25-dihydroxyvitamin D3 target genes. and decidua. American Journal of Pathology
Molecular Endocrinology 19, 26852695. 161, 105114.
Wang, T.T., Dabbas, B., Laperriere, D., Bitton, A.J., Ziegler, E.E. and Filer, L.J. Jr (1996) Present
Soualhine, H., Tavera-Mendoza, L.E., Dionne, Knowledge in Nutrition, 7th edn. ILSI Press,
S., Servant, M.J., Bitton, A., Seidman, E.G., Washington, DC.
12 Fine Tuning Host Responses in the
Face of Infection: Emerging Roles and
Clinical Applications of Host Defence
Peptides
Abstract
Host defence peptides (HDPs) are powerful modulators of human innate immunity, and can modify the
outcome of the endogenous host response to infection. The progressive development of pathogen
resistance to conventional antimicrobial agents has lead to a new appreciation of HDPs for their ability to
fight infection, enhance vaccine responses, limit inflammation and promote wound healing, within the
context of human disease. HDPs are a family of cationic, short, amphipathic peptides that include the
classical mammalian antimicrobial peptides, cathelicidins and defensins, as well as non-antimicrobial
peptides with similar immunomodulatory properties. This chapter reviews our current basic
understanding of the anti-infective and immunomodulatory properties of both endogenous HDPs and
synthetic derivatives (termed innate defence regulators) with regard to their ability to selectively fine
tune the responses of host cells and physiology. The clinical application of these molecules is also
discussed, with a focus on past and ongoing clinical trials of HDPs and innate defence regulators as novel
therapeutics for infectious and inflammatory diseases.
Infectious diseases have been the primary neoplastic disease) as the most significant
cause of morbidity and mortality for most of killers facing Western society (WHO, 2008).
human history. At the end of the 19th Following the introduction of penicillin
century, the average human life expectancy and the relatively rapid development of
was 25 years (Casanova and Abel, 2005). It other antibiotic classes, we have come to rely
was the development of Pasteurs insights on the availability of eective antibiotics to
into the nature and role of microbes in combat infections. However, having now
infections that paved the way for our first well and truly entered the era of antibiotic
medical breakthroughs in hygiene, vac- resistance, it is necessary to develop novel
cination and antibiotics. These discoveries antimicrobial therapies and strategies to
led to a rapid acceleration in life expectancy overcome the growing problem of multidrug-
around the world, and infectious causes of resistant bacterial strains. The discovery of
mortality have been falling behind the naturally occurring antimicrobial peptides
diseases of old age (cardiovascular and (AMPs) provided hope that these could
* Corresponding author.
become the basis for a new class of antibiotic from autotoxicity related to the strong
compounds, although clinical trial results to cationic charge and amphipathicity of the
date have not yet confirmed this potential. mature peptide (the latter also being
Research in this area has increasingly begun managed in part by sequestration into
to focus on the immunomodulatory prop- granules). Neutrophil -defensins are stored
erties of these peptides and their applications within azurophilic granules for release by
for human disease, specifically their use as fusion with cell phagosomes containing
novel anti-infective therapeutics that circum- ingested microbes; this results in very high
vent antibiotic resistance by selectively local concentrations of defensins and, despite
enhancing the beneficial aspects of the their relatively weak antimicrobial activity,
endogenous host response to pathogens. concentrations are sucient to mediate direct
microbicidal activity (Selsted and Ouellette,
2005). Intestinal -defensins are constitutively
12.1 Mammalian Host Defence expressed and secreted from Paneth cells,
(Antimicrobial) Peptides with secretion being enhanced by contact
with bacterial components such as CpG
Mammalian peptides with direct anti- oligodeoxynucleotides (ODNs). Contact with
microbial activity are generally classified into bacterial components can also stimulate
the two main families of cathelicidins and -defensin secretion by natural killer (NK)
defensins, although other peptides exist that cells (Agerberth et al., 2000), indicating that,
do not fall into these categories. The in addition to their intracellular antimicrobial
defensins are cationic peptides of 1845 function within neutrophils, such defensins
amino acids and are further divided into also have important extracellular functions.
three subfamilies: the -, - and -defensins. -Defensins are produced by a range of
The - and -defensins are genetically tissues, mainly in response to pro-
distinct, with similar structures dominated inflammatory cytokines induced by Toll-like
by -strands that dier in the placement of receptor (TLR) agonists. For example,
their six conserved cysteine residues that -defensins produced by keratinocytes play
form three intramolecular disulfide bridges important roles in the host defence functions
(Selsted and Ouellette, 2005). The -defensins of the skin and in wound healing (Niyonsaba
are cyclic in structure, are only found in non- et al., 2007).
human primates and have antiviral activity While the defensins can demonstrate
against human immunodeficiency virus-1 antibacterial activity in vitro at low micromolar
(Gallo et al., 2006). Defensins are produced concentrations, most - and -defensins are
by leukocytes in many mammals, including only weakly eective in the presence of salt
humans, rats and rabbits, but not mice. concentrations approaching those found in
However, mice, like humans, also produce vivo; this can be over-ridden by extremely
-defensins in their intestinal crypts and high concentrations of these molecules such
these include a subclass termed cryptdins as the mg ml1 concentrations found in
(Amid et al., 2009). Conversely, mice do not phagocytic granules or the crypts of the
produce leukocyte -defensins like humans, intestine. At least one -defensin, human
while bovines are devoid of all -defensins. -defensin (hBD)-3, is more cationic than the
others, less aected by cation antagonism and
probably has meaningful activity at the
12.1.1 Defensins surface of the skin. Similarly, the -defensins
are active at these higher salt concentrations,
The - and -defensins are produced as although only when in their cyclic form (Tang
prepropeptides that are cleaved to form et al., 1999). This suggests that, while direct
propeptides, which in turn require additional antimicrobial activity is important in some
processing to form the active peptide. The physiological niches, where the concentration
propiece, which is cleaved to release the of peptide is high enough to overcome
active peptide, acts to protect defensins from antagonism by salts, in many other anatomical
proteolysis inside cells and protect the cell locations the alternative, immunomodulatory
Clinical Applications of Host Defence Peptides 197
functions of these peptides probably pre- with hBD-1, also influences susceptibility to
dominate. Crohns disease, a microbial-induced inflam-
Defensins modulate the functions of matory condition of the intestines (Kocsis et
many cell types, influencing immune cell al., 2008). Similarly, a predisposition to
recruitment, activation and maturation, Crohns disease has been observed in indi-
wound healing and angiogenesis. For viduals with a lower copy number of the
example, human -defensins 13 have been hBD-2 gene, and a correspondingly lower
shown to influence the maturation and expression of hBD-2 at the mRNA level
dierentiation of monocyte-derived dendritic (Fellermann et al., 2006).
cells (DCs), with dierent outcomes induced
by either high or low peptide concentrations
(Rodriguez-Garcia et al., 2009). Human 12.1.2 Cathelicidins
neutrophil -defensins 13 act as direct
chemoattractants for T cells and DCs (Yang et Cathelicidins are structurally distinct from
al., 1999; Hubert et al., 2007), whereas hBD14 defensins, containing a characteristic cathelin
induce chemotaxis of macrophages and prodomain and a signal sequence. Despite
calcium flux in mast cells, while having no this related N-terminal region, cathelicidins
eect on T cells or DCs (Soruri et al., 2007). vary markedly in structure and include
-Defensins also have mitogenic functions, -helical, -hairpin, -turn and extended
promoting wound healing through the peptides. They are produced by a wide range
stimulation of fibroblast and epithelial cell of species, including mammals, birds and fish,
proliferation (Murphy et al., 1993; Nishimura with some species producing up to seven
et al., 2004). In addition to aecting mast cell dierent cathelicidins (Durr et al., 2006).
chemotaxis and calcium flux, hBD-3 and Humans have only one cathelicidin, which is
hBD-4 have also been shown to induce mast synthesized as the proprotein hCAP-18 and
cell degranulation and prostaglandin D2 cleaved extracellularly to form the active
production, with mast cell activation being protein known as LL-37. Mice also have a
dependent on signalling through the single cathelicidin, known as CRAMP, which
mitogen-activated protein kinase (MAPK) has 67% identity to LL-37. Cathelicidins are
p38 and extracellular signal-regulated kinase produced by a range of cell types, including
(ERK)1/2 pathways (Nishimura et al., 2004). epithelial cells and leukocytes such as
As these immunomodulatory functions are neutrophils, monocytes, T cells, B cells and
generally seen at peptide concentrations NK cells. They appear in many body locations
lower than those needed for direct (e.g. at mucosal and skin surfaces) and fluids
antimicrobial activity, and are not inhibited (e.g. gastric juices, saliva, semen, sweat,
by physiologically relevant salt concen- plasma, airway surface liquid and breast
trations (being evident under tissue culture milk). Like the defensins, cathelicidins exhibit
conditions and often in animal models), these a broad range of biological activities that
immunomodulatory functions are likely to include both direct antimicrobial activity and
be more broadly physiologically relevant immunomodulatory functions. LL-37 is
than direct antimicrobial activity. present in many dierent tissues and
The in vivo significance of defensins in secretions, including sweat and breast milk,
the context of innate immune functions is and is stored at high concentrations in its
supported by observations in humans with proform hCAP-18 in the azurophilic granules
genetic alterations in defensin production. of neutrophils. The physiological concen-
For example, while defensins are usually trations of LL-37 are estimated to be up to
induced in response to interferon (IFN)-, in 1 M (5 g ml1) in airway surface fluid and at
those with a particular single nucleotide mucosal surfaces, and under pathological
polymorphism expression of hBD-1 and conditions can increase dramatically, with
hBD-3 is constitutive and this confers concentrations up to 300 M reported in
enhanced protection from candidiasis (Kalus psoriasis (Ong et al., 2002; Schaller-Bals et al.,
et al., 2009). This same single nucleotide poly- 2002; Bals and Wilson, 2003). The biological
morphism, together with another associated functions of LL-37 have been proposed to
198 M.L. Mayer et al.
vary depending upon the local conditions; activated receptors epidermal growth factor
for example, direct antimicrobial activity receptor and nucleotide scavenging receptor
requires quite high concentrations of the P2X7 in various properties of LL-37 have been
peptide and/or low salt concentrations. described (Tjabringa et al., 2003; Tomasinsig
Conversely, wound-healing, angiogenic and et al., 2008). Recent studies (Zhang et al.,
anti-endotoxic activity and synergistic 2009) indicate that LL-37 can act as an
induction of certain chemokines together alternative ligand for the IL-8 receptor
with endogenous signalling molecules are (CXCR2) on neutrophils, while Mookherjee
all seen at very low (<1 M) peptide et al. (2009a) reported a physical and
concentrations. functional interaction between LL-37 and
The ability of LL-37 to augment many intracellular glyceraldehyde 3-phosphate
aspects of the innate immune response has dehydrogenase (GAPDH). Many other
been well established in the literature. For studies have described the signal trans-
example, LL-37 modulates the production of duction pathways, transcription factors and
interleukin (IL)-8 by keratinocytes and eector proteins and pathways involved in
epithelial cells (Filewod et al., 2009) and peptide activity (Mookherjee et al., 2009b).
inhibits the action of IFN- on monocytes, Studies on cathelicidin activity in vivo
macrophages, DCs and B lymphocytes (Nnik and ex vivo have been undertaken using
et al., 2009). Studies on human peripheral transgenic mice lacking CRAMP, with notable
blood mononuclear cells have shown that observations including that the peptide is
LL-37 acts in synergy with immune mediators involved in NK-cell-mediated antitumour
such as granulocytemacrophage colony- responses (Buchau et al., 2010), partial
stimulating factor and IL-1 to enhance the protection from bacterial infections (Nizet et
production of cytokines (e.g. IL-6 and -10) and al., 2001; Bra et al., 2005; Iimura et al., 2005;
chemokines (e.g. monocyte chemotactic Huang et al., 2007) and viral infections
protein-1 and -3). This synergy is mediated (Howell et al., 2006) and the antimicrobial
through modulation of intracellular signalling activity of mast cells (Di Nardo et al., 2003).
pathways, such as via phosphoinositide However, these studies have not always
3-kinase and MAPK, and subsequent changes discriminated between direct and indirect
in the activation of associated transcription (modulation of other immune mechanisms)
factors (Yu et al., 2007). The immune modes of action. Treatment with exogenous
modulation mediated by the cathelicidin leads CRAMP has also been shown to reduce the
to a more eective and balanced innate severity of colitis induced by dextran sulfate
response, with synergistic upregulation of the sodium ingestion, with enhanced intestinal
chemokine response accompanied by anti- mucus production and reduced apoptosis
endotoxic activity, mediated both by (Tai et al., 2007). In addition to the
lipopolysaccharide (LPS) binding and immunomodulatory eects it exerts on
modulation of TLR signalling cascades to mammalian cells, LL-37 is able to prevent
reduce the production of proinflammatory biofilm formation by the bacterial pathogen
cytokines such as tumour necrosis factor Pseudomonas aeruginosa at a concentration far
(TNF)-. The anti-endotoxic activity of LL-37 below (at least 1/16th) its minimal inhibitory
is supported by experiments showing that it concentration by specifically modulating
can protect mice from death after challenge bacterial gene expression (Overhage et al.,
with LPS alone (i.e. any bactericidal activity is 2008).
irrelevant) and that it can reduce leukocyte
responses to LPS ex vivo (Mookherjee et al.,
2007). The mechanism of action is as yet 12.2 The Role of Endogenous Host
incompletely understood, but several cellular Defence Peptides in the Response to
components have been proposed as specific Infection
interactors. The roles of surface formyl
peptide receptor-like 1 as a direct binding The natural human host response to infection
receptor in chemotaxis, of other undetermined cannot be modelled on an agar plate, tissue
G-protein-coupled receptors and of the trans- culture dish or nutrient-broth-filled test tube.
Clinical Applications of Host Defence Peptides 199
Rather, it is a dynamic and complex process defence against infection. Although the bulk
that involves all of the physiological systems of HDP research has focused on the direct
and organs necessary for human life. Cationic antimicrobial nature of these molecules,
peptides are responsible for fine tuning partly due to their location in professional
immunological and physiological homeostatic immune cells and the relative ease of
parameters in the context of the host response measuring such activities, other non-
to infectious diseases. In light of the evidence antimicrobial activities have recently gained
that such peptides have a broad range of attention for their role in orchestrating global
biological functions that go well beyond physiological and biochemical changes that
direct antimicrobial activity in vitro modulate the host response, hence the use of
(Oppenheim and Yang, 2005; Hancock and the broader designation HDP when consider-
Sahl, 2006; Mookherjee et al., 2006), they are ing activities other than direct actions on
perhaps more accurately termed host defence microbes.
peptides (HDPs) rather than AMPs (Box 12.1). Immunomodulation by HDPs is often
The creation of synthetic innate defence understood to occur at local tissue sites of
regulator (IDR) peptides that lack appreciable infection. Dierential production of defensins
antimicrobial activity yet show similar and cathelicidins has been described to occur
physical properties to AMPs and powerful in epithelial, connective tissue and leukocyte
(indirect) anti-infective ecacy mediated cells against the background of an
through immunomodulatory mechanisms inflammatory milieu, while local concen-
adds further support to this idea. trations can also increase rapidly by
The human innate immune response to degranulation of phagocytic cells at the site
infection is a complex physiological process of infection, suggesting that the immuno-
that orchestrates non-specific responses to modulatory function occurs in a paracrine or
infectious threats. Within this dynamic series autocrine manner. Numerous studies on
of events, HDPs play a critical role in endogenous HDPs and equivalent synthetic
coordinating and fine tuning cellular, tissue- IDR peptides have shown that administering
specific and global physiological changes these molecules in infection models, either
that accompany the innate immune response. locally or systemically, results in alterations
Considering the critical functions of en- in global immune function (Table 12.1). Other
do genous HDPs beyond direct antimicrobial host-produced peptides, classically grouped
killing is essential to an expanded under- together as hormones, play a similar role in
standing of the important roles played by the face of infection, allowing the integration
these molecules within the context of host of local and systemic host defences with
Table 12.1. Small cationic peptides that have no antimicrobial activity or activity only in very dilute
medium or buffer, but that appear to play a role in the human host response to infection.
HDP family Physical Site(s) of Role in host response and Cellular (immunomodulatory)
and prototype properties production therapeutic applications function
Histatins: 24 amino Parotid and Play a broad role in maintaining Attenuate IL-6- and IL-8-
histatin 5 acids, +5 sublingual oral cavity homeostasis in the mediated gingival
net charge salivary face of oral and periodontal inflammation (Imatani et al.,
glands pathogens such as 2000) induced by P.
Porphyromonas gingivalis. gingivalis and prevent LPS-
Potent mediators of mucosal induced apoptosis in gingival
wound healing and fibroblasts (Imatani et al.,
re-epithelialization. The 2004). Wound healing occurs
histatin-derived peptide P-113D via an ERK1/2-dependent
has shown efficacy in animal mechanism (Oudhoff et al.,
models of Pseudomonas 2008).
aeruginosa sepsis (Cirioni
et al., 2004), and has been in
Phase II clinical trials (Table
12.2).
Iron-binding 25 amino Liver Iron homeostasis is a Circulating proinflammatory
peptides: acids, +2 cornerstone of host defence cytokines induce hepatic
hepcidin net charge (Schaible and Kaufmann, synthesis (Nemeth et al.,
2004). Iron-binding peptides 2004). Phagocytic cells
imit the utility of bacterial produce the peptide following
haemolysins in iron acquisition, TLR2 or TLR4 activation via
while simultaneously activating STAT1 and NF-B (Sow et
host antibacterial defences al., 2009), leading to
such as phagocytic cells and improved innate immune
complement (Ganz, 2009). recognition of bacterial
pathogens (Wang et al.,
2009a). Leads to impaired
incorporation of iron into
haemoglobin (Rivera et al.,
2005) and inflammation-
associated anaemia
(Nemeth, 2010).
Melanocortins: -MSH is Anterior Interface endocrine system with Bind to MC receptors found
MSH 13 amino pituitary the resolution of inflammation. on most human cells, with
acids, +1 gland Enhance anti-inflammatory MC-1R mediating
net charge; programme in leukocytes and immunomodulation. MC-1R
-MSH is 11 lymphocytes (Sarkar et al., ligation leads to large
amino acids, 2003; Yoon et al., 2003; Cooper increases in cellular cAMP
+2 net et al., 2005), without impairing via PKA, activation of the
charge the ability to clear infection. MAPK pathway and altered
Exhibit significant anti-endotoxin NF-B responses. In PBMCs,
activity (Scholzen, 2003; Yoon MSH causes downregulation
et al., 2003) and promote of TNF-, IL-1, IFN-, IL-6
wound healing (Zou et al., and IL-8, with simultaneous
2004; Yoon et al., 2008). induction of IL-10 and NO.
The MSH-derived peptide The immunomodulatory
AP214 has shown efficacy in functions of MSH are diverse,
preventing organ failure in cell-type-dependent and well
animal models of sepsis (Doi reviewed elsewhere (Bhm
et al., 2008) and is in clinical et al., 2008).
trials (Table 12.2).
Clinical Applications of Host Defence Peptides 201
species, NPs are secreted as 126151 amino has been found to do just the opposite,
acid precursors and cleaved into 2232 amino increasing mobilization of polymorpho-
acid active peptide forms with net positive nuclear neutrophils and macrophages and
charges. In addition to their traditional roles, enhancing endothelialleukocyte interactions.
these peptides have now been identified as ANP is also able to broadly activate immune
playing a critical role in fine tuning cells, increasing leukocyte production of
cardiovascular and innate immune responses bactericidal reactive oxygen species and
in the face of severe infection and sepsis inducing the maturation and proliferation of
(Mohapatra, 2007; Eleuteri and Di Stefano, T cells and DCs (Potter et al., 2009).
2009; Vesely and de Bold, 2009). Conversely, ANP has been shown to have
One of the most devastating (and often more of an anti-inflammatory/immuno-
lethal) complications of sepsis is septic shock, suppressant eect in lung tissue, in both
consisting of haemodynamic and cardio- animal models (Wang et al., 2009c; Sekino et
vascular collapse leading to renal and al., 2010) and patient trials (Oda et al., 2009).
multiorgan failure. Consequently, elevated Mechanistically, ANP can enhance the barrier
BNP (which has the longest half-life of the eect of endothelial tight junctions (and thus
NP family members) has been identified as limit leukocyte recruitment) in response to
an early clinical marker of sepsis (Rudiger et both inflammatory (LPS) (Irwin et al., 2005)
al., 2006; Kandil et al., 2008). Surprisingly, and allergic (ovalbumin (OVA)) stimuli
elevated BNP in sepsis has been found to (Mohapatra, 2007), probably via the attenu-
precede the onset of septic shock (Vila et al., ation of LPS- and TNF--induced MAPK,
2008). This was correlated with increased ERK1/2, NF-B and Rho pathway signalling
systemic inflammatory cytokines rather than (Xing and Birukova, 2009).
haemodynamic changes, and thus constituted The NPs and other small cationic HDPs
part of the early systemic innate immune that exhibit no or minimal direct anti-
response to the infectious agent. This finding microbial activity (i.e. active only in dilute
was reiterated in vitro, with IL-1 and TNF- medium or buer), but play an important
inducing BNP production in cardiomyocytes role in the endogenous host response to
(Vesely and de Bold, 2009), and in animal infection, are listed in Table 12.1. These
models, where an ANP/BNP hybrid antagon- peptides and their IDR derivatives (Box 12.1)
ist was shown to enhance LPS- and IL-1- have formed the basis of many
induced fever (Miyoshi et al., 2006). LPS immunomodulatory peptide agents currently
injection in healthy humans was found to in preclinical or clinical trials (Table 12.2).
cause rapid and substantial increases in
circulating pro-BNP (Vila et al., 2008),
suggesting that this peptide is part of the 12.3 Potential Therapeutic Uses
early host response to sepsis, perhaps beyond Anti-infective Activity
alerting the host physiology to prepare for
impending septic shock. The range of potential therapeutic
Whereas BNP-directed immunomodula- applications of HDPs and their synthetic
tion has largely been studied in the context derivatives has been greatly broadened by
of cardiac inflammation, the related peptides the discovery of their immunomodulatory
ANP and CNP have demonstrated immuno- properties. Beyond their use as novel anti-
modulatory eects on vascular and lung infectives against bacteria (Scott et al., 2007),
tissue. CNP, which has a net charge of +2, viruses (Gallo et al., 2006; Falco et al., 2009) or
was shown to inhibit leukocyte recruitment fungi (Benincasa et al., 2006; Kollef et al.,
and rolling across endothelial surfaces that 2006), they also have significant potential as
was driven by IL-1 or histamine in mice, adjuvants (Garlapati et al., 2009), anti-
and to inhibit plateletleukocyte interactions, inflammatories and anti-endotoxic agents
primarily through the modulation of (Andra et al., 2006) and are showing
P-selectin (Scotland et al., 2005). Interestingly, substantial promise in wound-healing
ANP, which has a higher net charge of +6, applications (Steinstraesser et al., 2008).
Clinical Applications of Host Defence Peptides 203
Table 12.2. Immunomodulatory host defence peptides, innate defence regulators and their derivatives in
clinical trials.
Most
advanced References and
Peptide Description Intervention progress trial registriesa
AP214 (Action Synthetic derivative of the Sepsis and Phase II Doi et al. (2008);
Pharma) HDP -MSH (Table 12.1). postsurgical organ NCT00903604
Parent peptide is fused to a failure
hexalysine sequence at its
C-terminus
CD-NP Chimeric synthetic NP (37- Organ failure Phase II Rose (2010);
mer) able to bind to the NCT00482937
receptors for all three
endogenous NPs. Modified to
lack haemodynamic activity
DiaPep277 HSP60 derivative (24-mer Autoimmune- Phase III NCT00644501;
(DeveloGen) peptide) that induces mediated diabetes ISRCTN55429664
T-regulatory cells
EA-230 Oligopeptide fragment from Sepsis Phase II van den Berg et
(Exponential -hCG (4-mer, LQGV) al. (2009); NAb
Biotherapies)
Ghrelin Endogenous HDP Airway Phase II (all) Table 12.1; JPRN-
inflammation, UMIN000002599;
chronic respiratory JPRN-
infection, cystic UMIN000001598;
fibrosis NCT00763477
Glutoxim/NOV-002 Hexapeptide with a stabilized Tuberculosis, Market Sokolova et al.
(Pharma BAM/ disulfide bond (bis-(-L- myelodysplastic (Russia); (2002);
Novelos) glutamyl)-L-cysteinyl-bis- syndromes Phase III (N. NCT00960726
glycine disodium salt) America)
Heptapeptide-7 Peptide fragment (7-mer), Wound healing, Phase II Falla and Zhang
(Helix BioMedix) derived from the synthetic skin regeneration (2010); NR
prototype HB-107 (which is
itself derived from cecropin
B)
hLF111 Derivative of the cationic Bacteraemia and Phase I/II van der Does et
(AM-Pharma) AMP human lactoferricin fungal infections in Earlier anti- al. (2010);
(amino acids 111) immuno- infective trials NCT00509938;
compromised terminated NCT00509847
haematopoietic (withdrawn);
stem-cell transplant NCT00509834
recipients (terminated)
IMX942 Immunomodulatory peptide Nosocomial Phase IA NA
(Inimex) lacking antimicrobial activity, infections, febrile
derived from the prototype neutropenia
IDR-1 (which is itself derived
from indolicidin)
Iseganan (IB-367) Synthetic protegrin-1 Oral mucositis in Phase III NCT00022373;
(Ardea derivative (17 amino acids) radiation therapy Earlier anti- NCT00118781
Biosciences) patients infective trials (terminated)
terminated
Continued
204 M.L. Mayer et al.
Abbreviations: AMP, antimicrobial peptide; hCG, human chorionic gonadotrophin; HLA, human leukocyte antigen; hLF,
human lactoferrin; HDP, host defence peptide; HSP, heat-shock protein; IDR-1, immune defence regulator 1; LPS,
lipopolysaccharide; LTA, lipoteichoic acid; MSH, melanocyte-stimulating hormone; NA, not available; NP, natriuretic
peptide; NR, not registered.
that this involves modulation of p38 and wide ranging, giving a vast potential for
ERK1/2 MAPK pathways, but is independent designer synthetic peptide drugs targeting
of JNK (c-Jun N-terminal kinase) (Niyonsaba dierent clinical conditions.
et al., 2005). IL-18 is a pleiotropic cytokine
that is classically thought of as an inducer of
IFN-, but is also able to promote 12.4 Recent Clinical Advances in
angiogenesis (Park et al., 2001). LL-37 and Therapeutic Application of Host
hBD-24 also promote the proliferation and Defence Peptides
migration of keratinocytes and the
production of IL-6, IL-10, 10-kDa IFN-- The introduction of insulin in the 1920s
induced protein, monocyte chemotactic marked the beginning of a new age in
protein-1, macrophage inflammatory protein pharmaceutical drugs, where endogenous
(MIP)-3 and RANTES (regulated on agents (or their mimetics) could be
activation, normal T-cell expressed and administered with the therapeutic aim of
secreted) by these cells (Niyonsaba et al., modulating human physiology. These
2007). In a subsequent study, the HDP biopharmaceutical agents, typically proteins
dermcidin (DCD-1L) stimulated keratino-
or peptides and more commonly referred to
cytes to produce a similar range of cytokines,
as biologics, have become a mainstay in the
including TNF-, IL-8, IFN-inducible protein
treatment of endocrine, autoimmune and
10 (IP-10) and MIP-3 (Niyonsaba et al.,
other diseases with pathophysiology or
2009), demonstrating that a wide range of
dysfunction involving human systems with
HDPs are able to activate keratinocytes. In an
complex feedback and feedforward regu-
in vivo system, hBD-2 has also been found to
lation. At the forefront of cutting-edge
stimulate chemotaxis of endothelial cells and
research and development into novel
promote capillary-like tube formation as
biologics are compounds that act at multiple
eectively as vascular endothelial growth
points within the dynamic regulatory
factor (Baroni et al., 2009). This same HDP
networks underlying disease states, moving
also promotes the migration, but not
proliferation, of intestinal epithelial cells beyond the classic one drugone receptor
(Otte et al., 2008), as does LL-37 (Otte et al., model of traditional pharmaceuticals.
2009). A synthetic acetylated and amidated Peptide-based immunotherapies are still
24-mer derivate of LL-37, termed P60.4-Ac in their infancy, but the use of immune-
(Nell et al., 2006), has been shown to inhibit targeting therapeutics is well established
disruption of the respiratory epithelium within the practice of Western medicine
ultrastructure by bacterial pathogen- (Waldmann, 2003). To date, the majority of
associated molecular patterns in vitro (Vonk these biologicals with immunomodulatory
et al., 2008). Although some of these activities action have aimed at the suppression or
occur at quite high concentrations of disruption of normal immune function, rather
peptides, it has been demonstrated that very than its enhancement. As a result, these drugs
low concentrations of peptides synergize have become the mainstay of treatments for
with endogenous host molecules such as chronic inflammatory and autoimmune
IL-1 and granulocytemacrophage colony- diseases, but show little appreciable value as
stimulating factor, as well as with TLR adjuvants or therapeutics in treating infectious
agonists such as flagellin and poly-IC diseases. Such applications are also limited by
(Bowdish et al., 2005a,b; Filewod et al., 2009). an inability to selectively target the eector
Thus, the range of biological activities of subsets that mediate the necessary com-
HDPs extends well beyond direct bacterial ponents of innate and adaptive immunity
killing, with an ability to modulate (Feldmann and Steinman, 2005), resulting in
intracellular signalling and gene expression side eects that can include enhanced
in a wide range of cell types. The susceptibility to infections (Botsios, 2005),
downstream eects of such activities are also significantly high rates of anaphylaxis (Corren
Clinical Applications of Host Defence Peptides 207
et al., 2009) and, in extreme cases, toxic While no clinical data are yet publicly
cytokine storms (Suntharalingam et al., 2006). available, IMX942 (which is a derivative of
Immunostimulatory drugs are beginning IDR-1 and lacks direct antimicrobial activity)
to make inroads as viable options for the has completed Phase I safety trials in
treatment of infectious diseases, often as chemotherapy patients with febrile neutro-
adjunctive therapy to mainstay antibiotics. A penia (Table 12.2). In addition, OP-145, a
recent Cochrane clinical trial meta-analysis structural derivate of LL-37, has been
(Del-Rio-Navarro et al., 2006), examining the reported to show ecacy in clinical trials for
use of 21 dierent biological immuno- the treatment of chronic otitis media in adult
modulatory agents in the treatment of patients (Table 12.2). Whether immuno-
pediatric acute respiratory infections, modulatory (immunostimulatory) peptides
provided fascinating data on the safety and will become mainstays in the treatment of
ecacy of these drugs. The Cochrane review bacterial antibiotics remains to be seen.
found that the use of immunostimulatory However, the successes in clinical trials to
drugs reduced the incidence of respiratory date demonstrate significant promise for
tract infections by an average of 40% across these peptides in playing, at minimum, an
the studies, with adverse eects not being adjunctive role to conventional antibiotic
significantly dierent to those associated therapy.
with placebo groups. Many of the peptides with documented
Due to the complexities inherent in immunomodulatory and antimicrobial
studying innate immunity and the cor- activity that have entered clinical trials have
responding diculty in selecting appropriate done so as topical antimicrobials for
clinical readouts or endpoints in preclinical infectious, inflammatory or wound-healing
and Phase I trials, clinical trials of applications (Table 12.2). The choice of
immunomodulatory drugs often evaluate moving forward with these agents as topical
compounds in manners that avoid excessive interventions likely reflects diculties in
responses or systemic absorption. For selecting appropriate systemic biomarkers as
example, RDP58, which is under investi- safety readouts. Thus, a series of peptides
gation for the treatment of ulcerative colitis have been tested topically, including
(Travis et al., 2005), was structurally modified omiganan (MX-226), iseganan (IB-367),
to prevent both proteolytic degradation and human lactoferrin (hLF)111 and HB-107
systemic absorption (Holtmann, 2003). Two (Table 12.2), despite the fact that most of
Phase II trials of RDP58 in a combined cohort these peptides have demonstrated immuno-
of 127 patients found the drug to be well modulatory activity in vivo (Giacometti et al.,
tolerated (adverse eects equivalent to 2003; Lee et al., 2004; Falla and Zhang, 2010;
placebo) and ecacious in improving sig- van der Does et al., 2010). Interestingly, the
moidoscopy scores in colitis patients majority of the peptide (and peptide-derived)
receiving medium or high doses of the drug drugs under investigation as immuno-
(Travis et al., 2005). The pathogenesis of modulatory anti-infectives are the classical
ulcerative colitis is thought to be at least neuroendocrine peptides described in Table
partially due to a dysregulated host response 12.1. This is probably because these peptides
to intestinal microbiota (Xavier and Podolsky, have been under investigation for decades
2007) and, although RDP58 is being (albeit in other contexts) and have well-
investigated for its anti-inflammatory prop- defined receptors and cellular mechanisms.
erties, it may also alter host responses to This is in contrast to HDPs, which are
microbiota in a manner similar to HDPs and relatively new discoveries. Based on these
IDRs. observations, we feel there is a strong
An exciting area in which the dual anti- possibility that, in the future, immuno-
infective and anti-inflammatory actions of modulatory activity will play a substantial
peptide immunotherapy have potential is the role in any clinical benefit demonstrated by
treatment of bacteraemia and septicaemia. HDP- and IDR-based drugs.
208 M.L. Mayer et al.
12.5 Innate Defence Regulators as matory sequelae of severe acne and a non-
Anti-infective Therapeutics infectious inflammatory skin disease, rosacea,
demonstrating promise for the treatment of
The increased awareness of the natural inflammatory conditions independent of
immunomodulatory functions of HDPs and antimicrobial activity. Systemic safety has
the role they play in the endogenous anti- been shown in Phase I clinical trials for hLF1
infective response has created an exciting 11, a truncated form of lactoferrin, which was
new avenue for their application: the use of examined in immunocompromised haemato-
synthetic peptides that selectively modulate poietic stem-cell transplant recipients. Recent
the innate immune response as an anti- work with hLF111 in animal models has also
infective therapeutic strategy. One of the first demonstrated its ecacy as a systemic anti-
synthetic immunomodulatory peptides infective through immunomodulatory
based on a HDP, immune defence regulator 1 mechanisms (van der Does et al., 2010).
(IDR-1), is able to mediate in vivo protection OP-215, a synthetic 24 amino acid
against bacterial challenge in the absence of derivate of LL-37, has also been shown to be
direct antimicrobial activity (Scott et al., ecacious and safe (compared to placebo) in
2007). The sequence of IDR-1 is based on the a randomized, double-blind, placebo-
12 amino acid bovine cathelicidin bactenecin, controlled, multicentre Phase II study when
which has immunomodulatory activities that applied topically in ear drops to chronic
reflect a subset of those apparent in the 37 suppurative otitis media patients (F.A.W.
amino acid human peptide LL-37 (Bowdish Peek et al., 2009, unpublished results).
et al., 2005a). The functional mechanism of Although little information on this peptide is
action of IDR-1 in animal infection models available, it is claimed that it binds LPS and
appears to involve the concomitant lipoteichoic acid, degrades biofilms and has
enhancement of chemokine production and antimicrobial actions, while its direct
moderation of proinflammatory cytokines chemotactic activity is said to be low.
such as TNF-, leading to enhanced cellular Another peptide, RDP58, which is a cationic
clearance of bacteria without excessive peptide derived from the heavy chain of
inflammation. These eects appear to be human leukocyte antigen (HLA) class I
mediated through modulation of specific molecules, has also demonstrated safety in
intracellular signalling pathways via intra- clinical trials (Travis et al., 2005). RDP58
cellular binding partners such as sequesto- reduced the production of the pro-
some-1/p62 (Yu et al., 2009) or GAPDH, inflammatory cytokines TNF-, IFN- and
which leads to altered activation states of key IL-12, but did not aect the production of
transcription factors (Scott et al., 2007; several other cytokines including IL-4, -6, -8
Mookherjee et al., 2009a). Such activities and -10. The proposed mechanism of action
provide a powerful rationale for designing involves interference with intracellular
synthetic peptides with optimized immuno- signalling pathways; however, the specific
modulatory activities based upon HDPs (e.g. details and interactors are currently
Nnik et al., 2010) and demonstrate that unknown (Travis et al., 2005). IMX942, which
direct antimicrobial activity is not a necessary is based on IDR-1, has completed Phase I
characteristic for the ecacy of such safety trials in chemotherapy patients with
peptides. febrile neutropenia (see www.inimexpharma.
Five immunomodulatory cationic pep- com).
tides have already demonstrated safety in While IDR-1/IMX942 and RDP58 are
human clinical trials. Two of these, MX-226 both cationic peptides that modulate innate
and hLF111, were originally developed as immunity, they appear to do so via distinct
AMPs but also demonstrate immuno- mechanisms. Both peptides suppress the
modulatory activity (van der Does et al., induction of particular proinflammatory
2010). Topical delivery of MX-226 has cytokines, such as TNF-, but their eects on
demonstrated safety and ecacy in Phase II other cytokines vary. For example, RDP58
clinical trials that targeted both the inflam- does not aect IL-6 or -10 production, while
Clinical Applications of Host Defence Peptides 209
the treatment of cells with IDR-1 tends to in vivo, as isolated cell systems cannot
enhance the production of these two capture the complexity of the innate immune
cytokines. The distinct responses to these response. Thus, compared to testing of
peptides indicate the value of dierent antimicrobial activity, for which there is often
design strategies, since RDP58 is based upon a strong correlation between in vitro and in
HLA class I molecules while IDR-1 is a vivo activity, it is much more labour-intensive
derivative of a bovine cathelicidin. Import- and costly to generate the large datasets
antly, this also indicates that if we can necessary for applying computational
understand the intricacies of immuno- methods to immunomodulatory peptide
modulatory peptide interactions with host design.
cell components and pathways, it may be Despite this constraint, we now have a
possible to design peptides with customized few precedents where rational peptide
immunomodulatory eects to target dierent design based on structurefunction param-
diseases. eters has proven successful. For example,
synthetic endotoxin-binding peptides can be
optimized for protection in vivo (Dankesreiter
et al., 2000) and a fragment of LL-37 can
12.6 Rational Design of be optimized for anti-endotoxin activity
Immunomodulatory Peptides through appropriate amino acid substitutions
(Nagaoka et al., 2002). It is also possible to
The development and validation of screening create truncated peptides based on HDPs
techniques is an important aspect of the IDR such as LL-37 that retain either the parent
design process. High-throughput in vitro peptides antimicrobial or immuno-
screening of antibacterial activity has been modulatory properties (Bra et al., 2005;
used for the validation and iterative Chapter 9). Taken together, these findings
optimization of rationally designed AMPs demonstrate that particular domains are
(Cherkasov et al., 2009; Fjell et al., 2009). The necessary for these functions and suggest
technical simplicity of in vitro testing for that it will be possible to identify particular
direct antibacterial activity has made it characteristics associated with protection.
possible to collect large datasets using high- Other studies have optimized the activities
throughput screening techniques and use of smaller bactenecin- and indolicidin-like
this information to identify particular synthetic peptides as components of adjuvant
physical characteristics that correlate with formulations by screening for chemokine-
antimicrobial activity. By contrast, the inducing activity followed by in vivo analysis
complexity of the interactions between (Garlapati et al., 2009; Kindrachuk et al., 2009;
multiple cell types and eector molecules of Kovacs-Nolan et al., 2009a,c). An iterative
the immune system mean that it is not design process has also successfully
possible to thoroughly assess subtle immuno- produced new IDRs with greater anti-
modulatory activities in vitro, and simplified infective ecacy than IDR-1 (Nnik et al.,
markers of this activity must be used. For 2010).
example, IDR-1 was selected based on
markers such as synergistically enhanced
release of chemokines from LPS-stimulated 12.7 Limitations and Challenges
human peripheral blood mononuclear cells
(Scott et al., 2007) and chosen for further All drug candidates must overcome potential
analysis after demonstrating an ability to toxicities and questions of in vivo stability
protect mice against bacterial infections. and appropriate delivery routes. Clearly it is
Another possible screen would be the ability possible to design IDRs that protect without
to suppress TNF- induction in response to substantial in vitro or animal-model toxicity
treatment with TLR agonists (Mookherjee et (Scott et al., 2007; Nnik et al., 2010).
al., 2006). Even with the use of such markers, However, no systemic toxicology and
candidate peptides must ultimately be tested pharmacokinetic studies have been described
210 M.L. Mayer et al.
for any peptide and questions still remain proposed for enhancing the in vivo stability
regarding potential in vivo toxicities. This is and long-term shelf-life of peptides.
particularly important when considering the Additionally, the cost of manufacturing
transition from murine models to use in synthetic peptides is very high, so strategies
humans, considering the many dierences that enhance bioavailability or reduce the
between the rodent and human immune size or number of doses required for
systems and possible requirements for therapeutic benefit are also important for
multiple dosing. While small-animal models decreasing the cost of treatment.
provide very valuable information regarding One way of enhancing peptide stability
in vivo activity, dosing, appropriate for- is to induce chemical modifications that
mulation and routes of administration, the provide resistance to proteolytic digestion
intricate regulatory networks and feedback without altering the functional characteristics
loops that make up the mammalian immune of the peptide. For example, the use of
system are such that even small dierences d-amino acid isomers of peptides is one
between hosts can be magnified quite approach used for RDP58 (Table 12.2),
substantially. In cases where severe problems exploiting the specificity of proteases for
have occurred with immunomodulators l-amino acid forms of the peptide. Since the
(Ponce, 2008), few in vitro or ex vivo data three-dimensional structure of the peptides
involving human cells were available and the may be important for their interactions with
problems may have been predicted had such specific receptors, retro-inverse peptides
experiments been undertaken prior to human comprised of d-amino acids in the reversed
trials (Dayan and Wraith, 2008) or with better sequence have been investigated; this pre-
regulatory oversight (Gottlieb, 2008; Shuch- serves the spatial positions of the side chains
man, 2008). Several peptide-based drugs, and while maintaining the protease resistance of
many immunomodulatory drugs of varying the d-amino acid forms (Fischer, 2003).
compositions, have successfully passed Recently, a synthetic peptide, M33, was
clinical testing and entered the market, shown to resist proteolytic degradation when
demonstrating that it is possible to ensure constructed in a tetra-branched form (Pini et
the safety of such drugs. al., 2009). The peptide demonstrated anti-
The reduction of the in vivo stability of bacterial activity against a range of Gram-
peptides by proteolytic processing is also of negative species and protected against
concern. While small peptides are probably endotoxemia in mice. Conversely, creating a
degraded within minutes in many com- cyclic structure as for defensins may
partments of the host (e.g. blood, where enhance in vivo stability. The use of unnatural
proteases abound), results with IDR-1 amino acid side chains or modified peptide
indicate that treatments up to 48 h prior to or backbones (peptidomimetics) has substantial
6 h after bacterial challenge are protective in potential in this regard.
animal models (Scott et al., 2007). This raises A potential limitation of any novel anti-
the question of how a peptide that is rapidly infective is the development of microbial
degraded can have long-lasting eects and resistance. Thus, a very important char-
whether enhancing peptide stability is acteristic of IDRs is that there is a lower
desirable in this context. One possibility is likelihood of microorganisms developing
that the peptides prime immune cells; resistance to them than is the case with direct
indeed, their ability to readily translocate AMPs (Steinstraesser et al., 2009). For the
into immune cells (Lau et al., 2005) may AMPs, it has been argued that their physical
protect them from proteolytic degradation. action on membranes or their multiple
This is consistent with the observation that targets in a single cell make resistance
the peptides are active when delivered by unlikely. However, several studies have
many dierent routes, possibly indicating demonstrated resistance to AMPs mediated
that these locally primed cells (or cells by the dierential expression of a range of
containing peptide) migrate to the infection genes, often through blocking uptake
site. Regardless, several methods have been (McPhee et al., 2003; Kraus and Peschel, 2008;
Clinical Applications of Host Defence Peptides 211
Ernst et al., 2009; Majchrzykiewicz et al., 2010; antitumour activity, since some HDPs reduce
Warner and Levy, 2010). Nevertheless, IDR angiogenesis rather than promote it
peptides do not act directly on bacteria, but (Economopoulou et al., 2005; Krylov et al.,
rather through modulation of the host 2007), while others promote apoptosis of
immune system. Since the innate immune tumour cells or some have even demon-
system has co-evolved with pathogenic strated anti-proliferative activity (Suttmann
microbes, subtly augmenting this response et al., 2008). Current research is investigating
should not enhance the selective pressure on the feasibility of designing membrane-
bacterial immune evasion mechanisms. disrupting peptides that exploit dierences
Additionally, enhanced innate clearance of in membrane fluidity to permit them to be
microbes mediated by IDRs should avoid the cytotoxic to tumour cells but not to healthy
creation of bacterial debris that continues to cells (Mader and Hoskin, 2006; Fadnes et al.,
promote inflammation in the absence of live 2009). Indeed, many of the HDP-based
bacteria (Andra et al., 2006), providing yet immunotherapies in clinical trials are being
another advantage over traditional antibiotic explored as cancer therapies (Table 12.2).
therapies; indeed, some of these peptides Therefore, as with any novel immuno-
actually suppress such septic inflammatory modulatory drug, while it is certainly
responses. important to remain conscious of the
A potentially serious undesirable possibility of tumour promotion as a side
outcome of treatment with peptides based eect, with the appropriate testing and
upon LL-37 is the promotion of tumori- rational design it is possible to ensure that
genesis. As discussed previously, LL-37 has this scenario never becomes a reality.
demonstrated wound-healing activity, which
includes enhanced angiogenesis and
proliferation of keratinocytes and epithelial 12.8 Conclusions
cells. These functions alone suggest the
possibility of tumour promotion as a HDPs and their synthetic derivatives have
potential side eect of the inappropriate demonstrated significant potential as anti-
presence of LL-37 and such concerns are infective therapeutics and current research
compounded by the observation that LL-37 promises to reveal crucial mechanistic
can act as a growth factor for lung cancer information that should greatly enhance the
cells (von Haussen et al., 2008), promote a development of optimized peptides. These
metastatic phenotype in breast cancer cells peptides exert powerful actions in the human
(Weber et al., 2009) and influence the body, including modulation of innate
progression of ovarian tumours (Coelt et al., immunity, suppression of harmful inflam-
2009). However, the very nature of the mation and promotion of adaptive immunity,
altered characteristics of cancer cells means and roles in the enhancement of wound
that any immunomodulatory molecule could healing and resolution of the inflammatory
inadvertently become a growth factor for process. New insights and awareness about
such cells. It is not surprising that LL-37 structurefunction activities, cellular binding
could be co-opted in such a way, especially if partners and dynamic functional roles of
it is a ligand for CXCR2 considering that this these peptides will allow the rational design
receptor is involved in melanoma growth of synthetic peptides targeted for dierent
and invasion (Singh et al., 2009). However, purposes, not all of which will necessarily be
this limitation is not grounds to assume that anti-infective. The ability to subtly alter the
certain HDP-based treatments are inherently normal balance between protective innate
problematic. Rather, these concerns illustrate immune responses, such as chemokine
that tailored synthetic analogues with some, production and concomitant leukocyte
but not all, of the natural functions of recruitment, and the potentially harmful
particular HDPs should be considered the eects of excessive inflammation provides a
optimal design output. Indeed, it seems unique opportunity to promote eective
likely that one can design IDRs with innate immune defences against pathogens.
212 M.L. Mayer et al.
dynamics. Molecular Endocrinology 22, Corren, J., Casale, T.B., Lanier, B., Buhl, R.,
147155. Holgate, S. and Jimenez, P. (2009) Safety and
Cherkasov, A., Hilpert, K., Jenssen, H., Fjell, C.D., tolerability of omalizumab. Clinical and
Waldbrook, M., Mullaly, S.C., Volkmer, R. and Experimental Allergy 39, 788797.
Hancock, R.E. (2009) Use of artificial intelligence Dankesreiter, S., Hoess, A., Schneider-Mergener,
in the design of small peptide antibiotics effective J., Wagner, H. and Miethke, T. (2000) Synthetic
against a broad spectrum of highly antibiotic- endotoxin-binding peptides block endotoxin-
resistant superbugs. ACS Chemical Biology 4, triggered TNF- production by macrophages in
6574. vitro and in vivo and prevent endotoxin-mediated
Chorny, A. and Delgado, M. (2008) Neuropeptides toxic shock. Journal of Immunology 164,
rescue mice from lethal sepsis by down- 48044811.
regulating secretion of the late-acting Dayan, C.M. and Wraith, D.C. (2008) Preparing for
inflammatory mediator high mobility group box first-in-man studies: the challenges for trans-
1. American Journal of Pathology 172, 1297 lational immunology post-TGN1412. Clinical
1307. and Experimental Immunology 151, 231234.
Chorny, A., Aanderson, P., Gonzalez-Rey, E. and Delgado, M., Pozo, D. and Ganea, D. (2004) The
Delgado, M. (2008) Ghrelin protects against significance of vasoactive intestinal peptide in
experimental sepsis by inhibiting high-mobility immunomodulation. Pharmacological Reviews
group box 1 release and by killing bacteria. 56, 249290.
Journal of Immunology 180, 83698377. Del-Rio-Navarro, B.E., Espinosa Rosales, F.,
Cirioni, O., Giacometti, A., Ghiselli, R., Orlando, F., Flenady, V. and Sienra-Monge, J.J. (2006)
Kamysz, W., DAmato, G., Mocchegiani, F., Immunostimulants for preventing respiratory
Lukasiak, J., Silvestri, C., Saba, V. and Scalise, tract infection in children. Cochrane Database of
Systematic Reviews 4, CD004974.
G. (2004) Potential therapeutic role of histatin
Di Nardo, A., Vitiello, A. and Gallo, R.L. (2003)
derivative P-113d in experimental rat models of
Cutting edge: mast cell antimicrobial activity is
Pseudomonas aeruginosa sepsis. Journal of
mediated by expression of cathelicidin
Infectious Diseases 190, 356364.
antimicrobial peptide. Journal of Immunology
Coffelt, S.B., Marini, F.C., Watson, K., Zwezdaryk,
170, 22742278.
K.J., Dembinski, J.L. LaMarca, H.L., Tomchuck,
Doi, K., Hu, X., Leelahavanichkul, A., Yasuda, H.,
S.L., Honer zu Bentrup, K., Danka, E.S., Henkle,
Schnermann, J. and Nielsen, S. (2008) AP214,
S.L. and Scandurro, A.B. (2009) The pro-
an analogue of -melanocyte-stimulating
inflammatory peptide LL-37 promotes ovarian
hormone, ameliorates sepsis-induced acute
tumor progression through recruitment of
kidney injury and mortality. Kidney International
multipotent mesenchymal stromal cells.
73, 12661274.
Proceedings of the National Academy of
Dorschner, R.A., Pestonjamasp, V.K., Tamakuwala,
Science of the USA 106, 38063811.
S., Ohtake, T., Rudisill, J., Nizet, V., Agerberth,
Conlin, V.S., Wu, X., Nguyen, C., Dai, C., Vallance, B., Gudmundsson, G.H. and Gallo, R.L. (2001)
B., Buchan, M., Boyer, L. and Jacobson, K. Cutaneous injury induces the release of
(2009) Vasoactive intestinal peptide ameliorates cathelicidin anti-microbial peptides active
intestinal barrier disruption associated with against group A Streptococcus. Journal of
Citrobacter rodentium-induced colitis. American Investigative Dermatology 117, 9197.
Journal of Physiology Gastrointestinal and Dressel, S., Harder, J., Cordes, J., Wittersheim, M.,
Liver Physiology 297, G735G750. Meyer-Hoffert, U., Sunderkotter, C. and Glaser,
Cooper, A., Robinson, S.J., Pickard, C., Jackson, R. (2010) Differential expression of antimicrobial
C.L., Friedmann, P.S. and Healy, E. (2005) peptides in margins of chronic wounds.
-Melanocyte-stimulating hormone suppresses Experimental Dermatology 19, 628632.
antigen-induced lymphocyte proliferation in Durr, U.H., Sudheendra, U.S. and Ramamoorthy, A.
humans independently of melanocortin 1 (2006) LL-37, the only human member of the
receptor gene status. Journal of Immunology cathelicidin family of antimicrobial peptides.
175, 48064813. Biochimica et Biophysica Acta 1758,
Coron, E., Flamant, M., Aubert, P., Wedel, T., 14081425.
Pedron, T., Letessier, E., Galmiche, J.P., Economopoulou, M., Bdeir, K., Cines, D.B., Fogt,
Sansonetti, P.J. and Neunlist, M. (2009) F., Bdeir, Y., Lubkowski, J., Lu, W., Preissner,
Characterisation of early mucosal and neuronal K.T., Hammes, H.P. and Chavakis, T.M. (2005)
lesions following Shigella flexneri infection in Inhibition of pathologic retinal neovascularization
human colon. PLoS One 4, e4713. by -defensins. Blood 106, 38313838.
214 M.L. Mayer et al.
Eleuteri, E. and Di Stefano, A. (2009) Biomarkers in Ganea, D. (1996) Regulatory effects of vasoactive
heart failure. Minerva Cardioangiologica Nov intestinal peptide on cytokine production in
30 [epub ahead of print]. central and peripheral lymphoid organs.
Ernst, C.M., Staubitz, P., Mishra, N.N., Yang, S., Advances in Neuroimmunology 6, 6174.
Hornig, G., Kalbacher, H., Bayer, A.S., Kraus, D. Ganz, T. (2009) Iron in innate immunity: starve the
and Peschel, A. (2009) The bacterial defensin invaders. Current Opinion in Immunology 21,
resistance protein MprF consists of separable 6367.
domains for lipid lysinylation and antimicrobial Garlapati, S., Facci, M., Polewicz, M., Strom, S.,
peptide repulsion. PLoS Pathogens 5, Babiuk, L.A., Mutwiri, G., Hancock, R.E., Elliott,
e1000660. M.R. and Gerdts, V. (2009) Strategies to link
Fadnes, B., Rekdal, O. and Uhlin-Hansen, L. (2009) innate and adaptive immunity when designing
The anticancer activity of lytic peptides is vaccine adjuvants. Veterinary Immunology and
inhibited by heparan sulfate on the surface of Immunopathology 128, 184191.
the tumor cells. BMC Cancer 9, 183. Giacometti, A., Cirioni, O., Ghiselli, R., Mocchegiani,
Falco, A., Ortega-Villaizan, M., Chico, V., Brocal, I., F., Viticchi, C., Orlando, F., DAmato, G., Del
Perez, L., Coll, J.M. and Estepa, A. (2009) Prete, M.S., Kamysz, W., Lukasiak, J., Saba, V.
Antimicrobial peptides as model molecules for and Scalise, G. (2003) Antiendotoxin activity of
the development of novel antiviral agents in protegrin analog IB-367 alone or in combination
aquaculture. Mini Reviews in Medicinal with piperacillin in different animal models of
Chemistry 9, 11591164. septic shock. Peptides 24, 17471752.
Falla, T.J. and Zhang, L. (2010) Efficacy of Gottlieb, S. (2008) Biosimilars: policy, clinical, and
hexapeptide-7 on menopausal skin. Journal of regulatory considerations. American Journal of
Drugs in Dermatology 9, 4954. Health-System Pharmacy 65, S2S8.
Feldmann, M. and Steinman, L. (2005) Design of Hancock, R.E. and Sahl, H.G. (2006) Antimicrobial
effective immunotherapy for human auto-
and host-defense peptides as new anti-infective
immunity. Nature 435, 612619.
therapeutic strategies. Nature Biotechnology
Fellermann, K., Stange, D.E., Schaeffeler, E.,
24, 15511557.
Schmalzl, H., Wehkamp, J., Bevins, C.L.,
Harder, J., Dressel, S., Wittersheim, M., Cordes, J.,
Reinisch, W., Teml, A., Schwab, M., Lichter, P.,
Meyer-Hoffert, U., Mrowietz, U., Flster-Holst,
Radlwimmer, B. and Stange E.F. (2006) A
R., Proksch, E., Schrder, J.M., Schwarz, T. and
chromosome 8 gene-cluster polymorphism with
Glser, R. (2010) Enhanced expression and
low human -defensin 2 gene copy number
secretion of antimicrobial peptides in atopic
predisposes to Crohn disease of the colon.
dermatitis and after superficial skin injury.
American Journal of Human Genetics 79, 439
Journal of Investigative Dermatology 130,
448.
13551364.
Filewod, N.C., Pistolic, J. and Hancock, R.E. (2009)
Low concentrations of LL-37 alter IL-8 production Hirsch, T., Spielmann, M., Zuhaili, B., Fossum, M.,
by keratinocytes and bronchial epithelial cells in Metzig, M., Koehler, T., Steinau, H.U., Yao, F.,
response to proinflammatory stimuli. FEMS Onderdonk, A.B., Steinstraesser, L. and
Immunology and Medical Microbiology 56, 233 Eriksson, E. (2009) Human -defensin-3
240. promotes wound healing in infected diabetic
Fischer, P.M. (2003) The design, synthesis and wounds. Journal of Gene Medicine 11, 220
application of stereochemical and directional 228.
peptide isomers: a critical review. Current Holtmann, M.M. (2003) RDP-58 (SangStat Medical).
Protein and Peptide Science 4, 339356. IDrugs 6, 11881194.
Fjell, C.D., Jenssen, H., Hilpert, K., Cheung, W.A., Howell, M.D., Wollenberg, A., Gallo, R.L., Flaig, M.,
Pante, N., Hancock, R.E. and Cherkasov, A. Streib, J.E., Wong, C., Pavicic, T., Boguniewicz,
(2009) Identification of novel antibacterial M. and Leung, D.Y. (2006) Cathelicidin deficiency
peptides by chemoinformatics and machine predisposes to eczema herpeticum. Journal of
learning. Journal of Medicinal Chemistry 52, Allergy and Clinical Immunology 117, 836841.
20062015. Huang, C., Yuan, M., Huang, H., Wu, G., Liu, Y., Yu,
Gallo, S.A., Wang, W., Rawat, S.S., Jung, G., S., Li, H. and Wang, T. (2009) Ghrelin inhibits
Waring, A.J., Cole, A.M., Lu, H., Yan, X., Daly, post-infarct myocardial remodeling and improves
N.L., Craik, D.J, Jiang, S., Lehrer, R.I. and cardiac function through anti-inflammation
Blumenthal, R. (2006) -Defensins prevent effect. Peptides 30, 22862291.
HIV-1 Env-mediated fusion by binding gp41 and Huang, L.C., Reins, R.Y., Gallo, R.L. and McDermott,
blocking 6-helix bundle formation. Journal of A.M. (2007) Cathelicidin-deficient (Cnlp/) mice
Biological Chemistry 281, 1878718792. show increased susceptibility to Pseudomonas
Clinical Applications of Host Defence Peptides 215
aeruginosa keratitis. Investigative Ophthal- Kim, J.H., Lee, C., Yoon, H.I., Song, J., Shin, W.G.
mology and Vision Science 48, 44984508. and Lee, J.H. (2010) Relation of ghrelin, leptin
Hubert, P., Herman, L., Maillard, C., Caberg, J.H., and inflammatory markers to nutritional status
Nikkels, A., Pierard, G., Foidart, J.M, Noel, A., in active pulmonary tuberculosis. Clinical
Boniver, J. and Delvenne, P. (2007) Defensins Nutrition 29, 512518.
induce the recruitment of dendritic cells in Kindrachuk, J., Jenssen, H., Elliott, M., Townsend,
cervical human papillomavirus-associated (pre) R., Nijnik, A., Lee, S.F., Gerdts, V., Babiuk, L.A.,
neoplastic lesions formed in vitro and Halperin, S.A. and Hancock, R.E. (2009) A novel
transplanted in vivo. FASEB Journal 21, 2765 vaccine adjuvant comprised of a synthetic
2775. innate defence regulator peptide and CpG
Hurtado, P. and Peh, C.A. (2010) LL-37 promotes oligonucleotide links innate and adaptive
rapid sensing of CpG oligodeoxynucleotides by immunity. Vaccine 27, 46624471.
B lymphocytes and plasmacytoid dendritic cells. Kocsis, A.K., Lakatos, P.L., Somogyvri, F., Fuszek,
Journal of Immunology 184, 14251435. P., Papp, J., Fischer, S., Szamosi, T., Lakatos,
Iimura, M., Gallo, R.L., Hase, K., Miyamoto, Y., L., Kovacs, A., Hofner, P. and Mndi, Y. (2008)
Eckmann, L. and Kagnoff, M.F. (2005) Association of -defensin 1 single nucleotide
Cathelicidin mediates innate intestinal defense polymorphisms with Crohns disease. Scandin-
against colonization with epithelial adherent avian Journal of Gastroenterology 43, 299
bacterial pathogens. Journal of Immunology 307.
174, 49014907. Koczulla, R., von Degenfeld, G., Kupatt, C., Krtz,
Imatani, T., Kato, T., Minaguchi, K. and Okuda, K. F., Zahler, S., Gloe, T., Issbrcker, K.,
(2000) Histatin 5 inhibits inflammatory cytokine Unterberger, P., Zaiou, M., Lebherz, C., Karl, A.,
induction from human gingival fibroblasts by Raake, P., Pfosser, A., Boekstegers, P., Welsch,
U., Hiemstra, P.S., Vogelmeier, C., Gallo, R. L.,
Porphyromonas gingivalis. Oral Microbiology
Clauss, M. and Bals, R. (2003) An angiogenic
and Immunology 15, 378382.
role for the human peptide antibiotic LL-37/
Imatani, T., Kato, T., Okuda, K. and Yamashita, Y.
hCAP-18. Journal of Clinical Investigation 111,
(2004) Histatin 5 inhibits apoptosis in human
16651672.
gingival fibroblasts induced by Porphyromonas
Kodama, T., Ashitani, J., Matsumoto, N., Kangawa,
gingivalis cell-surface polysaccharide. European
K. and Nakazato, M. (2008) Ghrelin treatment
Journal of Medical Research 9, 528532.
suppresses neutrophil-dominant inflammation
Irwin, D.C., Tissot van Patot, M.C., Tucker, A. and
in airways of patients with chronic respiratory
Bowen, R. (2005) Direct ANP inhibition of
infection. Pulmonary Pharmacology and
hypoxia-induced inflammatory pathways in
Therapeutics 21, 774779.
pulmonary microvascular and macrovascular
Kollef, M., Pittet, D., Snchez-Garca, M., Chastre,
endothelial monolayers. American Journal of
J., Fagon, J.Y., Bonten, M., Hyzy, R., Fleming,
Physiology Lung Cellular and Molecular
T.R., Fuchs, H., Bellm, L., Mercat, A., Maez,
Physiology 288, L849L859. R., Martnez, A., Eggimann, P., Daguerre, M.,
Jacob, A., Rajan, D., Pathickal, B., Balouch, I., Luyt, C.E. and Prevention of Pneumonia Study
Hartman, A., Wu, R., Zhou, M. and Wang, P. (POPS-1) Trial Group (2006) A randomized
(2010) The inhibitory effect of ghrelin on sepsis- double-blind trial of iseganan in prevention of
induced inflammation is mediated by the MAPK ventilator-associated pneumonia. American
phosphatase-1. International Journal of Journal of Respiratory and Critical Care
Molecular Medicine 25, 159164. Medicine 173, 9197.
Kalus, A.A., Fredericks, L.P., Hacker, B.M., Kovacs-Nolan, J., Latimer, L., Landi, A., Jenssen,
Dommisch, H., Presland, R.B., Kimball, J.R. H., Hancock, R.E., Babiuk, L.A. and van Drunen
and Dale, B.A. (2009) Association of a genetic Littel-van den Hurk, S. (2009a) The novel
polymorphism (44 C/G SNP) in the human adjuvant combination of CpG ODN, indolicidin
DEFB1 gene with expression and inducibility of and polyphosphazene induces potent antibody-
multiple -defensins in gingival keratinocytes. and cell-mediated immune responses in mice.
BMC Oral Health 9, 21. Vaccine 27, 20552064.
Kandil, E., Burack, J., Sawas, A., Bibawy, H., Kovacs-Nolan, J., Mapletoft, J.W., Latimer, L.,
Schwartzman, A., Zenilman, M.E. and Bluth, Babiuk, L.A. and Hurk, S.D. (2009b) CpG
M.H. (2008) B-type natriuretic peptide: a oligonucleotide, host defense peptide and
biomarker for the diagnosis and risk stratification polyphosphazene act synergistically, inducing
of patients with septic shock. Archives of long-lasting, balanced immune responses in
Surgery 143, 242246. cattle. Vaccine 27, 20482054.
216 M.L. Mayer et al.
Kovacs-Nolan, J., Mapletoft, J.W., Lawman, Z., microbial Agents and Chemotherapy 54, 440
Babiuk, L.A. and van Drunen Littel-van den Hurk, 451.
S. (2009c) Formulation of bovine respiratory McPhee, J.B., Lewenza, S. and Hancock, R.E.
syncytial virus fusion protein with CpG (2003) Cationic antimicrobial peptides activate
oligodeoxynucleotide, cationic host defence a two-component regulatory system, PmrA
peptide and polyphosphazene enhances PmrB, that regulates resistance to polymyxin B
humoral and cellular responses and induces a and cationic antimicrobial peptides in
protective type 1 immune response in mice. Pseudomonas aeruginosa. Molecular Micro-
Journal of General Virology 90, 18921905. biology 50, 205217.
Kraus, D. and Peschel, A. (2008) Staphylococcus Miyoshi, M., Kitagawa, Y., Imoto, T. and Watanabe,
aureus evasion of innate antimicrobial defense. T. (2006) Effect of natriuretic peptide receptor
Future Microbiology 3, 437451. antagonist on lipopolysaccharide-induced fever
Krylov, A.V., Kisseleva, E.P., Aleshina, G.M., in rats: is natriuretic peptide an endogenous
Shamova, O.V. and Kokryakov, V.N. (2007) antipyretic? Journal of Pharmacology and
Effects of defensin and lactoferrin on functional Experimental Therapeutics 318, 11631170.
activity of endothelial cells in vitro. Bulletins in Mohapatra, S.S. (2007) Role of natriuretic peptide
Experimental Biological Medicine 144, 331 signaling in modulating asthma and inflam-
334. mation. Canadian Journal of Physiology and
Lau, Y.E., Rozek, A., Scott, M.G., Goosney, D.L., Pharmacology 85, 754759.
Davidson, D.J. and Hancock, R.E. (2005) Mookherjee, N., Brown, K.L., Bowdish, D.M., Doria,
Interaction and cellular localization of the human S., Falsafi, R., Hokamp, K., Roche, F.M., Mu, R.,
host defense peptide LL-37 with lung epithelial Doho, G.H., Pistolic, J., Powers, J.P., Bryan, J.,
cells. Infection and Immunity 73, 583591. Brinkman, F.S. and Hancock, R.E. (2006)
Lee, P.H., Rudisill, J.A., Lin, K.H., Zhang, L., Harris, Modulation of the TLR-mediated inflammatory
S.M., Falla, T.J. and Gallo, R.L. (2004) HB-107,
response by the endogenous human host
a nonbacteriostatic fragment of the antimicrobial
defense peptide LL-37. Journal of Immunology
peptide cecropin B, accelerates murine wound
176, 24552464.
repair. Wound Repair and Regeneration 12,
Mookherjee, N., Rehaume, L.M. and Hancock, R.E.
351358.
(2007) Cathelicidins and functional analogues
Lillard, J.W. Jr, Boyaka, P.N., Chertov, O.,
as antisepsis molecules. Expert Opinion in
Oppenheim, J.J. and McGhee, J.R. (1999)
Therapeutic Targets 11, 9931004.
Mechanisms for induction of acquired host
Mookherjee, N., Lippert, D.N., Hamill, P., Falsafi,
immunity by neutrophil peptide defensins. Pro-
R., Nijnik, A., Kindrachuk, J., Pistolic, J., Gardy,
ceedings of the National Academy of Science
J., Miri, P., Naseer, M., Foster, L.J, and Hancock,
USA 96, 651656.
R.E. (2009a) Intracellular receptor for human
Lingnau, K., Egyed, A., Schellack, C., Mattner, F.,
host defense peptide LL-37 in monocytes.
Buschle, M. and Schmidt, W. (2002) Poly-L-
arginine synergizes with oligodeoxynucleotides Journal of Immunology 183, 26882696.
containing CpG-motifs (CpG-ODN) for enhanced Mookherjee, N., Hamill, P., Gardy, J., Blimkie, D.,
and prolonged immune responses and prevents Falsafi, R., Chikatamarla, A., Arenillas, D.J.,
the CpG-ODN-induced systemic release of pro- Doria, S., Kollmann, T.R. and Hancock, R.E.
inflammatory cytokines. Vaccine 20, 3498 (2009b) Systems biology evaluation of immune
3508. responses induced by human host defence
Lv, B., Tang, Y., Chen, F. and Xiao, X. (2009) peptide LL-37 in mononuclear cells. Molecular
Vasoactive intestinal peptide and pituary Biosystems 5, 483496.
adenylate cyclase-activating polypeptide inhibit Murphy, C.J., Foster, B.A., Mannis, M.J., Selsted,
tissue factor expression in monocyte in vitro and M.E. and Reid, T.W. (1993) Defensins are
in vivo. Shock 31, 185191. mitogenic for epithelial cells and fibroblasts.
Mader, J.S. and Hoskin, D.W. (2006) Cationic Journal of Cellular Physiology 155, 408413.
antimicrobial peptides as novel cytotoxic agents Nagaoka, I., Hirota, S., Niyonsaba, F., Hirata, M.,
for cancer treatment. Expert Opinion in Adachi, Y., Tamura, H., Tanaka, S. and Heumann,
Investigational Drugs 15, 933946. D.(2002) Augmentation of the lipopolysaccharide-
Majchrzykiewicz, J.A., Kuipers, O.P. and Bijlsma, neutralizing activities of human cathelicidin
J.J. (2010) Generic and specific adaptive CAP18/LL-37-derived antimicrobial peptides by
responses of Streptococcus pneumoniae to replacement with hydrophobic and cationic
challenge with three distinct antimicrobial amino acid residues. Clinical and Diagnostic
peptides: bacitracin, LL-37 and nisin. Anti- Laboratory Immunology 9, 972982.
Clinical Applications of Host Defence Peptides 217
Nell, M.J., Tjabringa, G.S., Wafelman, A.R., Verrijk, human keratinocytes. British Journal of
R., Hiemstra, P.S., Drijfhout, J.W. and Grote, J.J. Dermatology 160, 243249.
(2006) Development of novel LL-37 derived Nizet, V., Ohtake, T., Lauth, X., Trowbridge, J.,
antimicrobial peptides with LPS and LTA Rudisill, J., Dorschner, R.A., Pestonjamasp, V.,
neutralizing and antimicrobial activities for Piraino, J., Huttner, K. and Gallo, R.L. (2001)
therapeutic application. Peptides 27, 649660. Innate antimicrobial peptide protects the skin
Nemeth, E. (2010) Targeting the hepcidin from invasive bacterial infection. Nature 414,
ferroportin axis in the diagnosis and treatment 454457.
of anemias. Advances in Hematology 2010, Oda, J., Kasai, K., Noborio, M., Aoki, Y., Yamashita,
750643. K., Inoue, T., Ueyama, M. and Yukioka, T. (2009)
Nemeth, E., Rivera, S., Gabayan, V., Keller, C., Effect of intravenous atrial natriuretic peptide on
Taudorf, S., Pedersen, B.K. and Ganz, T. (2004) pulmonary dysfunction and renal function
IL-6 mediates hypoferremia of inflammation by following burn shock. Journal of Trauma 66,
inducing the synthesis of the iron regulatory 12811285.
hormone hepcidin. Journal of Clinical Ong, P.Y., Ohtake, T., Brandt, C., Strickland, I.,
Investigation 113, 12711276. Boguniewicz, M., Ganz, T., Gallo, R.L. and
Nicholls, E.F., Madera, L. and Hancock, R.E. (2010) Leung, D.Y. (2002) Endogenous antimicrobial
Immunomodulators as adjuvants for vaccines peptides and skin infections in atopic dermatitis.
and antimicrobial therapy. Annals of the New New England Journal of Medicine 347, 1151
York Academy of Sciences (in press). 1160.
Nijnik, A., Pistolic, J., Wyatt, A., Tam, S. and Oppenheim, J.J. and Yang, D. (2005) Alarmins:
chemotactic activators of immune responses.
Hancock, R.E. (2009) Human cathelicidin
Current Opinion in Immunology 17, 359365.
peptide LL-37 modulates the effects of IFN- on
Otte, J.M., Werner, I., Brand, S., Chromik, A.M.,
APCs. Journal of Immunology 183, 57885798.
Schmitz, F., Kleine, M. and Schmidt, W.E. (2008)
Nijnik, A., Madera, L., Ma, S., Waldbrook, M., Elliott,
Human beta defensin 2 promotes intestinal
M.R., Easton, D.M., Mayer, M.L., Mullaly, S.C.,
wound healing in vitro. Journal of Cell
Kindrachuk, J., Jenssen, H. and Hancock, R.E.
Biochemistry 104, 22862297.
(2010) Synthetic cationic peptide IDR-1002
Otte, J.M., Zdebik, A.E., Brand, S., Chromik, A.M.,
provides protection against bacterial infections
Strauss, S., Schmitz, F., Steinstraesser, L. and
through chemokine induction and enhanced
Schmidt, W.E. (2009) Effects of the cathelicidin
leukocyte recruitment. Journal of Immunology
LL-37 on intestinal epithelial barrier integrity.
184, 25392550. Regulatory Peptides 156, 104117.
Nishimura, M., Abiko, Y., Kurashige, Y., Takeshima, Oudhoff, M.J., Bolscher, J.G., Nazmi, K., Kalay, H.,
M., Yamazaki, M., Kusano, K., Saitoh, M., vant Hof, W., Amerongen, A.V. and Veerman,
Nakashima, K., Inoue, T. and Kaku, T. (2004) E.C. (2008) Histatins are the major wound-
Effect of defensin peptides on eukaryotic cells: closure stimulating factors in human saliva as
primary epithelial cells, fibroblasts and identified in a cell culture assay. FASEB Journal
squamous cell carcinoma cell lines. Journal of 22, 38053812.
Dermatological Science 36, 8795. Overhage, J., Campisano, A., Bains, M., Torfs, E.C.,
Niyonsaba, F., Ushio, H., Nagaoka, I., Okumura, K. Rehm, B.H. and Hancock, R.E. (2008) Human
and Ogawa, H. (2005) The human -defensins host defense peptide LL-37 prevents bacterial
(-1, -2, -3, -4) and cathelicidin LL-37 induce biofilm formation. Infection and Immunity 76,
IL-18 secretion through p38 and ERK MAPK 41764182.
activation in primary human keratinocytes. Park, C.C., Morel, J.C., Amin, M.A., Connors, M.A.,
Journal of Immunology 175, 17761784. Harlow, L.A. and Koch, A.E. (2001) Evidence of
Niyonsaba, F., Ushio, H., Nakano, N., Ng, W., IL-18 as a novel angiogenic mediator. Journal of
Sayama, K., Hashimoto, K., Nagaoka, I., Immunology 167, 16441653.
Okumura, K. and Ogawa, H. (2007) Antimicrobial Pini, A., Falciani, C., Mantengoli, E., Bindi, S.,
peptides human -defensins stimulate epidermal Brunetti, J., Iozzi, S., Maria Rossolini, G. and
keratinocyte migration, proliferation and Bracci, L. (2009) A novel tetrabranched
production of proinflammatory cytokines and antimicrobial peptide that neutralizes bacterial
chemokines. Journal of Investigative lipopolysaccharide and prevents septic shock in
Dermatology 127, 594604. vivo. FASEB Journal 24, 10151022.
Niyonsaba, F., Suzuki, A., Ushio, H., Nagaoka, I., Ponce, R. (2008) Adverse consequences of
Ogawa, H. and Okumura, K. (2009) The human immunostimulation. Journal of Immunotoxicology
antimicrobial peptide dermcidin activates normal 5, 3341.
218 M.L. Mayer et al.
Potter, L.R., Yoder, A.R., Flora, D.R., Antos, L.K. Proceedings of the National Academy of
and Dickey, D.M. (2009) Natriuretic peptides: Sciences of the USA 102, 1445214457.
their structures, receptors, physiologic functions Scott, M.G., Dullaghan, E., Mookherjee, N., Glavas,
and therapeutic applications. Handbook of N., Waldbrook, M., Thompson, A., Wang, A.,
Experimental Pharmacology 341366. Lee, K., Doria, S., Hamill, P., Yu, J.J., Li, Y.,
Rivera, S., Nemeth, E., Gabayan, V., Lopez, M.A., Donini, O., Guarna, M.M., Finlay, B.B, North,
Farshidi, D. and Ganz, T. (2005) Synthetic J.R. and Hancock, R.E. (2007) An anti-infective
hepcidin causes rapid dose-dependent peptide that selectively modulates the innate
hypoferremia and is concentrated in ferroportin- immune response. Nature Biotechnology 25,
containing organs. Blood 106, 21962199. 465472.
Rodriguez-Garcia, M., Oliva, H., Climent, N., Sekino, M., Makita, T., Ureshino, H., Sungsam, C.
Escribese, M.M., Garcia, F., Moran, T.M., Gatell, and Sumikawa, K. (2010) Synthetic atrial
J.M. and Gallart, T. (2009) Impact of natriuretic peptide improves systemic and
-defensins13 on the maturation and splanchnic circulation and has a lung-protective
differentiation of human monocyte-derived DCs. effect during endotoxemia in pigs. Anesthesia
Concentration-dependent opposite dual effects. and Analgesia 110, 141147.
Clinical Immunology 131, 374384. Selsted, M.E. and Ouellette, A.J. (2005) Mammalian
Rose, R.A. (2010) CD-NP, a chimeric natriuretic defensins in the antimicrobial immune response.
peptide for the treatment of heart failure. Current Nature Immunology 6, 551557.
Opinion in Investigational Drugs 11, 349356. Shuchman, M. (2008) Clinical trials regulation
Rudiger, A., Gasser, S., Fischler, M., Hornemann, how Canada compares. Canadian Medical
T., von Eckardstein, A. and Maggiorini, M. (2006) Association Journal 179, 635638.
Comparable increase of B-type natriuretic Singh, S., Nannuru, K.C., Sadanandam, A., Varney,
peptide and amino-terminal pro-B-type M.L. and Singh, R.K. (2009) CXCR1 and CXCR2
natriuretic peptide levels in patients with severe
enhances human melanoma tumourigenesis,
sepsis, septic shock, and acute heart failure.
growth and invasion. British Journal of Cancer
Critical Care Medicine 34, 21402144.
100, 16381646.
Sarkar, A., Sreenivasan, Y. and Manna, S.K. (2003)
Sokolova, G.B., Sinitsyn, M.V., Kozhemiakin, L.A.
-Melanocyte-stimulating hormone induces cell
and Perelman, M.I. (2002) Glutoxim in the
death in mast cells: involvement of NF-B. FEBS
complex treatment of tuberculosis. Antibiotiki i
Letters 549, 8793.
Khimioterapiia 47, 2023.
Schaible, U.E. and Kaufmann, S.H. (2004) Iron and
Soruri, A., Grigat, J., Forssmann, U., Riggert, J.
microbial infection. Nature Reviews Microbiology
and Zwirner, J. (2007) -Defensins chemoattract
2, 946953.
macrophages and mast cells but not
Schaller-Bals, S., Schulze, A. and Bals, R. (2002)
lymphocytes and dendritic cells: CCR6 is not
Increased levels of antimicrobial peptides in
tracheal aspirates of newborn infants during involved. European Journal of Immunology 37,
infection. American Journal of Respiratory and 24742486.
Critical Care Medicine 165, 992995. Sow, F.B., Alvarez, G.R., Gross, R.P., Satoskar,
Schellack, C., Prinz, K., Egyed, A., Fritz, J.H., A.R., Schlesinger, L.S., Zwilling, B.S. and
Wittmann, B., Ginzler, M., Swatosch, G., Zauner, Lafuse, W.P. (2009) Role of STAT1, NF-B, and
W., Kast, C., Akira, S., von Gabain, A., Buschle, C/EBP in the macrophage transcriptional
M. and Lingnau, K. (2006) IC31, a novel adjuvant regulation of hepcidin by mycobacterial infection
signaling via TLR9, induces potent cellular and and IFN-gamma. Journal of Leukocyte Biology
humoral immune responses. Vaccine 24, 5461 86, 12471258.
5472. Steinstraesser, L., Ring, A., Bals, R., Steinau, H.U.
Scholzen, T.E., Sunderktten, C., Kalden, D.-H., and Langer, S. (2006) The human host defense
Brzoska, T., Fastrich, M., Fisbeck, T., Armstrong, peptide LL37/hCAP accelerates angiogenesis
C.A., Ansel, J.C. and Luger, T.A. (2003) in PEGT/PBT biopolymers. Annals of Plastic
-Melanocyte stimulating hormone prevents Surgery 56, 9398.
lipopolysaccharide-induced vasculitis by down- Steinstraesser, L., Koehler, T., Jacobsen, F.,
regulating endothelial cell adhesion molecule Daigeler, A., Goertz, O., Langer, S., Kesting, M.,
expression. Endocrinology 144, 360370. Steinau, H., Eriksson, E. and Hirsch, T. (2008)
Scotland, R.S., Cohen, M., Foster, P., Lovell, M., Host defense peptides in wound healing.
Mathur, A., Ahluwalia, A. and Hobbs, A.J. (2005) Molecular Medicine 14, 528537.
C-type natriuretic peptide inhibits leukocyte Steinstraesser, L., Kraneburg, U.M., Hirsch, T.,
recruitment and plateletleukocyte interactions Kesting, M., Steinau, H.U., Jacobsen, F. and
via suppression of P-selectin expression. Al-Benna, S. (2009) Host defense peptides as
Clinical Applications of Host Defence Peptides 219
effector molecules of the innate immune therapy for ulcerative colitis. Inflammatory Bowel
response: a sledgehammer for drug resistance? Disease 11, 713719.
International Journal of Molecular Science 10, van den Berg, H.R., Khan, N.A., van der Zee, M.,
39513970. Bonthuis, F., IJzermans, J.N., Dik, W.A., de
Suntharalingam, G., Perry, M.R., Ward, S., Brett, Bruin, R.W. and Benner, R. (2009) Synthetic
S.J., Castello-Cortes, A., Brunner, M.D. and oligopeptides related to the -subunit of human
Panoskaltsis, N. (2006) Cytokine storm in a chorionic gonadotropin attenuate inflammation
phase 1 trial of the anti-CD28 monoclonal and liver damage after (trauma) hemorrhagic
antibody TGN1412. New England Journal of shock and resuscitation. Shock 31, 285291.
Medicine 355, 10181028. van der Does, A.M., Bogaards, S.J., Ravensbergen,
Suttmann, H., Retz, M., Paulsen, F., Harder, J., B., Beekhuizen, H., van Dissel, J.T. and
Zwergel, U., Kamradt, J., Wullich, B., Unteregger, Nibbering, P.H. (2010) Antimicrobial peptide
G., Stckle, M. and Lehmann, J. (2008) hLF111 directs granulocytemacrophage
Antimicrobial peptides of the cecropin-family colony-stimulating factor-driven monocyte
show potent antitumor activity against bladder differentiation toward macrophages with
cancer cells. BMC Urology 8, 5. enhanced recognition and clearance of
Szliter, E.A., Lighvani, S., Barrett, R.P. and Hazlett, pathogens. Antimicrobial Agents and Chemo-
L.D. (2007) Vasoactive intestinal peptide therapy 54, 811816.
balances pro- and anti-inflammatory cytokines Vesely, D.L. and de Bold, A.J. (2009) Cardiac
in the Pseudomonas aeruginosa-infected natriuretic peptides gene expression and
cornea and protects against corneal perforation. secretion in inflammation. Journal of Investigative
Journal of Immunology 178, 11051114. Medicine 57, 2932.
Tai, E.K., Wu, W.K., Wong, H.P., Lam, E.K., Yu, L. Vila, G., Resl, M., Stelzeneder, D., Struck, J., Maier,
and Cho, C.H. (2007) A new role for cathelicidin C., Riedl, M., Hlsmann, M., Pacher, R., Luger,
in ulcerative colitis in mice. Experimental A. and Clodi, M. (2008) Plasma NT-proBNP
Biological Medicine 232, 799808. increases in response to LPS administration in
Tang, Y.Q., Yuan, J., Osapay, G., Osapay, K., Tran, healthy men. Journal of Applied Physiology 105,
D., Miller, C.J., Ouellette, A.J. and Selsted, M.E. 17411745.
(1999) A cyclic antimicrobial peptide produced von Haussen, J., Koczulla, R., Shaykhiev, R., Herr,
in primate leukocytes by the ligation of two C., Pinkenburg, O., Reimer, D., Wiewrodt, R.,
truncated -defensins. Science 286, 498502. Biesterfeld, S., Aigner, A., Czubayko, F. and
Tani, K., Murphy, W.J., Chertov, O., Salcedo, R., Bals, R. (2008) The host defence peptide LL-37/
Koh, C.Y., Utsunomiya, I., Funakoshi, S., Asai, hCAP-18 is a growth factor for lung cancer cells.
O., Herrmann, S.H., Wang, J.M., Kwak, L.W., Lung Cancer 59, 1223.
and Oppenheim, J.J. (2000) Defensins act as Vonk, M.J., Hiemstra, P.S. and Grote, J.J. (2008) An
potent adjuvants that promote cellular and antimicrobial peptide modulates epithelial
humoral immune responses in mice to a responses to bacterial products. The
lymphoma idiotype and carrier antigens. Laryngoscope 118, 816820.
International Immunology 12, 691700. Waldmann, T.A. (2003) Immunotherapy: past,
Tjabringa, G.S., Aarbiou, J., Ninaber, D.K., Drijfhout, present and future. Nature Medicine 9,
J.W., Sorensen, O.E., Borregaard, N., Rabe, 269277.
K.F. and Hiemstra, P.S. (2003) The antimicrobial Wang, L., Harrington, L., Trebicka, E., Shi, H.N.,
peptide LL-37 activates innate immunity at the Kagan, J.C., Hong, C.C., Lin, H.Y., Babitt, J.L.
airway epithelial surface by transactivation of and Cherayil, B.J. (2009) Selective modulation
the epidermal growth factor receptor. Journal of of TLR4-activated inflammatory responses by
Immunology 171, 66906696. altered iron homeostasis in mice. Journal of
Tomasinsig, L., Pizzirani, C., Skerlavaj, B., Clinical Investigation 119, 33223328.
Pellegatti, P., Gulinelli, S., Tossi, A., Di Virgilio, F. Wang, W., Bansal, S., Falk, S., Ljubanovic, D. and
and Zanetti, M. (2008) The human cathelicidin Schrier, R. (2009) Ghrelin protects mice against
LL-37 modulates the activities of the P2X7 endotoxemia-induced acute kidney injury.
receptor in a structure-dependent manner. American Journal of Physiology Renal
Journal of Biological Chemistry 283, Physiology 297, F1032F1037.
3047130481. Wang, X., Xu, W., Kong, X., Chen, D., Hellermann,
Travis, S., Yap, L.M., Hawkey, C., Warren, B., G., Ahlert, T.A., Giaimo, J.D., Cormier, S.A., Li,
Lazarov, M., Fong, T. and Tesi, R.J. (2005) X., Lockey, R.F., Mohapatra, S. and Mohapatra,
RDP58 is a novel and potentially effective oral S.S. (2009) Modulation of lung inflammation by
220 M.L. Mayer et al.
Note: page numbers in italic indicate tables, page numbers in bold indicate figures. For antimicrobial
peptides not listed, please visit the online database at http://aps.unmc.edu/AP/main.html.
221
222 Index