What is anthrax?

Anthrax is an infection caused by bacteria (a type of germ) called Bacillus anthracis (say:
buh-sil-us an-thray-sus). These bacteria make spores, a form of the germ covered by a
protective shell. The spores can live for years in the soil, and they cause anthrax when
they enter the body. Although the disease is most common in farm animals — like sheep,
cows, and goats — there's a small chance that people can get it as well, usually from
some type of contact with an animal or part of an animal that had anthrax.

Ref:http://kidshealth.org/kid/health_problems/infection/anthrax.html

Or

Anthrax is an acute disease caused by the bacterium Bacillus anthracis. Most forms of
the disease are lethal, and it affects both humans and other animals. There are effective
vaccines against anthrax, and some forms of the disease respond well to antibiotic
treatment.

Like many other members of the genus Bacillus, Bacillus anthracis can form dormant
endospores (often referred to as "spores" for short, but not to be confused with fungal
spores) that are able to survive in harsh conditions for decades or even centuries.[1] Such
spores can be found on all continents, even Antarctica.[2] When spores are inhaled,
ingested, or come into contact with a skin lesion on a host they may reactivate and
multiply rapidly.

Anthrax commonly infects wild and domesticated herbivorous mammals that ingest or
inhale the spores while grazing. Ingestion is thought to be the most common route by
which herbivores contract anthrax. Carnivores living in the same environment may
become infected by consuming infected animals. Diseased animals can spread anthrax to
humans, either by direct contact (e.g., inoculation of infected blood to broken skin) or by
consumption of a diseased animal's flesh.

Anthrax spores can be produced in vitro and used as a biological weapon. Anthrax does
not spread directly from one infected animal or person to another; it is spread by spores.
These spores can be transported by clothing or shoes. The body of an animal that had
active anthrax at the time of death can also be a source of anthrax spores.

Ref: http://en.wikipedia.org/wiki/Anthrax
organism responsible for production of anthrax
toxin
Bacterial Protein Toxins

© 2011 Kenneth Todar, PhD

Bacterial Toxigenesis

Toxigenesis, or the ability to produce toxins, is an underlying mechanism by which many bacterial pathogens
produce disease. At a chemical level, there are two main types of bacterial toxins, lipopolysaccharides, which
are associated with the cell wall of Gram-negative bacteria, and proteins, which are released from bacterial cells
and may act at tissue sites removed from the site of bacterial growth. The cell-associated toxins are referred to
as endotoxins and the extracellular diffusible toxins are referred to as exotoxins.

Endotoxins are cell-associated substances that are structural components of bacteria. Most endotoxins are
located in the cell envelope. In the context of this article, endotoxin refers specifically to the lipopolysaccharide
(LPS) or lipooligosaccharide (LOS) located in the outer membrane of Gram-negative bacteria. Although structural
components of cells, soluble endotoxins may be released from growing bacteria or from cells that are lysed as a
result of effective host defense mechanisms or by the activities of certain antibiotics. Endotoxins generally act in
the vicinity of bacterial growth or presence.

Exotoxins are usually secreted by bacteria and act at a site removed from bacterial growth. However, in some
cases, exotoxins are only released by lysis of the bacterial cell. Exotoxins are usually proteins, minimally
polypeptides, that act enzymatically or through direct action with host cells and stimulate a variety of host
responses. Most exotoxins act at tissue sites remote from the original point of bacterial invasion or growth.
However, some bacterial exotoxins act at the site of pathogen colonization and may play a role in invasion.

BACTERIAL PROTEIN TOXINS

Exotoxins are usually secreted by living bacteria during exponential growth. The production of the toxin is
generally specific to a particular bacterial species that produces the disease associated with the toxin (e.g. only
Clostridium tetani produces tetanus toxin; only Corynebacterium diphtheriae produces the diphtheria toxin).
Usually, virulent strains of the bacterium produce the toxin while nonvirulent strains do not, and the toxin is the
major determinant of virulence (e.g. tetanus and diphtheria). At one time, it was thought that exotoxin
production was limited mainly to Gram-positive bacteria, but clearly both Gram-positive and Gram-negative
bacteria produce soluble protein toxins.

Bacterial protein toxins are the most powerful human poisons known and retain high activity at very high
dilutions. The lethality of the most potent bacterial exotoxins is compared to the lethality of strychnine, snake
venom, and endotoxin in Table 1 below.

TABLE 1. LETHALITY OF BACTERIAL PROTEIN TOXINS

Toxin Toxic Dose Host Lethal compared

(mg) toxicity with:
Endotoxin Snake
Strychnine
(LPS) Venom
Botulinum
0.8x10-8 Mouse 3x106 3x107 3x105
toxin
Tetanus toxin 4x10-8 Mouse 1x106 1x107 1x105
Shiga toxin 2.3x10-6 Rabbit 1x106 1x107 1x105
Diphtheria Guinea
6x10-5 2x103 2x104 2x102
toxin pig
Usually the site of damage caused by an exotoxin indicates the location for activity of that toxin. Terms such as
enterotoxin, neurotoxin, leukocidin or hemolysin are descriptive terms that indicate the target site of some
well-defined protein toxins. A few bacterial toxins that obviously bring about the death of an animal are known
simply as lethal toxins, and even though the tissues affected and the target site or substrate may be known,
the precise mechanism by which death occurs is not clear (e.g. anthrax LF).
Some bacterial toxins are utilized as invasins because they act locally to promote bacterial invasion. Examples
are extracellular enzymes that degrade tissue matrices or fibrin, allowing the bacteria to spread. This includes
collagenase, hyaluronidase and streptokinase. Other toxins, also considered invasins, degrade membrane
components, such as phospholipases and lecithinases. The pore-forming toxins that insert a pore into eucaryotic
membranes are considered as invasins, as well, but they will be reviewed here.

Some protein toxins have very specific cytotoxic activity (i.e., they attack specific types of cells). For example,
tetanus and botulinum toxins attack only neurons. But some toxins (as produced by staphylococci, streptococci,
clostridia, etc.) have fairly broad cytotoxic activity and cause nonspecific death of various types of cells or
damage to tissues, eventually resulting in necrosis. Toxins that are phospholipases act in this way. This is also
true of pore-forming hemolysins and leukocidins.

Bacterial protein toxins are strongly antigenic. In vivo, specific antibody neutralizes the toxicity of these
bacterial exotoxins (antitoxin). However, in vitro, specific antitoxin may not fully inhibit their activity. This
suggests that the antigenic determinant of the toxin may be distinct from the active portion of the protein
molecule. The degree of neutralization of the active site may depend on the distance from the antigenic site on
the molecule. However, since the toxin is fully neutralized in vivo, this suggests that other host factors must play
a role in toxin neutralization in nature.

Protein exotoxins are inherently unstable. In time they lose their toxic properties but retain their antigenic ones.
This was first discovered by Ehrlich who coined the term "toxoid" for this product. Toxoids are detoxified toxins
which retain their antigenicity and their immunizing capacity. The formation of toxoids can be accelerated by
treating toxins with a variety of reagents including formalin, iodine, pepsin, ascorbic acid, ketones, etc. The
mixture is maintained at 37 degrees at pH range 6 to 9 for several weeks. The resulting toxoids can be used for
artificial immunization against diseases caused by pathogens where the primary determinant of bacterial
virulence is toxin production. Toxoids are effective immunizing agents against diphtheria and tetanus that are
part of the DPT (DTP) vaccine.

Toxins with Enzymatic Activity

As proteins, many bacterial toxins resemble enzymes in a number of ways. Like enzymes, they are denatured
by heat, acid and proteolytic enzymes, they act catalytically, and they exhibit specificity of action. The
substrate (in the host) may be a component of tissue cells, organs or body fluid.

A plus B Subunit Arrangement

Many protein toxins, notably those that act intracellularly (with regard to host cells), consist of two components:
one component (subunit A) is responsible for the enzymatic activity of the toxin; the other component
(subunit B) is concerned with binding to a specific receptor on the host cell membrane and transferring the
enzyme across the membrane. The enzymatic component is not active until it is released from the native (A+B)
toxin. Isolated A subunits are enzymatically active but lack binding and cell entry capability. Isolated B subunits
may bind to target cells (and even block the binding of the native toxin), but they are nontoxic.

There are a variety of ways that toxin subunits may be synthesized and arranged: A + B indicates that the toxin
is synthesized and secreted as two separate protein subunits that interact at the target cell surface; A-B or A-5B
indicates that the A and B subunits are synthesized separately, but associated by noncovalent bonds during
secretion and binding to their target; 5B indicates that the binding domain of the protein is composed of 5
identical subunits. A/B denotes a toxin synthesized as a single polypeptide, divided into A and B domains that
may be separated by proteolytic cleavage.

Attachment and Entry of Toxins

There are at least two mechanisms of toxin entry into target cells.

In one mechanism called direct entry, the B subunit of the native (A+B) toxin binds to a specific receptor on the
target cell and induces the formation of a pore in the membrane through which the A subunit is transferred into
the cell cytoplasm.

In an alternative mechanism, the native toxin binds to the target cell and the A+B structure is taken into the cell
by the process of receptor-mediated endocytosis (RME). The toxin is internalized in the cell in a membrane-
enclosed vesicle called an endosome. H+ ions enter the endosome lowering the internal pH which causes the
A+B subunits to separate. The B subunit affects the release of the A subunit from the endosome so that it will
reach its target in the cell cytoplasm. The B subunit remains in the endosome and is recycled to the cell surface.
In both cases above, a large protein molecule must insert into and cross a membrane lipid bilayer, either the cell
membrane or the endosome membrane. This activity is reflected in the ability of most A+B or A/B toxins, or their
B components, to insert into artificial lipid bilayers, creating ion permeable pathways. If the B subunit contains a
hydrophobic region (of amino acids) that insert into the membrane (as in the case of the diphtheria toxin), it may
be referred to as the T (translocation) domain of the toxin.

A few bacterial toxins (e.g. diphtheria) are known to utilize both direct entry and RME to enter into host cells,
which is not surprising since both mechanisms are variations on a theme. Bacterial toxins with similar enzymatic
mechanisms may enter their target cells by different mechanisms. Thus, the diphtheria toxin and Pseudomonas
exotoxin A, which have identical mechanisms of enzymatic activity, enter their host cells in slightly different
ways. The adenylate cyclase toxin of Bordetella pertussis (pertussis AC) and anthrax EF produced by Bacillus
anthracis, act similarly to catalyze the production of cAMP from host cell intracellular ATP reserves. However, the
anthrax toxin enters cells by receptor mediated endocytosis, whereas the pertussis adenylate cyclase traverses
the cell membrane directly.

The specific receptors for the B subunit of toxins on target cells or tissues are usually sialogangliosides
(glycoproteins) called G-proteins on the cell membrane. For example, the cholera toxin utilizes the ganglioside
GM1, and tetanus toxin utilizes ganglioside GT1 and/or GD1b as receptors on host cells.

Diphtheria Toxin

The best known and studied bacterial toxin is the diphtheria toxin, produced by Corynebacterium diphtheriae.
Diphtheria toxin is a bacterial exotoxin of the A/B prototype. It is produced as single polypeptide chain with a
molecular weight of 60,000 daltons. The function of the protein is distinguishable into two parts: subunit A, with
a m.w. of 21,000 daltons, contains the enzymatic activity for inhibition of elongation factor-2 involved in host
protein synthesis; subunit B, with a m.w. of 39,000 daltons, is responsible for binding to the membrane of a
susceptible host cell. The B subunit possesses a region T (translocation) domain which inserts into the endosome
membrane thus securing the release of the enzymatic component into the cytoplasm.

Figure 1. Diphtheria Toxin (Dtx). A (red) is the catalytic domain; B (yellow) is the binding domain
which displays the receptor for cell attachment; T (blue) is the hydrophobic domain responsible for
insertion into the endosome membrane to secure the release of A. The protein is illustrated in its
"closed" configuration.

In vitro, the native toxin is produced in an inactive form which can be activated by the proteolytic enzyme trypsin
in the presence of thiol (reducing agent). The enzymatic activity of Fragment A is masked in the intact toxin.
Fragment B is required to enable to enable Fragment A to reach the cytoplasm of susceptible cells. The C
terminal end of Fragment B is hydrophilic and contains determinants that interact with specific membrane
receptors on sensitive cell membranes and the N-terminal end of Fragment B (called the T domain) is strongly
hydrophobic. The specific membrane receptor for the B fragment has been shown to be a transmembranous
heparin-binding protein on the susceptible cell's surface.

The diphtheria toxin enters its target cells by either direct entry or receptor mediated endocytosis. The first step
is the irreversible binding of the C-terminal hydrophilic portion of Fragment B (AA 432-535) to the receptor.
During RME, the whole toxin is then taken up in an endocytic vesicle. In the vesicle, the pH drops to about 5
which allows unfolding of the A and B chains. This exposes hydrophobic regions of both the A and B chains that
can insert into the vesicle membrane. The result is exposure of the A chain to the cytoplasmic side of the
membrane. There, reduction and proteolytic cleavage releases the A chain in the cytoplasm. The A fragment is
released as an extended chain but regains its active (enzymatic) globular conformation in the cytoplasm. The A
chain catalyzes the ADP ribosylation of elongation factor-2 (EF-2) as shown in Figure 2.
Figure 2. Entry and activity of diphtheria toxin (Dtx) in susceptible cells. The B domain of the toxin
binds to a cognate receptor on a susceptible cell. The toxin is taken up in an endosome by receptor
mediated encocytosis. Acidification of the endocytic vesicle allows unfolding of the A and B chains
exposing the hydrophobic T domain of the toxin. The T domain inserts into the endosome membrane
translocating the A fragment into the cytoplasm where it regains its enzymatic configuration. The
enzymatic A component utilizes NAD as a substrate. It catalyzes the attachment of the ADP-ribose
portion of NAD to elongation factor (EF-2) which inactivates its function in protein synthesis.

Table 2 describes several bacterial toxins with known enzymatic activity and the biological effects of the toxins in
humans.

TABLE 2. BIOLOGICAL EFFECTS OF SOME BACTERIAL EXOTOXINS WITH ENZYMATIC ACTIVITY

TOXIN (subunit
ENZYMATIC ACTIVITY BIOLOGICAL EFFECTS
arr)*
Activates adenylate cyclase;
ADP ribosylates eucaryotic increased level of intracellular
Cholera toxin (A-
adenylate cyclase Gs cAMP promote secretion of fluid
5B)
regulatory protein and electrolytes in intestinal
epithelium leading to diarrhea

Diphtheria toxin ADP ribosylates elongation Inhibits protein synthesis in

(A/B) factor 2 animal cells resulting in death of
the cells

Blocks inhibition of adenylate

Pertussis toxin (A- ADP ribosylates adenylate
cyclase; increased levels of
5B) cyclase Gi regulatory protein
cAMP affect hormone activity
and reduce phagocytic activity
E. coli heat-labile ADP ribosylates adenylate Similar or identical to cholera
toxin LT (A-5B) cyclase Gs regulatory protein toxin

Inactivates the mammalian 60S

ribosomal subunit and leads to
Glycosidase cleavage of
inhibition of protein synthesis
ribosomal RNA (cleaves a
Shiga toxin (A/5B and death of the susceptible cells;
single Adenine base from the
pathology is diarrhea,
28S rRNA)
hemorrhagic colitis (HC) and/or
hemolytic uremic syndrome
(HUS)
ADP ribosylates elongation
Pseudomonas Inhibits protein synthesis in
factor-2 analogous to
Exotoxin A (A/B) susceptible cells, resulting in
diphtheria toxin
death of the cells

Zn++ dependent protease acts Inhibits presynaptic

Botulinum toxin
on synaptobrevin at motor acetylycholine release from
(A/B)
neuron ganglioside peripheral cholinergic neurons
resulting in flaccid paralysis
Zn++ dependent protease acts
Inhibits neurotransmitter release
Tetanus toxin (A/B) on synaptobrevin in central
from inhibitory neurons in the
nervous system
CNS resulting in spastic paralysis
Anthrax toxin LF Metallo protease that cleaves
(A2+B) MAPKK (mitogen-activated Combined with the B subunit
protein kinase kinase) (PA), LF induces cytokine
 release and death of target cells
enzymes
or experimental animals

Calmodulin-regulated
adenylate cyclases that Increases cAMP in phagocytes
Bordetella pertussis
catalyze the formation of leading to inhibition of
AC toxin (A/B) and
cyclic AMP from ATP in phagocytosis by neutrophils and
Bacillus anthracis
susceptible cells, as well as macrophages; also causes
EF (A1+B)
the formation of ion- hemolysis and leukolysis
permeable pores in cell
membranes
Cleaves desmoglein 1, a
Separation of the stratum
Staphylococcus cadherin found in
granulosum of the epidermis,
aureus Exfoliatin B desmosomes in the epidermis
between the living layers and the
(also a superantigen)
superficial dead layers.
* toxin subunit arrangements: A-B or A-5B indicates subunits synthesized separately and associated by
noncovalent bonds; A/B denotes subunit domains of a single protein that may be separated by proteolytic
cleavage; A+B indicates subunits synthesized and secreted as separate protein subunits that interact at the
target cell surface; 5B indicates that the binding domain is composed of 5 identical subunits.

Pore-forming Toxins

Pore-forming toxins, as the name suggests, insert a transmembranous pore into a host cell membrane, thereby
disrupting the selective influx and efflux of ions across the membrane. This group of toxins includes the RTX
toxins of Gram-negative bacteria, streptolysin O produced by S. pyogenes, and S. aureus alpha toxin. Generally,
these toxins are produced as subunits that self-assemble as a pore on the eucaryotic membrane.

S. aureus alpha-toxin is considered the model of oligomerizing pore-forming cytotoxins. The alpha-toxin is
synthesized as a 319 amino acid precursor molecule that contains an N-terminal signal sequence of 26 amino
acids. The secreted mature toxin, or protomer, is a hydrophilic molecule with a molecular weight of 33 kDa.
Seven toxin protomers assemble to form a 232 kDa mushroom-shaped heptamer comprising three distinct
domains. The cap and rim domains of the heptamer are situated at the surface of the plasma membrane, while
the stem domain serves as a transmembranous ion channel through the membrane.

TABLE 3. SOME PORE-FORMING BACTERIAL TOXINS

Toxin Bacterial source Target Disease

perfringiolysin O Clostridium cholesterol gas gangrene
perfringens
hemolysin Escherichia coli cell membrane UTI
listeriolysin Listeria cholesterol systemic; meningitis
monocytogenes
anthrax EF Bacillus anthracis cell membrane anthrax (edema)
alpha toxin Staphylococcus aureus cell membrane abcesses
pneumolysin Streptococcus cholesterol pneumonia; otitis
pneumoniae media
streptolysin O Streptococcus cholesterol strep throat
pyogenes
leukocidin Staphylococcus aureus phagocyte pyogenic infections
membrane

Superantigens: Toxins that Stimulate the Immune System

Several bacterial toxins can act directly on the T cells and antigen-presenting cells of the immune system.
Impairment of the immunologic functions of these cells by toxin can lead to human disease. One large family of
toxins in this category are the so-called pyrogenic exotoxins produced by staphylococci and streptococci,
whose biological activities include potent stimulation of the immune system, pyrogenicity, and enhancement of
endotoxin shock.

Pyrogenic exotoxins are secreted toxins of 22 kDa to 30 kDa, and include staphylococcal enterotoxins serotypes
A-E, G, and H; group A streptococcal pyrogenic exotoxins A-C; staphylococcal exfoliatin toxin; and staphylococcal
TSST-1.

In general, the potent immunostimulatory properties of superantigens are a direct result of toxin binding to
distinct regions outside the peptide binding cleft of the major histocompatibility class II molecules (MHC II),
expressed on the surface of antigen-presenting cells, and to specific Vß elements on the T-cell receptor of T-
lymphocytes. This results in a massive proliferation of up to 20% of peripheral T cells. Concomitant to T-cell
proliferation is a massive release of cytokines from lymphocytes (e.g. interleukin-2, tumor necrosis factor ß,
gamma interferon) and monocytes (e.g. IL-1, IL-6, tumor necrosis factor a). These cytokines serve as mediators
of the hypotension, high fever, and diffuse erythematous rash that are characteristic of toxic-shock syndrome.

The staphylococcal enterotoxins are superantigens, but it is not known if this activity contributes to vomiting or
diarrhea characteristic of staphylococcal food poisoning.

Control of Synthesis and the Release of Protein Toxins

The regulation of synthesis and secretion of many bacterial toxins is tightly controlled by regulatory elements
that are sensitive to environmental signals. For example, the production of diphtheria toxin is totally repressed by
the availability of adequate amounts of iron in the medium for bacterial growth. Only under conditions of limiting
amounts of iron in the growth medium does toxin production become derepressed. The expression of cholera
toxin and related virulence factors (adhesins) is controlled by environmental osmolarity and temperature. In B.
pertussis, induction of different virulence components is staggered, such that attachment factors are produced
initially to establish the infection, and toxins are synthesized and released later to counter the host defenses and
promote bacterial survival.

The processes by which protein toxins are assembled and secreted by bacterial cells are also variable. Many of
the classic exotoxins are synthesized with an NH terminal leader (signal) sequence consisting of a few (1-3)
charged amino acids and a stretch of (14-20) hydrophobic amino acids. The signal sequence may bind and insert
into the cytoplasmic membrane during translation such that the polypeptide is secreted while being synthesized.
The signal peptide is cleaved as the toxin (protein) is released into the periplasm. Alternatively, the toxin may be
synthesized intracytoplasmically, then bound to a leader sequence for passage across the membrane. Frequently,
chaperone proteins are required to guide this process. Some multicomponent toxins, such as the cholera toxin,
have their subunits synthesized and secreted separately into the periplasm where they are assembled. In Gram-
negative bacteria, the outer membrane poses an additional permeability barrier that a protein toxin usually has
to mediate if it is to be released in a soluble form. It has been proposed that some Gram-negative exotoxins (e.g.
E. coli ST enterotoxin) might be released in membrane vesicles composed of outer membrane components. Since
these vesicles possibly possess outer membrane-associated attachment factors, they could act as "smart bombs"
capable of specifically interacting with and possibly entering target cells to release their contents of toxin.

Other considerations

The genetic ability to produce a toxin, including regulatory genes, may be found on the bacxterial chromosome,
plasmids and lysogenic bacteriophages. Sometimes they occur within pathogenicity islands. In any case, the
processes of genetic exchange in bacteria, notably conjugation and transduction, can mobilize genetic
elements between strains and species of bacteria. Horizontal gene transfer (HGT) of genes that encode
virulence is known to occur between species of bacteria. This explains how E. coli and Vibrio cholerae produce a
nearly identical diarrhea-inducing toxin, as well as how E. coli O157:H7 acquired ability to produce shiga toxin
form Shigella dysenteriae. The intestinal tract is probably an ideal habitat for bacteria to undergo HGT with one
another.

There is conclusive evidence for the pathogenic role of diphtheria, tetanus and botulinum toxins, various
enterotoxins, staphylococcal toxic shock syndrome toxin, and streptococcal pyrogenic exotoxins. And there is
good evidence for the pathological involvement of pertussis toxin, anthrax toxin, shiga toxin and the necrotizing
toxins of clostridia, in bacterial disease. But why certain bacteria produce such potent toxins is mysterious and is
analogous to asking why an organism should produce an antibiotic. The production of a toxin may play a role in
adapting a bacterium to a particular niche, but it is not essential to the viability of the organism. Most toxigenic
bacteria are free-living in nature and in associations with humans in a form which is phenotypically identical to
the toxigenic strain but lacking the ability to produce the toxin.
A summary of bacterial protein toxins and their activities is given in Tables 4. Details of the mechanisms of action
of these toxins and their involvement in the pathogenesis of disease is discussed in chapters with the specific
bacterial pathogens.

For more information and references on bacterial toxins go to this website: Bacterial Toxins: Friends or Foes?

TABLE 4. SUMMARY: ACTIVITIES OF EXTRACELLULAR BACTERIAL TOXINS

NAME OF BACTERIA
ACTIVITY
TOXIN INVOLVED
An adenylate cyclase enzyme that increases
levels in intracellular cyclic AMP in
Anthrax toxin (EF) Bacillus anthracis phagocytes and formation of ion-permeable
pores in cell membrane. Leads to edema and
decreased phagocytic responses

Acts locally to increase levels of cyclic

Adenylate cyclase Bordetella
AMP in phagocytes and formation of ion-
toxin (pertussis AC) pertussis
permeable pores in cell membranes

Alpha toxin Staphylococcus Protein subunits assemble into an

aureus oligomeric structure that forms an ion
channel (pore) in the cell plasma membrane

ADP ribosylation of G proteins stimulates

Cholera enterotoxin
Vibrio cholerae adenlyate cyclase and increases cAMP in
(Ctx)
cells of the GI tract, causing secretion of
water and electrolytes leading to diarrhea

E. coli LT toxin Escherichia coli Similar to cholera toxin

Binding of the heat-stable enterotoxins (ST)

to a guanylate cyclase receptor results in an
E. coli ST toxins Escherichia coli increase in cyclic GMP (cGMP) that
adversely effects electrolyte flux. Promotes
secretion of water and electrolytes from
intestinal epithelium leading to diarrhea.

Enzymatically cleaves eucaryotic 28S rRNA

Shigella resulting in inhibition of protein synthesis in
Shiga toxin
dysenteriae susceptible cells. Results in diarrhea,
E. coli O157:H7 hemorrhagic colitis (HC) and hemolytic
uremic syndrome (HUS)

Perfringens Stimulates adenylate cyclase leading to

Clostridium
enterotoxin increased cAMP in epithelial cells. Result is
perfringens
diarrhea

Modifies Rho, a subfamily of small GTP-

ToxinA/ToxinB Clostridium binding proteins that are regulators of the
difficile actin cytoskeleton. Deamidation of the
glutamine residue at position 63 of Rho to a
glutamic acid produces a dominant active
Rho protein unable to hydrolyze bound
GTP. Pathological result is cell necrosis and
 bloody diarrhea associated with colitis

Zn++ dependent protease that inhibits

Botulinum toxin Clostridium
neurotransmission at neuromuscular
botulinum
synapses resulting in flaccid paralysis

Zn++ dependent protease that Inhibits

Tetanus toxin Clostridium tetani
neurotransmission at inhibitory synapses
resulting in spastic paralysis

Diphtheria toxin ADP ribosylation of elongation factor 2

Corynebacterium
(Dtx) leads to inhibition of protein synthesis in
diphtheriae
target cells
Inhibits protein synthesis; similar to
Exotoxin A Pseudomonas
diphtheria toxin
aeruginosa

Lethal Factor (LF) is a Zn++ dependent

Anthrax toxin (LF) Bacillus anthracis protease that induces cytokine release and is
cytotoxic to cells by an unknown
mechanism

ADP ribosylation of G proteins blocks

Pertussis toxin (Ptx) Bordetella
inhibition of adenylate cyclase in
pertussis
susceptible cells

Exfoliatin toxin* Staphylococcus Cleavage within epidermal cells

aureus (intraepidermal separation); also acts as a
superantigen

Superantigen causes massive activation of

Staphylococcus Staphylococcus
the immune system, including lymphocytes
enterotoxins* aureus
and macrophages; exact role in in emesis
not not known

Toxic shock
Staphylococcus Superantigen acts on the vascular system
syndrome toxin
aureus causing inflammation, fever and shock
(TSST-1)*
Super antigen same as TSST -
Erythrogenic toxin
Streptococcus inflammation, fever and shock; can cause
[streptococcal
pyogenes localized erythematous reactions (scarlet
pyrogenic exotoxin
fever)
(SPE)]*

Ref: http://www.textbookofbacteriology.net/proteintoxins.html

virulence factor of anthrax

Bacillus anthracis virulence factors
Anthrax is a disease caused by Bacillus anthracis, a spore-forming, Gram positive, rod-
shaped bacterium (Fig. 1). The lethality of the disease owes itself to the bacterium's two
principal virulence factors: (i) the polyglutamic acid capsule, which is anti-phagocytic,
and (ii) the tripartite protein toxin, called anthrax toxin. Anthrax toxin is a mixture of
three protein components: (i) protective antigen (PA), (ii) edema factor (EF), and (iii)
lethal factor (LF).
Ref: http://en.wikipedia.org/wiki/Anthrax_toxin

Anthrax: Virulence and Vaccines:

1. W. Edmund Farrar, MD

+ Author Affiliations

1. Medical University of South Carolina; Charleston, SC 29425

Anthrax is an infectious disease that has afflicted humans and their domestic livestock
since ancient times. Although in many industrialized countries the disease is controlled
by vaccination and good practices in rearing livestock, it remains a serious problem in
many less developed regions of the world.

The causative organism, Bacillus anthracis, is a spore-forming, rod-shaped bacterium

that inhabits the soil. Although the causal relation between the organism and anthrax has
been known for more than a century, since the time of Koch and Pasteur, the specific
factors responsible for the virulence of the organism have been identified and
characterized during the last 30 years. The precise molecular basis for virulence has been
elucidated only during the past decade.

Fully virulent strains of Bacillus anthracis possess two unique virulence factors: a poly-
D-glutamic acid capsule that inhibits phagocytosis [1] and a tripartite toxin composed of
protective antigen, edema factor, and lethal factor [2]. Capsules are produced by virulent
strains of Bacillus anthracis growing in vivo and by cells grown on media containing
serum or bicarbonate or both and incubated in a CO2-enriched atmosphere.

The existence of an anthrax toxin was first demonstrated in 1955 in experiments that
showed that injection of sterile plasma from infected guinea pigs resulted in local edema
and death. Studies by American and British investigators during the ensuing decade
showed that the toxin contained three separate components. The individual toxin
components have no known biological effects when administered alone, but edema factor
injected with protective antigen into the skin of rabbits or guinea pigs causes local edema,
and protective antigen injected with lethal factor into rats causes death in as little as 60
minutes. Protective antigen, so called because its injection into experimental animals
results in protective immunity, binds to cell-surface receptors to produce an uptake
system that can be used by both the edema factor and lethal factor to gain access to the
cytoplasm. “Edema toxin” (edema factor and protective antigen) and “lethal toxin” (lethal
factor and protective antigen) thus resemble the A-B enzyme-binding structures
characteristic of many well-studied bacterial toxins. After protective antigen, which is
analogous to the B chain, binds to a specific membrane receptor on the surface of a
eukaryotic cell, it is cleaved at a single site, exposing a binding site for the other toxin
component. The membrane-bound fragment of the protective antigen then binds to the
edema factor or lethal factor and mediates the entry of the active moiety into the cell.

The edema factor has been found to be a calmodulin-dependent adenylate cyclase that
elevates cyclic adenosine monophosphate (AMP) levels approximately 200-fold greater
than normal in Chinese hamster ovary cells. Local edema, a typical sign of anthrax, may
be directly related to adenylate cyclase activity associated with the edema factor. The
increase in intracellular cyclic AMP caused by this toxin may lead to edema in a manner
analogous to the loss of water into the intestinal lumen caused by cholera toxin, which
also increases intracellular cyclic AMP [3]. Edema factor and the protective antigen also
inhibit phagocytosis of anthrax bacilli by polymorphonuclear leukocytes, and this effect
may further increase host susceptibility to anthrax. The dependence of edema factor
activity on calmodulin, a substance found only in eukaryotic cells, suggests that the
edema factor did not evolve from a bacterial enzyme but from a eukaryotic adenylate
cyclase, the gene for which was adventitiously transferred into Bacillus anthracis and
retained because it made the bacteria more virulent [3].

The mechanism of action of lethal factor is poorly understood, but it is lethal for many
species of experimental animals and is assumed to be the major factor causing death in
anthrax. No enzymatic activity has yet been associated with the lethal factor, and the
nature of its intracellular target is unknown.

Virulent strains of Bacillus anthracis contain two large plasmids, pX01 and pX02 [1, 4,
5]. Both plasmids are required for full pathogenicity, and strains that contain only one of
these plasmids are avirulent. The plasmid pX01 encodes all three components of the
anthrax toxin, and pX02 encodes the poly-D-glutamic acid capsule. These facts provide
the basis for effective vaccines, the first of which was developed by Pasteur in 1881 and
used in a brilliantly successful field trial in sheep at the village of Pouilly-le-Fort. The
avirulent Sterne vaccine strain, which is pX01+/pX02-, produces toxin but no capsule and
is used effectively as a live veterinary vaccine [6]. The nonencapsulated strain used in
production of the acellular vaccine licensed for human use in the United States produces
primarily protective antigen [6]. The heat-attenuated Pasteur vaccine strains form
capsules but cannot produce toxin. Pasteur probably cured his strains of plasmid pX01 by
heat attenuation to produce his vaccine for immunization of cows and sheep. Although
the Pasteur-type vaccine provides a much lower level of protective immunity than the
toxigenic vaccine strains [7], it was still good enough to save all the sheep on that fateful
day at Pouilly-le-Fort. (The modest immunogenicity of Pasteur's vaccine may well have
been caused by the persistence of a small proportion of pX01+/pX02+ cells, which thus
remained toxigenic and fully virulent.)
The genes encoding the capsule and each of the three toxin components have been cloned
in Escherichia coli [8-11], and the base sequences have been determined [12-14]. The
protective antigen gene has been cloned in Bacillus subtilis, and immunization with the
live recombinant strain protects guinea pigs from lethal challenge with virulent Bacillus
anthracis spores [15].

None of the currently available anthrax vaccines is ideal, and efforts to develop better
vaccines, with the use of newer molecular methods, represent an active area of research
[16, 17]. Although the Sterne vaccine is effective and safe for use in many domestic
animals (cattle, sheep, pigs, camels, buffaloes, and elephants), progressive disease caused
by the vaccine strain has been observed in goats and llamas. The duration of protection
conferred by the Sterne vaccine is somewhat limited, and the necessity for administration
by injection is a disadvantage in many developing countries. The vaccine used in humans
in the United States produces high titers of antibody to protective antigen in guinea pigs,
but only animals vaccinated with the Sterne strain are completely protected against
challenge with highly virulent strains of Bacillus anthracis.

Several different approaches are being used in the development of improved anthrax
vaccines for humans. Purified protective antigen vaccines are being combined with
adjuvants derived from the cell wall of the BCG strain of the tubercle bacillus [18] or
with killed cells of Bordetella pertussis [19] to enhance the cellular immune response to
the protective antigen. The Bacillus subtilis strain into which the protective antigen gene
has been cloned, mentioned above [15], is a recombinant vaccine that does not contain
the Bacillus anthracis genome. Transposon-induced mutagenesis has been used to
produce mutant vaccine strains that cannot synthesize essential aromatic amino acids
unavailable from the mammalian host and that thus cannot replicate for more than a few
cycles in the mammalian host [16, 20]. Oral vaccines might consist of immunizing
antigens from Bacillus anthracis cloned into appropriate bacterial vectors such as
Salmonella species [17].

Anthrax has been known in humans and their animals for more than 7000 years, and the
association between Bacillus anthracis and its hosts probably existed for millennia before
it was recognized and recorded. In many countries, vaccination of animals and humans
and improved methods of rearing livestock have effectively controlled the disease.
Recent advances in molecular biology have allowed the elucidation of the precise
mechanisms of virulence in Bacillus anthracis and give promise of even more effective
vaccines. But it is difficult to imagine that the vast reservoir of Bacillus anthracis in the
soil can ever be eradicated. The anthrax bacillus will surely endure; its place on our
planet seems at least as secure as our own.

• Copyright ©2004 by the American College of Physicians

Previous Section

References
1. 1.↵

Green BD, Battisti L, Koehler TM, Thorne CB, Ivins BE. Demonstration of a
capsule plasmid in Bacillus anthracis. Infect Immun. 1985; 49:291-7.

2. 2.↵

Leppla SH. Production and purification of anthrax toxin. Methods Enzymol.

1988; 165:103-16.

3. 3.↵

Leppla SH. Bacillus anthracis calmodulin-dependent adenylate cyclase: chemical

and enzymatic properties and interactions with eucaryotic cells. Adv Cyclic
Nucleotide Protein Phosphorylation Res. 1984; 17:189-98.

4. 4.↵

Mikesell P, Ivins BE, Ristroph JD, Dreier TM. Evidence for plasmid-mediated
toxin production in Bacillus anthracis. Infect Immun. 1983; 39:371-6.

5. 5.↵

Kaspar RL, Robertson DL. Purification and physical analysis of Bacillus

anthracis plasmids pX01 and pX02. Biochem Biophys Res Commun. 1987;
149:362-8.

6. 6.↵

Hambleton P, Carman JA, Melling J. Anthrax: the disease in relation to

vaccines. Vaccine. 1984; 2:125-32.

7. 7.↵

Ivins BE, Ezzell JW Jr, Jemski J, Hedlund KW, Ristroph JD, Leppla SH.
Immunization studies with attenuated strains of Bacillus anthracis. Infect Immun.
1986; 52:454-8.

8. 8.↵

Uchida I, Makino S, Sasakawa C, Terakado N, Yoshikawa M. Cloning of the

genetic region for encapsulation of Bacillus anthracis. Salisbury Medical
Bulletin. Special Supplement. 1990; 68:62.

9. 9.↵
 Vodkin MH, Leppla SH. Cloning of the protective antigen gene of Bacillus
anthracis. Cell. 1983; 34:693-7.

10. 10.↵

Mock M, Labruyere E, Glaser P, Danchin A, Ullmann A. Cloning and

expression of the calmodulin-sensitive Bacillus anthracis adenylate cyclase in
Escherichia coli. Gene. 1988; 64:277-84.

11. 11.↵

Robertson DL, Leppla SH. Molecular cloning and expression in Escherichia

coli of the lethal factor gene of Bacillus anthracis. Gene. 1986; 44:71-8.

12. 12.↵

Welkos SL, Lowe JR, Eden-McCutchan F, Vodkin M, Leppla SH, Schmidt

JJ. Sequence and analysis of the DNA encoding protective antigen of Bacillus
anthracis. Gene. 1988; 69:287-300.

13. 13.↵

Robertson DL, Tippetts MT, Leppla SH. Nucleotide sequence of the Bacillus
anthracis edema factor gene (cya): a calmodulin-dependent adenylate cyclase.
Gene. 1988; 73:363-71.

14. 14.↵

Robertson DL, Bragg TS. Nucleotide sequence of the lethal factor (lef) and
edema factor (cya) genes from Bacillus anthracis: elucidation of the EF and LF
functional domains. Salisbury Medical Bulletin. Special Supplement. 1990; 68:59.

15. 15.↵

Ivins BE, Welkos SL. Cloning and expression of the Bacillus anthracis
protective antigen gene in Bacillus subtilis. Infect Immun. 1986; 545:537-42.

16. 16.↵

Ivins BE, Welkos SL, Little SF, Knudson GB. Cloned protective activity and
progress in development of improved anthrax vaccines. Salisbury Medical
Bulletin. Special Supplement. 1990; 68:86-8.

17. 17.↵

Turnbull PC. Anthrax vaccines: past, present and future. Vaccine. 1991; 9:533-9.
 18. 18.↵

Ivins BE, Welkos SL, Little SF, Crumrine MH, Nelson GO. Immunization
against anthrax with Bacillus anthracis protective antigen combined with
adjuvants. Infect Immun. 1992; 60:662-8.

19. 19.↵

Turnbull PC, Quinn CP, Hewson R, Stockbridge MC, Melling J. Protection

conferred by microbially-supplemented UK and purified PA vaccines. Salisbury
Medical Bulletin. Special Supplement. 1990; 68:89-91.

20. 20.↵

Ivins BE, Welkos SL, Knudson GB, LeBlanc DJ. Transposon Tn916
mutagenesis in Bacillus anthracis. Infect Immun. 1988; 56:176-81.

Ref: http://www.annals.org/content/121/5/379.full

type of anthrax vaccine:

VACCINE TYPE:

Anthrax Vaccine (AVA, BIOTHRAX ), killed vaccine made from the cell-free filtrate of
nonencapsulated, attenuated strain of B. anthracis culture that contains no dead or live
bacteria (note: a live attenuated vaccine was manufactured in the former USSR, but no
longer available).

INDICATIONS
ACIP RECOMMENDATIONS

• Routine vaccination with anthrax vaccine is indicated for persons engaged a) in

work involving production quantities or concentrations of B. anthracis cultures
and b) in activities with a high potential for aerosol production. Laboratory
personnel using standard Biosafety Level 2 practices in the routine processing of
clinical samples are not at increased risk for exposure to B. anthracis spores.
• Routine vaccination of veterinarians in the U.S. is not recommended because of
the low incidence of animal cases. Vaccination may be considered in high-risk
persons handling potentially infected animals in areas with a high incidence of
anthrax cases.
• Routine vaccination is not recommended for bioterrorism preparedness (e.g., first
responders, federal responders, medical practitioners, and private citizens), but
The Working Group on Civilian Biodefense does recommend vaccination of
 exposed persons following a biological attack in conjunction with abx
administration for 60 days following exposure.
• ACIP recommends that If antimicrobial prophylaxis is administered in
combination with post-exposure vaccination, it is prudent to continue antibiotics
until 7-14 days after the third vaccine dose.

OTHER INFORMATION

• Post-exposure prophylaxis: AVA given as a sole agent was not effective in one
primate study. AVA was effective if abx with activity against anthrax was given
concurrently. The duration of post-exposure antimicrobial prophylaxis should be
60 days if used alone for PEP of unvaccinated exposed persons.
• Department of Defense has mandated all US military active- and reserve-duty
personnel receive pre-exposure (vaccine) prophylaxis as an adjunct to prolonged
post-exposure antibiotic prophylaxis.
• Pre-exposure vaccination of some persons deemed to be in high-risk groups
should also be considered.

FORMS
brand
preparation manufacturer route form dosage^ cost*
name
Anthrax Vaccine Bioport Corp, Lansing, 10 doses/ $900 per
BioThrax SQ Vial
Adsorbed Michigan. vial vial

*Prices represent cost per unit specified, are representative of "Average Wholesale Price"
(AWP).
^Dosage is indicated in mg unless otherwise noted.

PATHOGEN DIRECTED PROTECTION

• B. anthracis

DOSE/ADMINISTRATION
PRIMARY SERIES

Dose (pre-exposure prophylaxis): 0.5 ml SQ x 6 doses (0, 2 and 4 wks followed by

injections at 6, 12 and 18 mos). Six dose regimen used, because this regimen won FDA
approval during registration trials in the 1950’s (Brachman).

BOOSTER

Revaccination: yearly booster dose (0.5ml) required to maintain immunity.

ADVERSE DRUG REACTIONS
GENERAL

• No long-term sequelae reported

• Generally well tolerated

COMMON

• Injection site nodule: most frequently reported local reaction and more common
in women for unexplained reasons (60% vs 30% in men).
• About 4% develop extensive erythema and swelling that may extend to the
antecubital fossa --often misdiagnosed as bacterial cellulitis.

OCCASIONAL

• Headache (0.4%)
• Myalgia or arthralgia
• Headache
• Fatigue

RARE

• Anaphylaxis

VACCINE/DRUG INTERACTIONS
• No known drug interaction

CONTRAINDICATIONS
• History of an anaphylactic reaction to the vaccine. Previous anthrax infection (re:
more severe adverse events among recipients with a vaccine history of anthrax).

IMMUNE RESPONSE
Relationship between immunity and quantitative antibody levels has not been evaluated.
Onset of protection: antibody titer increased 3-4x approximately 7d after the second dose
(3-4 wks from first dose), however clear minimum therapeutic antibody response has not
been established, but appears to be sufficient to prevent development of disease once
antibiotics were discontinued. An estimated 83% of human vaccinees develop a vaccine-
induced immune response after two doses of the vaccine and >95% develop a four fold
rise in antibody titer after three doses.
CLINICAL EFFICACY
• Effective in a placebo-controlled human trial against cutaneous anthrax. Primate
models showed that antibiotic prevented inhalation anthrax, but were not
protected from rechallenge.
• However, all animals given vaccine PLUS antibiotic were protected with re-
challenge[Friedlander, 1993].

OTHER INFORMATION
• Vaccine indicated only for risk of inhalation anthrax, but prevents cutaneous
anthrax as well. Until ample reserve stockpiles of vaccine are available, reliance
must be placed upon abx protection.
• The risk for persons who come in contact in the workplace with imported animal
hides, furs, bone meal, wool, animal hair, or bristles has been reduced by changes
in industry standards and import restrictions. Routine preexposure vaccination is
recommended only for persons in this group for whom these standards and
restrictions are insufficient to prevent exposure to anthrax spores.
• Safety in Pregnancy: Category D. Earlier unpublished study of infants born to
women in the U.S. military service worldwide in 1998 and 1999 suggest that the
vaccine may be linked with an increase in the number of birth defects. However, a
published study found no effect on pregnancy or adverse birth outcomes in a
cohort involving 4092 women[Wiesen, 2002].
• Potentially exposed persons should be observed for signs of febrile illness .

Basis for recommendation

• Centers for Disease Control and Prevention (CDC): Use of anthrax vaccine in
response to terrorism: supplemental recommendations of the Advisory Committee
on Immunization Practices. MMWR Morb Mortal Wkly Rep
51:1024, 2002 Nov. 15 [PMID:12458919]

Comment: ACIP recommendations

Rating: Basis for recommendation

• Advisory Committee on Immunization Practices: Use of anthrax vaccine in the

United States. MMWR Recomm Rep 49:1, 2000 Dec. 15 [PMID:11145529]

Comment: ACIP recommendations

Rating: Basis for recommendation

References
 1. Wiesen AR, Littell CT: Relationship between prepregnancy anthrax vaccination
and pregnancy and birth outcomes among US Army women. JAMA
287:1556, 2002 March 27 [PMID:11911758]
2. Friedlander AM et al: Postexposure prophylaxis against experimental inhalation
anthrax. J Infect Dis 167:1239, 1993 [PMID:8486963]

Ref:http://www.hopkinsguides.com/hopkins/ub/view/Johns_Hopkins_ABX
_Guide/540030/all/Anthrax_vaccine

mechanism of production of anthrax

toxin:
Anthrax Toxin Mechanism of Action

One of the key causes of anthrax virulence is the production of three specific factors
by the gram-positive spore forming bacteria Bacillus anthracis. Even with successful
antibiotic treatment, anthrax toxins can remain in the circulation and cause
lethality. The toxins produced by anthrax bacteria are derived from three genes:
lethal factor (LF), protective antigen (PA) and edema factor (EF). The entry of toxin
into cells begins with the recognition of a recently identified cellular receptor in the
plasma membrane by PA. Proteolytic cleavage of cell-bound PA creates a smaller
fragment that then multimerizes into a pore-like structure in the plasma
membrane. The LF and EF proteins bind to the PA pre-pore, followed by
internalization of the entire structure through receptor-mediated endocytosis. In the
endosomal compartment, the acidic pH causes a conformational change that inserts
PA fragments and releases LF and EF into the cytoplasm. In the cytoplasm, LF acts
as a protease that cleaves MAP kinase kinase (MAPKK 1 and MAPKK 2), inhibiting
pathways that rely on this kinase family and causing cell death. Edema factor is an
adenylate cyclase that inhibits the immune response, including phagocytosis by
macrophages. Several potential mechanisms could be used to block anthrax toxin
action, one of which was demonstrated by the design of a multivalent protein
inhibitor of toxin interaction with PA.

Glenn Croston, PhD

 Beauregard, K.E., Collier, R.J., Swanson, J.A. Proteolytic activation of receptor-
bound anthrax protective antigen on macrophages promotes its internalization. Cell.
Microbiol. 2000, 2(3), 251-8

Bradley, KA. et al. Identification of the cellular receptor for anthrax toxin. Nature
2001, 414(6860), 225-9

Duesbery, N.S. et al. Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal

factor. Science 1998, 280(5364), 734-7

Leppla, S.H. Anthrax toxin edema factor: a bacterial adenylate cyclase that
increases cyclic AMP concentrations of eukaryotic cells. Proc. Natl. Acad. Sci. U. S.
A. 1982, 79(10), 3162-6

Mourez, M. et al. Designing a polyvalent inhibitor of anthrax toxin. Nat. Biotechnol.

2001, (10), 958-61

Ref: http://www.biocarta.com/pathfiles/h_anthraxPathway.asp

Control of toxin production in Bacillus anthracis :

Willem van Schaik

The bacterium Bacillus anthracis causes the disease anthrax. This disease primarily
affects herbivores in tropical regions of the world. Humans can also contract anthrax,
either by accidental exposure to B. anthracis or as a consequence of bioterrorist activities.

The most important factors that determine the capacity to cause disease of B. anthracis
are the anthrax toxin and a capsule. By producing these so-called virulence factors B.
anthracis can evade the immune system of the host, thereby allowing the proliferation of
the bacteria in the body.
This study aimed to elucidate the mechanisms which govern the production of the
virulence factors in B. anthracis. In general, production of virulence factors in bacteria is
tightly controlled as it is a waste of valuable energy to produce virulence factors under
conditions when they are not needed, for example outside the host’s body. In B.
anthracis it was already known that the anthrax toxin and capsule are maximally
produced at 37°C and in the presence of carbon dioxide. Both these conditions occur in
mammalian hosts. Previous research by a number of groups has shown that a protein,
called AtxA, controls the production of both anthrax toxin and capsule.
In this study we have found that another protein, CodY, controls the production of B.
anthracis toxins but not of capsule. The CodY protein was found to  respond to the
presence of several metabolites in the bacterial cell. This indicates that CodY is an
important link between the metabolic state of the cell and the production of virulence
factors by B. anthracis. We also found that CodY acts independent of AtxA, thereby
creating an additional level of complexity in the regulation of the production of virulence
factors in B. anthracis. All these findings broaden our knowledge of B. anthracis and
may provide clues for the development of novel treatment strategies against anthrax. Â
Anthrax

A Reamerging Fear
Anthrax became a household word when letters
intentionally laced with a white powder were mailed to
senators and journalists last year. U.S. government
officials now realize a large-scale anthrax attack could
become a reality and scientists race to find new ways to Bacillus anthracis: the
treat the disease. What makes anthrax such an ideal rod-shaped
biological weapon? How do the bacterial spores invade the bacterium that
host? And why is anthrax toxin so deadly? causes anthrax
(stained black)
Despite its recent infamy, in current times anthrax is
and spores
actually a relatively rare disease–especially in humans.
Anthrax is the disease caused by the rod-shaped bacterium, (colored red).
Bacillus anthracis. Spores of B. anthracis occur naturally in the soil where they can lay
dormant for decades. Waiting for a grazing animal–such as a cow, horse, or sheep–to
accidentally ingest it. Once inside the animal, the spore germinates, switches to its
virulent form, and produces the anthrax toxin, which eventually kills the host.
Historically, people only became infected after working with infected livestock products
or eating contaminated meat.

History
Anthrax is a disease that has plagued man and his livestock for centuries, causing it to be
one of the most well-studied and understood diseases. The name anthrax is derived from
the Greek word for coal, anthracis, because of one of the symptoms of infection is the
formation of black ulcers on the skin. Over one
hundred years ago B. anthracis became the first
bacterium to be shown to cause disease when Robert
Koch injected live B. anthracis culture into laboratory
animals. Shortly after being injected with the
bacterium, the animals showed symptoms of anthrax
infection and later died.

Using anthrax as a biological weapon is not a new

concept. During the second half of the 20th century
several governments investigated using the anthrax
bacterium as a biological weapon, including the
United States and the former Soviet Union. Of more This black skin ulcer
typically seen in
anthrax skin
infections.
recent concern is the belief that several well-organized extremist groups are developing
anthrax to wage large-scale biological warfare. This is no surprise to scientists who work
on the microbe. B. anthracis spores are virtually indestructible, remaining viable after
being exposed to extreme temperatures and ultra-violet radiation. The spores are also
light, odorless, and colorless, and can easily infect a large population quickly and without
being detected. In short, anthrax makes the perfect deadly biological weapon.

Silent Killer
There are three ways to acquire anthrax. The most common and least serious is called
cutaneous (or skin) anthrax and occurs when B. anthracis spores make contact with
abrased skin. This form of the disease results in the formation of black ulcers on the skin
and is usually treatable with antibiotics. The skin form of anthrax can turn deadly if the
microbes invade the blood stream and the infection becomes systemic. Anthrax can also
be acquired by ingesting improperly cooked meat from an infected animal. This type of
infection is extremely rare, especially in North America and Europe, but results in a
gastrointestinal infection that is often lethal even after antibiotic treatment. The third and
most deadly type of infection is inhalation anthrax. Infection occurs when 10,000 to
50,000 spores are inhaled into the lungs. Once in the lungs, the spores germinate, form
active bacteria, and migrate to the lymph nodes where the bacteria get inside
macrophages–the host’s immune system cells that normally seek and destroy bacteria.
Later, the bacteria multiply and convert into a virulent form that produces the anthrax
toxin. Anthrax toxin causes internal bleeding and destroys the body’s immune system,
eventually causing septic shock and killing the host.

It is possible to be exposed to the B. anthracis microbe and never

develop an anthrax infection. Last year, several government
Inhalation workers tested positive for exposure to the microbe but never
anthrax acquired the disease. Unfortunately anthrax is difficult to diagnose
is during the initial stages of the flu-like infection when antibiotics
deadliest such as Cipro are most effective in treating the disease. Because
form current therapies are not potent enough to be effective at later
stages of infection is important to understand the anthrax toxin on
the molecular level. Knowing how anthrax toxin works to evade
and destroy the host’s immune system cells has enabled researchers to develop novel,
more potent therapeutics.

Infection on the Molecular Level

After the anthracis spores germinate
inside the host, becoming active bacteria,
they switch from their avirulent (non-
toxic) to virulent (toxic) form. The
virulent form of the bacteria expresses
new proteins that were previously not
expressed, resulting in the production of The anthrax bacterium converts
a polypeptide coat and the anthrax toxin. from its rough avirulent
These virulence factors help the bacteria (right) form to its virulent
to evade the host’s immune system and smooth form (left). The smooth
eventually destroy it. appearance is due to a
polypeptide coat which helps
The polypeptide coat protects the
microbes from being ingested by the the bacterium evade the host’s
host’s bactericidal macrophages. This immune system.
coat is composed entirely of poly-D-
glutamate and serves to hide the bacteria’s proteins, which the host’s immune system
normally recognizes and attacks. In this way the bacteria can exist without being detected
or destroyed by macrophages. The B. anthracis cells look rough in their avirulent state
but adopt a smooth appearance once they surround themselves with this protective
polypeptide coat.

Death from virulent B. anthracis is due to the production of a toxin that shuts down the
host’s immune system and causes cell death. This deadly toxin is called anthrax toxin.
The anthrax toxin has three components, all of which are fairly large, soluble proteins.
Two of the components, edema factor (EF) and lethal factor (LF), cause either swelling
of the cells (edema) or cell lysis (death) within the host’s cells. The third component,
protective antigen (PA), binds to the host cell receptor and facilitates delivery of EF and
LF into the cell. Separately these components are non-toxic, but PA combined with either
EF or LF forms either edema toxin or lethal toxin, respectively.

Toxin Cellular effects

Compon
ents
PA non-toxic
EF non-toxic
LF non-toxic
EF + LF non-toxic
PA + EF form edema toxin and cause cell swelling
PA + LF form lethal toxin and cause cell lysis (death)
The mechanism of anthrax toxin entry into the cell has been thoroughly studied. In 2001,
it was discovered that the protective antigen (PA) binds a receptor called the anthrax
toxin receptor (ATR) present on the mammalian cell membrane. Once bound to the ATR,
a host protease protease cleaves off the N-terminus of PA, inducing a switch to its
activated form. The activated PA binds to six other activated PAs, forming a heptamer on
the surface of the mammalian cell. This heptameric complex then binds either EF or LF.
The mammalian cell draws in the PA/EF (or LF) complex through endocytosis, a process
where the cell ingests receptor-bound molecules by pinching off internalized parts of the
membrane and forming an endosome inside the cell. The environment inside the
endosome becomes more acidic, causing the PA molecules to change shape. This
conformational change in the PA molecules forms a pore in the endosomal membrane
and either EF or LF gets injected into the cytosol of the cell. Once in the cytosol, the EF
and LF components disrupt the cell by affecting cell signaling.

EF, which is referred to as edema toxin once inside the cell, is an

adenylate cyclase, an enzyme, which catalyzes the conversion of
Anthrax toxin is ATP to cyclic AMP (cAMP). Because cAMP is an important
the regulatory molecule in the cell, production of more cAMP by
cause of edema toxin disrupts normal cell function. One of these functions
disease's of cAMP in the cell is to maintain proper osmotic pressure by
deadly regulating the flow of small ions and water in and out of the cell.
sympto An increase in the amount of cAMP causes the cell to swell.
Additionally, since edema toxin uses ATP to form cAMP, the cell
ms becomes depleted of ATP. Macrophages need the energy-rich ATP
in order to engulf and destroy bacteria. As a result, infected
macrophages become bloated and useless.

The lethal factor, or lethal toxin, disrupts normal cell function by another route. Lethal
toxin is a zinc protease that targets members of the mitogen-activated protein kinase
kinase (MAPKK) family. This leads to inhibition of several cell signaling pathways and
results in an increased amount of cytokines, protein that act as cell mediators. While the
exact role these cytokines play in causing cell lysis remains unknown, it is believed that
high cytokine levels cause an increase in harmful
oxidative molecules. If the concentration of these
harmful molecules gets too high within the cell, the
macrophage ruptures and dies.

Treating Anthrax: How Cipro

Works
Cipro, or ciprofloxacin“ (Bayer Pharmaceutical), is a
powerful broad-spectrum antibiotic used to treat many
diverse bacterial infections, including anthrax. One of Cipro is an antibiotic
the ways bacteria and mammalian cells differ is that commonly used to
bacteria store their DNA in a circular plasmid form. treat anthrax. It
prevents the
bacteria from
multiplying by
damaging their
genomic DNA.
This plasmid is too large for the bacterial cell in its uncoiled form so bacteria enlist an
enzyme called DNA gyrase to twist the DNA into a more compact, supercoiled form.
Gyrase cuts one strand of the double stranded DNA, winds the DNA around itself and
pastes the DNA back together to form supercoiled DNA. Cipro blocks the reannealing
stage of this process by sitting in the active site of the DNA gyrase, leaving the double-
stranded DNA broken. With their genomic DNA damaged, the bacteria cannot replicate.
Therefore Cipro is only really effective in stopping the bacteria from multiplying, but
does little to kill the bacteria or to stop the production of the anthrax toxin.

New Approaches–Antitoxin Therapeutics

Since it is unfeasible to vaccinate the entire population against anthrax and antibiotics are
often ineffective in treating a severe anthrax infection, new “antitoxin” therapies are
currently being investigated. Scientists are developing new therapies to treat anthrax by
blocking the anthrax toxin from ever entering the mammalian cell.

One antitoxin approach is to block the PA component from binding ATR on the cell.
Soluble anthrax toxin receptor (sATR) is being investigated as a decoy to keep PA away
from its intended target on the surface of the cell. Perhaps a more powerful blocking
agent has been designed by scientists at the University of Texas at Austin who use
genetically engineered antibodies that bind PA fifty times more tightly than ATR.

A second antitoxin approach is to block the assembly of the PA heptamer. A group at

Harvard University has developed a peptide that tightly binds the PA monomer,
rendering it unable to form a heptamer with other PA monomers. Additionally, there has
been some investigation into the design of small molecules or peptides to sit on the
surface of the PA heptamer so that the EF and LF are blocked from binding the complex
and therefore cannot enter the cell.

Conclusion
The prevalence of anthrax in the news and support from government-funded agencies are
fostering the development of novel antitoxin therapeutics at an ever-increasing rate.
While we are years away from seeing any of these therapeutics available for use by the
public, we are getting closer to finding a cure. In the event of a large-scale bioterrorist
attack, the key to effectively treating anthrax may be to use an antibiotic, such as Cipro,
in combination with a novel antitoxin therapeutic. This “drug cocktail” would
simultaneously kill the bacteria and render their deadly toxins impotent.

Ref:http://www.wiley.com/college/boyer/0470003790/cutting_edge/anthrax/
anthrax.htm

anthrax toxin producing effect in to the host

cell:
Ken Bradley

Host Interactions with Bacterial Toxins

The research in our laboratory is inspired by the long-term goal of understanding how
microbial pathogens interact with their hosts to promote disease. Specifically, we are
interested in studying host genes that are required for bacterial toxins to exert their
effects. An example of such a host factor would be a receptor that allows for toxin
binding and entry into host cells. We have used anthrax toxin as our model system, but
are also beginning to study toxins produced by other pathogenic bacteria such as
cholesterol dependent cytolysins (CDCs) and cytolethal distending toxins (CDTs). To
achieve this goal, we employ a variety of forward genetic techniques in the host,
including somatic cell genetics, chemical genetics, and traditional mouse genetics. Once
identified, novel host-pathogen interactions are characterized using biochemical and cell
biological strategies to gain a deeper understanding of how these host factors interact
with their pathogen-encoded counterparts.

ANTHRAX TOXIN
Bacillus anthracis produces two major virulence factors, a tripartite exotoxin referred to
as anthrax toxin, and an antiphagocytic capsule. These virulence factors mediate
pathogen survival and, in the case of the toxin, directly induce damage to the host.
Anthrax toxin is an atypical AB toxin in that two distinct catalytic “A” moieties associate
with a single class of binding “B” moiety. As a result, two separate enzymatic activities
are associated with anthrax toxin. The enzymatic subunits are lethal factor (LF), a zinc-
dependent metalloproteinase, and edema factor (EF), a calmodulin-dependent adenylate
cyclase. LF and EF gain access to the host cytosol via the binding and translocation
properties of the shared binding subunit, protective angtigen (PA). Two distinct toxins
can be generated by combining either LF or EF with PA. The combination of LF and PA
is called lethal toxin (LT), and this toxin inactivates MAPK signaling in the host. Edema
toxin, formed by the combination of EF and PA, induces high cAMP levels in host cells.
Interestingly, we recently found that ET induces upregulation of anthrax toxin receptors
(ANTXRs), thereby inducing a positive feedback loop resulting in increased sensitivity of
macrophages and dendritic cells to both LT and ET.

Somatic Cell Genetic Approach in Macrophages. Isolation of a receptor-deficient CHO

cell line in our original ANTXR cloning efforts {Bradley, 2001} enabled subsequent
structure-function studies on ANTXRs. However, macrophages are more likely to be a
physiologically relevant target and these cells respond quite differently to LT in culture.
Thus, in order to study LT-host cell interactions, we sought to perform somatic cell
genetics using a macrophage cell line, RAW264.7. These efforts were successful, and we
obtained a macrophage clone that lacks anthrax toxin receptor expression following
chemical mutagenesis {Banks, 2005}. This finding demonstrated that forward genetic
screening can be accomplished in a macrophage cell line, a cell type which many
pathogens have evolved to interact with. In addition, the isolation of the receptor-mutant
clone has created opportunities to study the importance of toxin-macrophage interactions
during anthrax infection. For example, we were able to show that mutant cells lacking
toxin receptors are not only insensitive to purified toxin, but were also able to clear
anthrax spores when challenged with a high multiplicity of infection {Banks, 2005; Cote,
2008}. This addressed a long-standing debate about whether toxin production is
important during early outgrowth of spores, which occurs within phagolysosomal
compartments in the macrophages. ANTXRs were found to co-localize with spores in
phagolysosomes, supporting the conclusion that toxin production in this compartment
may be important for successful bacterial outgrowth. In collaboration with researches at
USAMRIID/Ft. Detrick, we have shown that mice supplemented with macrophages that
lack anthrax toxin receptor expression are able to clear a lethal dose of fully virulent B.
anthracis spores (Ames strain), while mice supplemented with wild-type or receptor-
complemented mutant macrophages cannot {Cote, 2008}. This demonstrated for the first
time that toxin targeting of macrophages plays a significant role in vivo for the outcome
of an anthrax infection.

Development of a conditional, insertion-based somatic cell genetic toolset.

While chemical mutagenesis of mammalian cells has proven an effective means to isolate
pathogen- and/or toxin-resistant cells {Banks, 2005; Bradley, 2001; Bruce, 2005}, gene
identification usually requires cDNA complementation. However, the complementation
phenotype is not always easily assayed and/or the gene of interest may not be represented
in the cDNA library used. Additionally, with the exception of temperature-sensitive
screens, it is not possible to obtain mutant cells that lack a gene essential for cell viability.
To overcome these limitations, we developed a novel forward genetic system that allows
for conditional, epigenetic control of host gene transcription. We call this system
SILENCE for Silencing Induced by Long Terminal Repeat (LTR) Encoded Cis-acting
response Element {Banks, 2007}.

Chemical Genetics and High Throughput Screening.

In addition to somatic cell genetics, we employ chemical genetic approaches to identify
host factors involved in anthrax toxin susceptibility. This work is done in conjunction
with the Molecular Screening Shared Resource, a high throughput screening (HTS)
facility at UCLA. Dr. Bradley currently serves as Director of the MSSR, which has
initiated over 150 screening projects for researchers across the United States since 2003.
The goal of the chemical screening approach in our lab is two-fold: identify small
molecules that can serve as probes to understand toxin action on host cells, and identify
small molecules that can serve as lead compounds for development of therapeutics to
treat anthrax. To this end, we have reported that two FDA-approved drugs used to treat
cardiac arrhythmia or angina block the entry of anthrax toxin at concentrations similar to
those found in the serum of human patients {Sanchez, 2007}. These compounds came out
of a primary screen of ~500 compounds that represent molecules with known bioactive
properties. Based on the initial success of this approach, we expanded our screen to
include ~70k small molecules, representing several libraries of diverse chemical
structures that are uncharacterized with respect to biological activity. We are currently
characterizing a number of hits that resulted from this larger screen. Finally, the HTS
capabilities of the MSSR are being utilized to screen genome-wide siRNA and shRNA
libraries for genes required for sensitivity to anthrax toxin.

CYTOLETHAL DISTENDING TOXINS

Cytolethal distending toxins (CDTs) are members of an emerging group of bacterial
toxins and effectors called “cyclomodulins” that interfere with the eukaryotic cell cycle.
CDTs intoxicate many cell types and induce G2/M cell cycle arrest, resulting in slow
cellular distension and ultimately in cell death. Interfering with the eukaryotic cell cycle
has significant implications for pathogens that interact with host cells undergoing rapid
division, such as gut epithelium and lymphocytes. CDTs are produced by several
unrelated Gram-negative pathogens that induce a wide variety of diseases, including
typhoid fever, hemolytic uremic syndrome, and periodontitis. The role of CDT in
virulence is poorly understood, but the presence of this toxin results in increased
invasiveness, persistence, and/or severity of symptoms. To determine the mechanism by
which CDTs interact with mammalian cells, we are undertaking somatic cell genetic and
HTS approaches to identify host cell pathways and genes required for efficient toxin
uptake and action.

Publications
Terra JK, Cote CK, France B, Jenkins AL, Bozue JA, Welkos SL, LeVine SM, Bradley
KA. (2010) Cutting edge: resistance to Bacillus anthracis infection mediated by a lethal
toxin sensitive allele of Nalp1b/Nlrp1b J Immunol. 184(1), 17-20. [link]
Saji George, Suman Pokhrel, Tian Xia, Benjamin Gilbert, Zhaoxia Ji, Marco Schowalter,
Andreas Rosenauer, Robert Damoiseaux, Kenneth A. Bradley, Lutz Madler and Andre
Nel (2010) Use of a Rapid Cytotoxicity Screening Approach To Engineer a Safer Zinc
Oxide Nanoparticle through Iron Doping ACS Nano 4(1), 15–29. [link]
Maldonado-Arocho, F. J. Bradley, K. A. (2009) Anthrax Edema Toxin Induces
Maturation of Dendritic Cells and Enhances Chemotaxis towards Macrophage
Inflammatory Protein 3 beta Infection and Immunity 77(5), 2036-2042. [link]
Liong, M., Bradley, K.A., Zink, J.I. (2009) Antimicrobial Activity of Silver Nanocrystals
Encapsulated in Mesoporous Silica Nanoparticles Adv. Mater 21(17), 1684-1689. [link]
Lehrer RI, Jung G, Ruchala P, Wang W, Micewicz ED, Waring AJ, Gillespie EJ, Bradley
KA, Ratner AJ, Rest RF, Lu W. (2009) Human alpha-defensins inhibit hemolysis
mediated by cholesterol-dependent cytolysins Infect Immun. 77(9), 4028-40. [link]
Godwin HA, Chopra K, Bradley KA, Cohen Y, Harthorn BH, Hoek EM, Holden P,
Keller AA, Lenihan HS, Nisbet RM, Nel AE. (2009) The University of California Center
for the Environmental Implications of Nanotechnology Environ Sci Technol. 43(17),
6453-7. [link]
Visnyei, Koppany Saxe, Jonathan P. Huang, Jing Damoiseaux, Robert Bradley, Kenneth
A. Prins, Robert M. Kornblum, Harley I. (2007) Identification of small molecule
inhibitors targeting brain tumor stem cells Proceedings of the American Association for
Cancer Research Annual Meeting 48, 1312-1313.
Poulos, J. L. Jeon, T. J. Damoiseaux, R. Gillespie, E. J. Bradley, K. A. Schmidt, J. J.
(2009) Ion channel and toxin measurement using a high throughput lipid membrane
platform Biosensors and Bioelectronics 24(6), 1806-1810. [link]
Lee Shaoying, Deng Hongyu, Yu Fuqu, Melega William P, Damoiseaux Robert, Bradley
Kenneth A, Sun Ren (2008) Regulation of Kaposi's sarcoma-associated herpesvirus
reactivation by dopamine receptor-mediated signaling pathways. Journal of acquired
immune deficiency syndromes (1999) 48(5), 531-40. [link]
Cote Christopher K, DiMezzo Tracy L, Banks David J, France Bryan, Bradley Kenneth
A, Welkos Susan L (2008) Early interactions between fully virulent Bacillus anthracis
and macrophages that influence the balance between spore clearance and development of
a lethal infection. Microbes and infection / Institut Pasteur 10(6), 613-9. [link]
Sanchez Ana M, Thomas Diane, Gillespie Eugene J, Damoiseaux Robert, Rogers Joseph,
Saxe Jonathan P, Huang Jing, Manchester Marianne, Bradley Kenneth A (2007)
Amiodarone and bepridil inhibit anthrax toxin entry into host cells. Antimicrobial agents
and chemotherapy 51(7), 2403-11. [link]
Nassanian Hoorig, Sanchez Ana M, Lo Alice, Bradley Kenneth A, Lee Benhur (2007)
Efficient construction of an inverted minimal H1 promoter driven siRNA expression
cassette: facilitation of promoter and siRNA sequence exchange. PLoS ONE 2(1), e767.
[link]
Banks David J, Bradley Kenneth A (2007) SILENCE: a new forward genetic technology.
Nature methods 4(1), 51-3. [link]
Wang Wei, Mulakala Chandrika, Ward Sabrina C, Jung Grace, Luong Hai, Pham Duy,
Waring Alan J, Kaznessis Yiannis, Lu Wuyuan, Bradley Kenneth A, Lehrer Robert I
(2006) Retrocyclins kill bacilli and germinating spores of Bacillus anthracis and
inactivate anthrax lethal toxin. The Journal of biological chemistry 281(43), 32755-64.
[link]
Ding Zhiping, Bradley Kenneth A, Amin Arnaout M, Xiong Jian-Ping (2006) Expression
and purification of functional human anthrax toxin receptor (ATR/TEM8) binding
domain from Escherichia coli. Protein expression and purification 49(1), 121-8. [link]
Salles Isabelle I, Voth Daniel E, Ward Sabrina C, Averette Kathleen M, Tweten Rodney
K, Bradley Kenneth A, Ballard Jimmy D (2006) Cytotoxic activity of Bacillus anthracis
protective antigen observed in a macrophage cell line overexpressing ANTXR1. Cellular
microbiology 8(8), 1272-81. [link]
Christman Karen L, Requa Michael V, Enriquez-Rios Vanessa D, Ward Sabrina C,
Bradley Kenneth A, Turner Kimberly L, Maynard Heather D (2006) Submicron
streptavidin patterns for protein assembly. Langmuir : the ACS journal of surfaces and
colloids 22(17), 7444-50. [link]
Maldonado-Arocho Francisco J, Fulcher Jennifer A, Lee Benhur, Bradley Kenneth A
(2006) Anthrax oedema toxin induces anthrax toxin receptor expression in monocyte-
derived cells. Molecular microbiology 61(2), 324-37. [link]
Banks David J, Ward Sabrina C, Bradley Kenneth A (2006) New insights into the
functions of anthrax toxin. Expert reviews in molecular medicine 8(7), 1-18. [link]
Bruce James W, Bradley Kenneth A, Ahlquist Paul, Young John A T (2005) Isolation of
cell lines that show novel, murine leukemia virus-specific blocks to early steps of
retroviral replication. Journal of virology 79(20), 12969-78. [link]
Banks David J, Barnajian Moshe, Maldonado-Arocho Francisco J, Sanchez Ana M,
Bradley Kenneth A (2005) Anthrax toxin receptor 2 mediates Bacillus anthracis killing of
macrophages following spore challenge. Cellular microbiology 7(8), 1173-85. [link]
Sánchez Ana M, Bradley Kenneth A (2004) Anthrax toxin: can a little be a good thing?
Trends in microbiology 12(4), 143-5. [link]
Bradley Kenneth A, Mogridge Jeremy, Jonah G, Rainey A, Batty Sarah, Young John A T
(2003) Binding of anthrax toxin to its receptor is similar to alpha integrin-ligand
interactions. The Journal of biological chemistry 278(49), 49342-7. [link]
Scobie Heather M, Rainey G Jonah A, Bradley Kenneth A, Young John A T (2003)
Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor.
Proceedings of the National Academy of Sciences of the United States of America 100(9),
5170-4. [link]
Bradley, K. A. Mogridge, J. Mourez, M. Collier, R. J. Young, J. A. (2001) Identification
of the cellular receptor for anthrax toxin Nature 414(6860), 225-9. [link]

Ref: https://www.uclaaccess.ucla.edu/Faculty.aspx?rv_FacultyId=3517

Our research group is interested in various aspects of the structure-function

relationship of metalloproteins. Particular focus is currently directed towards
the elucidation of the metal ion requirement and mechanism of Anthrax
Lethal Factor, a zinc-dependent metallo-endopeptidase, which constitutes
one of the three protein components of the anthrax toxin.

Anthrax
Anthrax is an infectious disease
caused by the rod-shaped, gram-
positive bacterium Bacillus
anthracis. The disease mainly
affects animals (mainly cattle and
other herbivores), but it can
occasionally also be transferred to
humans (click here for a 'Brief
History of Anthrax' and its use as a
biological weapon). The most deadly
form of anthrax is associated with
spores of B. anthracis. The spores Bacillus anthracis
are very resistant to exposure to
heat, radiation and chemical
reagents, and therefore remain
intact in a natural environment for
extended periods of time. After
inhalation by the host, spores
become lodged in the lungs where
they are readily picked up by
macrophages. Germination of the
spores and subsequent replication
of B. anthracis occur after their
transport to the lymph nodes,
where the bacteria quickly reach
very high titers (ultimately leading
to death of the host). Treatment of
inhalation anthrax is difficult since
symptoms usually evade early
detection for treatment with
antibiotics.

In addition to inhalation anthrax,

there are two other forms of the
disease: cutaneous (i.e. skin)
anthrax and gastrointestinal
anthrax. Cutaneous anthrax is
rarely fatal as it can be usually
treated with antibiotics (e.g.
ciprofloxacin, penicillins,
doxycycline). It is characterized by
the occurrence of black, necrotic
lesions (click here to view an image
of such a lesion), which give the
disease its name (anthrakis, Greek
word for coal). The third,
gastrointestinal form of anthrax is
caused by ingestion of bacterial
spores from a contaminated source
(e.g. infected cattle). This form of
the disease is extremely rare, but
often fatal.
Mechanism of LF and EF translocation (click called pXO1. In addition to
image to enlarge)
Anthrax Toxin
The toxic effects associated
with anthrax are a consequence
of the production and secretion
of three proteins which
constitute the so-called anthrax
toxin. These are: the protective
antigen (PA), the lethal factor
(LF), and the edema factor
(EF). While the combination of
PA and LF is termed lethal
toxin, the combination of PA
with EF is referred to as edema
toxin. The genes encoding
these proteins are located on
an extrachromosomal plasmid
pXO1, B. anthracis harbours
another plasmid (pXO2)
encoding a poly-glutamic acid
capsule, which contributes to
the bacteria's resistance to
adverse conditions. The
protective antigen, so-called for
its use in vaccines, is
responsible for the translocation
of LF and EF into the host cell
cytosol where these proteins
function enzymatically. The
figure on the right/left
illustrates some of the steps
involved in transport of LF and
EF into the cytosol. After
binding to the anthrax toxin
receptor (ATR) on the host cell
surface, PA is proteolytically
cleaved into two fragments by
the membrane bound protease
furin. The larger fragment (63
kDa) remains bound to the
receptor where it participates in
the formation of a heptamer of
PA protein molecules.
Heptameric PA can now bind up
to three molecules of LF and/or
EF. The protein complex then
 enters the cell via receptor-
mediated endocytosis. The
lower pH of the endosome
facilitates pore formation by PA,
releasing LF/EF into an
intraluminal vesicle found
within the endosome. Finally,
LF/EF is exocytosed into the
host cell cytoplasm where EF
functions as a Ca(II) and
calmodulin-dependant adenylyl
cyclase, catalyzing the
conversion of ATP to cyclic AMP.
The increase in cAMP levels
results in an impairment of
water homeostasis, and thus
leads to the occurrence of
edema.

Lethal Factor
The lethal factor is a zinc-
dependent metallo-
endopeptidase (90 kDa), which
catalyzes the cleavage of
mitogen-activated protein
kinase kinases (MAPKKs) within
their N-termini. Proteolysis of
MAPKKs leads to a disruption of
major cell signaling pathways
(with deadly consequences). LF
contains four distinct domains.
While domain 1 is responsible
for binding to PA during
translocation, domains 2 to 4
are involved in substrate
recognition and binding.
Furthermore, domain 4
harbours the active site of LF,
i.e. the site where the target
substrates (MAPKKs) are Crystal structure of LF
cleaved near their N-termini.
The 'heart' of the active site of
LF is comprised of a single
Zn(II) ion, which is
tetrahedrally coordinated by
three amino acid residues
(His686, His690, Glu735) and a
single water molecule (or
hydroxide ion). The Zn(II) ion
activates the water/hydroxide
ligand for nucleophilic attack on
the substrate peptide bond to
be cleaved. A glutamate and a
tyrosine residue are also
thought to be involved in the
catalytic cycle. While Glu687
may act as a general base,
Tyr728 appears to function as a
general acid (protonation of the
amine leaving group). Finally, it
has been shown that, in
addition to zinc ions, LF
requires calcium and
magnesium ions for full
catalytic function in vitro. The
molecular basis for this peculiar
metal ion dependence is
currently unknown.
Ref: http://oldwebsite.laurentian.ca/chem/ssiemann/research/research.html
Mechanisms of Bacterial Pathogenicity (page 4)

(This chapter has 8 pages)

© 2011 Kenneth Todar, PhD

INVASION

The invasion of a host by a pathogen may be aided by the production of bacterial extracellular substances which
act against the host by breaking down primary or secondary defenses of the body. Medical microbiologists have
long referred to these substances as invasins. Most invasins are proteins (enzymes) that act locally to damage
host cells and/or have the immediate effect of facilitating the growth and spread of the pathogen. The damage to
the host as a result of this invasive activity may become part of the pathology of an infectious disease.

The extracellular proteins produced by bacteria which promote their invasion are not clearly distinguished from
some extracellular protein toxins ("exotoxins") which also damage the host. Invasins usually act at a short range
(in the immediate vicinity of bacterial growth) and may not actually kill cells as part of their range of activity;
exotoxins are often cytotoxic and may act at remote sites (removed from the site of bacterial growth). Also,
exotoxins typically are more specific and more potent in their activity than invasins. Even so, some classic
exotoxins (e.g. diphtheria toxin, anthrax toxin) may play some role in colonization or invasion in the early stages
of an infection, and some invasins (e.g. staphylococcal leukocidin) have a relatively specific cytopathic effect.

A Survey of Bacterial Invasins

Spreading Factors

"Spreading Factors" is a descriptive term for a family of bacterial enzymes that affect the physical properties of
tissue matrices and intercellular spaces, thereby promoting the spread of the pathogen.

Hyaluronidase. is the original spreading factor. It is produced by streptococci. staphylococci, and clostridia. The
enzyme attacks the interstitial cement ("ground substance") of connective tissue by depolymerizing hyaluronic
acid.

Collagenase is produced by Clostridium histolyticum and Clostridium perfringens. It breaks down collagen, the
framework of muscles, which facilitates gas gangrene due to these organisms.

Neuraminidase is produced by intestinal pathogens such as Vibrio cholerae and Shigella dysenteriae. It
degrades neuraminic acid (also called sialic acid), an intercellular cement of the epithelial cells of the intestinal
mucosa.
Streptokinase and staphylokinase are produced by streptococci and staphylococci, respectively. Kinase
enzymes convert inactive plasminogen to plasmin which digests fibrin and prevents clotting of the blood. The
relative absence of fibrin in spreading bacterial lesions allows more rapid diffusion of the infectious bacteria.

Enzymes that Cause Hemolysis and/or Leucolysis

These enzymes usually act on the animal cell membrane by insertion into the membrane (forming a pore that
results in cell lysis), or by enzymatic attack on phospholipids, which destabilizes the membrane. They may be
referred to as lecithinases or phospholipases, and if they lyse red blood cells they are sometimes called
hemolysins. Leukocidins, produced by staphylococci and streptolysin produced by streptococci specifically
lyse phagocytes and their granules. These latter two enzymes are also considered to be bacterial exotoxins.

Phospholipases, produced by Clostridium perfringens (i.e., alpha toxin), hydrolyze phospholipids in cell
membranes by removal of polar head groups.

Lecithinases, also produced by Clostridium perfringens, destroy lecithin (phosphatidylcholine) in cell

membranes.

Hemolysins, notably produced by staphylococci (i.e., alpha toxin), streptococci (i.e., streptolysin) and various
clostridia, may be channel-forming proteins or phospholipases or lecithinases that destroy red blood cells and
other cells (i.e., phagocytes) by lysis.

Beta-hemolytic Streptococcus. This is the characteristic appearance of a blood agar plate culture of
the bacterium. Note the translucency around the bacterial colonies, representing hemolysis of the red
cells in the culture medium due to production of a diffusible hemolysin (streptolysin).

Staphylococcal coagulase

Coagulase, formed by Staphylococcus aureus, is a cell-associated and diffusible enzyme that converts fibrinogen
to fibrin which causes clotting. Coagulase activity is almost always associated with pathogenic S. aureus and
almost never associated with nonpathogenic S. epidermidis, which has led to much speculation as to its role as a
determinant of virulence. Possibly, cell bound coagulase could provide an antigenic disguise if it clotted fibrin on
the cell surface. Or a staphylococcal lesion encased in fibrin (e.g. a boil or pimple) could make the bacterial cells
resistant to phagocytes or tissue bactericides or even drugs which might be unable to diffuse to their bacterial
target.

Extracellular Digestive Enzymes

Heterotrophic bacteria, in general, produce a wide variety of extracellular enzymes including proteases, lipases,
glycohydrolases, nucleases, etc., which are not clearly shown to have a direct role in invasion or
pathogenesis. These enzymes presumably have other functions related to bacterial nutrition or metabolism, but
may aid in invasion either directly or indirectly.

Toxins With Short-Range Effects Related to Invasion

Bacterial protein toxins which have adenylate cyclase activity, are thought to have immediate effects on host cells
that promote bacterial invasion. One component of the anthrax toxin (EF or Edema Factor) is an adenylate
cyclase that acts on nearby cells to cause increased levels of cyclic AMP and disruption of cell permeability. One
of the toxins of Bordetella pertussis, the agent of whooping cough, has a similar effect. These toxins may
contribute to invasion through their effects on macrophages or lymphocytes in the vicinity which are playing an
essential role to contain the infection. For example, since they use ATP as a substrate, they may deplete
phagocyte reserves of energy needed for ingestion. Edema is seen as a pathology because the increase in cAMP
in affected cells disrupts equilibrium.

Gelatinous edema seen in a cutaneous anthrax lesion. CDC.

The following table summarizes the activities of many bacterial proteins that are noted for their contribution to
bacterial invasion of tissues.

TABLE 3. SOME EXTRACELLULAR BACTERIAL PROTEINS THAT ARE CONSIDERED

INVASINS

Invasin Bacteria Involved Activity

Streptococci,
Hyaluronidase staphylococci and Degrades hyaluronic of connective tissue
clostridia
Collagenase Clostridium species Dissolves collagen framework of muscles
Vibrio cholerae and Degrades neuraminic acid of intestinal
Neuraminidase
Shigella dysenteriae mucosa
Converts fibrinogen to fibrin which causes
Coagulase Staphylococcus aureus
clotting
Staphylococci and Converts plasminogen to plasmin which
Kinases
streptococci digests fibrin
Leukocidin Staphylococcus aureus Disrupts neutrophil membranes and causes
 discharge of lysosomal granules
Repels phagocytes and disrupts phagocyte
Streptococcus
Streptolysin membrane and causes discharge of
pyogenes
lysosomal granules
Streptococci,
Phospholipases or lecithinases that destroy
Hemolysins staphylococci and
red blood cells (and other cells) by lysis
clostridia
Clostridium
Lecithinases Destroy lecithin in cell membranes
perfringens
Clostridium
Phospholipases Destroy phospholipids in cell membrane
perfringens
One component (EF) is an adenylate
Anthrax EF Bacillus anthracis cyclase which causes increased levels of
intracellular cyclic AMP
One toxin component is an adenylate
Pertussis AC Bordetella pertussis cyclase that acts locally producing an
increase in intracellular cyclic AMP
Ref: http://www.textbookofbacteriology.net/pathogenesis_4.html

diagram of action of secreted anthrax vaccine:

The immune response generated in rats by the new agent protects against lethal toxin
exposure after only one injection, and is faster and stronger than any currently available
vaccine.

The new study, led by Scripps Research scientists Anette Schneemann and Marianne
Manchester, and Salk Institute Professor John A.T. Young, was published in the October
5 issue of the journal PLoS Pathogens (Volume 3, Issue 10).

“The new anti-anthrax agent that we developed is an important and potentially critical
development for anyone who works with the bacterium or those who might be exposed to
it in a bioterrorism attack,” Schneemann said. “While other strategies are being pursued
to develop improved anthrax vaccines, none of these offer the distinct advantage of
combining the function of a vaccine with a potent antitoxin.”

Concerns about anthrax-a potentially fatal disease caused by the spore-forming, gram-
positive bacterium Bacillus anthracis-as a weapon of bioterrorism has prompted increased
efforts to develop better antitoxins and vaccines. The current vaccine, which was
developed in the 1950s, is safe and effective, but requires multiple injections followed by
annual boosters. Current anthrax treatment involves antibiotics such as ciprofloxacin and
doxycycline that attack the bacteria but provide no protection against the dangerous
toxins secreted by the bacteria.
The new study introduces a highly effective dual-action compound that leapfrogs current
efforts to develop a second-generation anthrax vaccine. In the research, the scientists
created a “multivalent display,” with several sites of attachment for recombinant
protective antigen protein (PA), the primary component of the current anthrax vaccine,
rather than only one. Virus-like particles coated with PA were found to produce a potent
toxin-neutralizing antibody response that protected rats from the lethal anthrax toxin after
only a single immunization.

The antitoxin strategy arose from the discovery of the anthrax toxin receptor, ANTXR2,
in the Young lab. “The new anti-anthrax agent is based on a multivalent display of
ANTXR2 on the surface of an insect virus,” explains Schneemann. “Our approach was
based on the assumption that a multivalent display of recombinant protective antigen
protein would induce a far more potent immune response. That turned out to be correct.”

Specifically, the new vaccine-antitoxin combination is based on the multivalent display

(180 copies) of the PA-binding von Willebrand A (VWA) domain of the ANTXR2
cellular receptor on the Flock House virus. The chimeric virus-like particle platform,
which produces protective immunity and has been shown to be safe, inhibited lethal toxin
action in in vitro and in vivo models of anthrax infection.

In fact, rats survived exposure to the toxin four weeks after a single injection of the new
double-acting agent. This result suggests an extremely rapid production of neutralizing
antibodies without the use of an adjuvant, a secondary agent that helps stimulate the
immune system and is often used to increase the vaccine response-key goals for the
development of third-generation anthrax vaccines.

In addition to its use against anthrax, Schneemann notes that creating a multivalent
platform may also have the potential to work against other infectious agents.

“One important reason for the success of this project is that it arose from the
multidisciplinary and highly collaborative efforts of our team of microbiologists,
structural biologists, and immunologists,” said Manchester, who headed a National
Institutes of Health (NIH)-funded program project grant that supported the work.

Source : Scripps Research Institute

Ref:
http://www.biologynews.net/archives/2007/10/05/scripps_research_scientist
s_develop_innovative_dual_action_anthrax_vaccineantitoxin_combination.h
tml

Innovative dual action anthrax vaccine-antitoxin

combination
October 05, 2007
La Jolla, CA – A collaboration between scientists at the Salk Institute for Biological
Studies and The Scripps Research Institute led to the development of a new and highly
effective agent that provides protection against anthrax by combining a fast-acting
anthrax toxin inhibitor with a vaccine in a single compound.

The immune response generated in rats by the new agent protects against lethal toxin
exposure after only one injection, and is faster and stronger than any currently available
vaccine.

Top: An insect virus (shown in green) acts as a carrier for anthrax toxin receptor
molecules (shown in yellow), which act like a sponge to soak up free toxin.

Bottom: When coated with toxin (shown in purple), the virus-like particles produced a
potent toxin-neutralizing antibody response that protected rats from the lethal anthrax
toxin after only a single immunization.

Image: Courtesy of Dr. Vijay S. Reddy, The Scripps Research Institute

The new study, led by Scripps research scientists Anette Schneemann, Ph.D., and
Marianne Manchester, Ph.D., and John A.T. Young, Ph.D., a professor in the Infectious
Disease Laboratory at Salk, was published in the October issue of the journal PLoS
Pathogens.

"The new anti-anthrax agent that we developed is an important and potentially critical
development for anyone who works with the bacterium or those who might be exposed to
it in a bioterrorism attack," Schneemann said. "While other strategies are being pursued
to develop improved anthrax vaccines, none of these offer the distinct advantage of
combining the function of a vaccine with a potent antitoxin."

Concerns about anthrax-a potentially fatal disease caused by the spore-forming, gram-
positive bacterium Bacillus anthracis-as a weapon of bioterrorism has prompted
increased efforts to develop better antitoxins and vaccines. The current vaccine, which
was developed in the 1950s, is safe and effective, but requires multiple injections
followed by annual boosters. Current anthrax treatment involves antibiotics such as
ciprofloxacin and doxycycline that attack the bacteria but provide no protection against
the dangerous toxins secreted by the bacteria.

The new study introduces a highly effective dual-action compound that leapfrogs current
efforts to develop a second-generation anthrax vaccine. In the research, the scientists
created a "multivalent display," with multiple sites of attachment for recombinant
protective antigen protein (PA), the primary component of the current anthrax vaccine,
rather than only one. Virus-like particles coated with PA were found to produce a potent
toxin-neutralizing antibody response that protected rats from the lethal anthrax toxin after
only a single immunization.

The antitoxin strategy arose from the discovery and characterization of both known
anthrax toxin receptors, ANTXR1 and ANTXR2, in the Young lab. "We have taken our
knowledge of how anthrax toxin normally binds ANTXR2 on cell surfaces and used it to
develop a platform that displays multiple copies of the receptor," explains Young. "The
receptor molecules can either act like a sponge to soak up free toxin, or as a potent
vaccine when it is pre-bound to the toxin," he adds.

The new anti-anthrax agent uses an insect virus as a carrier to display multiple ANTXR2
molecules on its surface. "Our approach was based on the assumption that a multivalent
display of recombinant protective antigen protein would induce a far more potent
immune response. That turned out to be correct," explains Schneemann.

Specifically, the new vaccine-antitoxin combination is based on the multivalent display

(180 copies) of the PA-binding von Willebrand A (VWA) domain of the ANTXR2
cellular receptor on the Flock House virus. The chimeric virus-like particle platform,
which produces protective immunity and has been shown to be safe, inhibited lethal toxin
action in in vitro and in vivo models of anthrax infection.

In fact, rats survived exposure to the toxin four weeks after a single injection of the new
double-acting agent. This result suggests an extremely rapid production of neutralizing
antibodies without the use of an adjuvant, a secondary agent that helps stimulate the
immune system and is often used to increase the vaccine response-key goals for the
development of third-generation anthrax vaccines.

"One important reason for the success of this project is that it arose from the
multidisciplinary and highly collaborative efforts of our team of microbiologists,
structural biologists, and immunologists," said Manchester, who headed a NIH-funded
Program Project grant that supported the work.

In addition to its use against anthrax, Schneemann notes that creating a multivalent
platform may also have the potential to work against other infectious agents.

Other authors of the study, "A Viral Nanoparticle with Dual Function as an Anthrax
Antitoxin and Vaccine," are Darly J. Manayani, Vijay Reddy, Michael E. Pique, Marc E.
Siladi, Diane Thomas, Kelly A. Dryden, and Marianne Manchester of The Scripps
Research Institute; John M. Marlett, G. Jonah A. Rainey, Heather M. Scobie, and John
A.T.Young of the Salk Institute for Biological Studies; and Mark Yeager of The Scripps
Research Institute and the Scripps Clinic.

The study was supported by the National Institutes of Health.

The Salk Institute for Biological Studies in La Jolla, California, is an independent

nonprofit organization dedicated to fundamental discoveries in the life sciences, the
improvement of human health and the training of future generations of researchers. Jonas
Salk, M.D., whose polio vaccine all but eradicated the crippling disease poliomyelitis in
1955, opened the Institute in 1965 with a gift of land from the City of San Diego and the
financial support of the March of Dimes.

Ref: http://www.salk.edu/news/pressrelease_details.php?press_id=181

Scientists Develop Innovative Dual Action Anthrax

Vaccine-Antitoxin Combination
By Eric Sauter

Scientists at The Scripps Research Institute and The Salk Institute for Biological Studies
have developed a new and highly effective agent that provides protection against anthrax
by combining a fast-acting anthrax toxin inhibitor with a vaccine in a single compound.
The immune response generated in rats by the new agent protects against lethal toxin
exposure after only one injection, and is faster and stronger than any currently available
vaccine.

The new study, led by Scripps Research scientists Anette Schneemann and Marianne
Manchester, and Salk Institute Professor John A.T. Young, was published in the October
5 issue of the journal PLoS Pathogens (Volume 3, Issue 10).

"The new anti-anthrax agent that we developed is an important and potentially critical
development for anyone who works with the bacterium or those who might be exposed to
it in a bioterrorism attack," Schneemann said. "While other strategies are being pursued
to develop improved anthrax vaccines, none of these offer the distinct advantage of
combining the function of a vaccine with a potent antitoxin."

Concerns about anthrax—a potentially fatal disease caused by the spore-forming, gram-
positive bacterium Bacillus anthracis—as a weapon of bioterrorism has prompted
increased efforts to develop better antitoxins and vaccines. The current vaccine, which
was developed in the 1950s, is safe and effective, but requires multiple injections
followed by annual boosters. Current anthrax treatment involves antibiotics such as
ciprofloxacin and doxycycline that attack the bacteria but provide no protection against
the dangerous toxins secreted by the bacteria.

The new study introduces a highly effective dual-action compound that leapfrogs current
efforts to develop a second-generation anthrax vaccine. In the research, the scientists
created a "multivalent display," with several sites of attachment for recombinant
protective antigen protein (PA), the primary component of the current anthrax vaccine,
rather than only one. Virus-like particles coated with PA were found to produce a potent
toxin-neutralizing antibody response that protected rats from the lethal anthrax toxin after
only a single immunization.

The antitoxin strategy arose from the discovery of the anthrax toxin receptor, ANTXR2,
in the Young lab. "The new anti-anthrax agent is based on a multivalent display of
ANTXR2 on the surface of an insect virus," explains Schneemann. "Our approach was
based on the assumption that a multivalent display of recombinant protective antigen
protein would induce a far more potent immune response. That turned out to be correct."

Specifically, the new vaccine-antitoxin combination is based on the multivalent display

(180 copies) of the PA-binding von Willebrand A (VWA) domain of the ANTXR2
cellular receptor on the Flock House virus. The chimeric virus-like particle platform,
which produces protective immunity and has been shown to be safe, inhibited lethal toxin
action in in vitro and in vivo models of anthrax infection.

In fact, rats survived exposure to the toxin four weeks after a single injection of the new
double-acting agent. This result suggests an extremely rapid production of neutralizing
antibodies without the use of an adjuvant, a secondary agent that helps stimulate the
immune system and is often used to increase the vaccine response—key goals for the
development of third-generation anthrax vaccines.

In addition to its use against anthrax, Schneemann notes that creating a multivalent
platform may also have the potential to work against other infectious agents.

"One important reason for the success of this project is that it arose from the
multidisciplinary and highly collaborative efforts of our team of microbiologists,
structural biologists, and immunologists," said Manchester, who headed a National
Institutes of Health (NIH)-funded program project grant that supported the work.

Other authors of the study, "A Viral Nanoparticle with Dual Function as an Anthrax
Antitoxin and Vaccine," are Darly J. Manayani, Vijay Reddy, Michael E. Pique, Marc E.
Siladi, Diane Thomas,Kelly A. Dryden, and Marianne Manchester of The Scripps
Research Institute; John M. Marlett, G. Jonah A. Rainey,Heather M. Scobie,and John
A.T.Young of The Salk Institute for Biological Studies; and Mark Yeager of The Scripps
Research Institute and the Scripps Clinic.

Ref: http://www.scripps.edu/newsandviews/e_20071008/antitoxin.html

Detections of anthrax vaccine

Detection of anthrax vaccine virulence factors by polymerase chain reaction

Antonio Fasanellaa, , , Stefania Lositoa, Teresa Trottaa, Rosanna Adoneb,

Salvatore Massac, Franco Ciuchinib and Doriano Chioccoa
a
Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Via Manfredonia
20, 71100 Foggia, Italy
b
Istituto Superiore di Sanità, Laboratorio di MedicinaVeterinaria, Viale Regina Elena,
299 Rome, Italy
c
Istituto di Produzioni e Tecnologia Alimentari, Facoltà di Agraria, Università di Foggia,
Foggia, Italy

Received 10 October 2000;

revised 29 March 2001;
accepted 23 April 2001.
Available online 10 July 2001.
Abstract

In Italy, an attenuated Bacillus anthracis strain, named ‘Carbosap’, is used for

immunization against ovine and bovine anthrax. Analysis on ‘Carbosap’, Sterne vaccine
strain F34 and Pasteur vaccine strain SS104, were performed using primers specific for
the sequences, encoding the toxic factors, located on plasmids pXO1 and pXO2 and
primers specific for the chromosome. The results obtained from polymerase chain
reaction (PCR) assay revealed the presence of both plasmids pXO1 and pXO2 in
‘Carbosap’ strain. This study showed that the ‘Carbosap’ vaccine strain has a different
plasmid pattern in comparison to Pasteur vaccine strain SS104 and Sterne vaccine strain
F34.

Keywords: Anthrax; PCR; Vaccine

Article Outline
Detection of anthrax vaccine virulence factors by polymerase chain reaction

Purchase
$ 31.50

Antonio Fasanellaa, , , Stefania Lositoa, Teresa Trottaa, Rosanna Adoneb,

Salvatore Massac, Franco Ciuchinib and Doriano Chioccoa
a
Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Via Manfredonia
20, 71100 Foggia, Italy
b
Istituto Superiore di Sanità, Laboratorio di MedicinaVeterinaria, Viale Regina Elena,
299 Rome, Italy
c
Istituto di Produzioni e Tecnologia Alimentari, Facoltà di Agraria, Università di Foggia,
Foggia, Italy

Received 10 October 2000;

revised 29 March 2001;
accepted 23 April 2001.
Available online 10 July 2001.

Abstract

In Italy, an attenuated Bacillus anthracis strain, named ‘Carbosap’, is used for

immunization against ovine and bovine anthrax. Analysis on ‘Carbosap’, Sterne vaccine
strain F34 and Pasteur vaccine strain SS104, were performed using primers specific for
the sequences, encoding the toxic factors, located on plasmids pXO1 and pXO2 and
primers specific for the chromosome. The results obtained from polymerase chain
reaction (PCR) assay revealed the presence of both plasmids pXO1 and pXO2 in
‘Carbosap’ strain. This study showed that the ‘Carbosap’ vaccine strain has a different
plasmid pattern in comparison to Pasteur vaccine strain SS104 and Sterne vaccine strain
F34.

Keywords: Anthrax; PCR; Vaccine

Detection of anthrax vaccine virulence factors by

polymerase chain reaction.
Fasanella A, Losito S, Trotta T, Adone R, Massa S, Ciuchini F, Chiocco D.

Source

Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Via Manfredonia 20,
71100, Foggia, Italy. izsfoggia@isnet.it

Abstract

In Italy, an attenuated Bacillus anthracis strain, named 'Carbosap', is used for

immunization against ovine and bovine anthrax. Analysis on 'Carbosap', Sterne vaccine
strain F34 and Pasteur vaccine strain SS104, were performed using primers specific for
the sequences, encoding the toxic factors, located on plasmids pXO1 and pXO2 and
primers specific for the chromosome. The results obtained from polymerase chain
reaction (PCR) assay revealed the presence of both plasmids pXO1 and pXO2 in
'Carbosap' strain. This study showed that the 'Carbosap' vaccine strain has a different
plasmid pattern in comparison to Pasteur vaccine strain SS104 and Sterne vaccine strain
F34.

Detection of Anthrax Toxin by an Ultrasensitive

Immunoassay Using Europium Nanoparticles
Shixing Tang,1* Mahtab Moayeri,2 Zhaochun Chen,3 Harri Harma,4 Jiangqin Zhao,1
Haijing Hu,2 Robert H. Purcell,3 Stephen H. Leppla,2 and Indira K. Hewlett1*

Laboratory of Molecular Virology, Center for Biologics Evaluation and Research, Food
and Drug Administration, Bethesda, Maryland 20892,1 Laboratory of Bacterial Diseases,
National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, Maryland 20892,2 Laboratory of Infectious Diseases, National Institute of
Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
20892,3 Laboratory of Biophysics, University of Turku, Turku FIN-20520, Finland4
Received 10 November 2008/ Returned for modification 8 December 2008/ Accepted 17
December 2008

We developed a europium nanoparticle-based immunoassay (ENIA) for the sensitive

detection of anthrax protective antigen (PA). The ENIA exhibited a linear dose-dependent
pattern within the detection range of 0.01 to 100 ng/ml and was approximately 100-fold
more sensitive than enzyme-linked immunosorbent assay (ELISA). False-positive results
were not observed with serum samples from healthy adults, mouse plasma without PA, or
plasma samples collected from mice injected with anthrax lethal factor or edema factor
alone. For the detection of plasma samples spiked with PA, the detection sensitivities for
ENIA and ELISA were 100% (11/11 samples) and 36.4% (4/11 samples), respectively.
The assay exhibited a linear but qualitative correlation between the PA injected and the
PA detected in murine blood (r = 0.97731; P < 0.0001). Anthrax PA was also detected in
the circulation of mice infected with spores from a toxigenic Sterne-like strain of Bacillus
anthracis, but only in the later stages of infection. These results indicate that the universal
labeling technology based on europium nanoparticles and its application may provide a
rapid and sensitive testing platform for clinical diagnosis and laboratory research.

* Corresponding author. Mailing address: Laboratory of Molecular Virology, Center for

Biologics Evaluation and Research, Food and Drug Administration, Building 29B, Room
4NN16, 8800 Rockville Pike, Bethesda, MD 20892. Phone: (301) 827-0795. Fax: (301)
480-7928. E-mail for Shixing Tang: Shixing.tang@fda.hhs.gov. E-mail for Indira K.
Hewlett: Indira.hewlett@fda.hhs.gov

Published ahead of print on 7 January 2009.

Clinical and Vaccine Immunology, March 2009, p. 408-413, Vol. 16, No. 3
1071-412X/09/$08.00+0 doi:10.1128/CVI.00412-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

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Nattel, S., Cook, J. L., Levitan, I. (2011). Anthrax Lethal Factor Activates K+
Channels To Induce IL-1{beta} Secretion in Macrophages. J. Immunol. 186:
5236-5243 [Abstract] [Full Text]
• Choi, J. S., Kim, S. G., Lahousse, M., Park, H.-Y., Park, H.-C., Jeong, B., Kim, J.,
Kim, S.-K., Yoon, M.-Y. (2011). Screening and Characterization of High-Affinity
ssDNA Aptamers against Anthrax Protective Antigen. J Biomol Screen 16: 266-
271 [Abstract] [Full Text]
• Tang, S., Zhao, J., Wang, A., Viswanath, R., Harma, H., Little, R. F., Yarchoan,
R., Stramer, S. L., Nyambi, P. N., Lee, S., Wood, O., Wong, E. Y., Wang, X.,
 Hewlett, I. K. (2010). Characterization of Immune Responses to Capsid Protein
p24 of Human Immunodeficiency Virus Type 1 and Implications for Detection.
CVI 17: 1244-1251 [Abstract] [Full Text]
• Tang, S., Hewlett, I. (2010). Nanoparticle-based immunoassays for sensitive and
early detection of HIV-1 capsid (p24) antigen. The Journal of Infectious Disease
201: S59-S64 [Abstract] [Full Text]
• Boyer, A. E., Quinn, C. P., Hoffmaster, A. R., Kozel, T. R., Saile, E., Marston, C.
K., Percival, A., Plikaytis, B. D., Woolfitt, A. R., Gallegos, M., Sabourin, P.,
McWilliams, L. G., Pirkle, J. L., Barr, J. R. (2009). Kinetics of Lethal Factor and
Poly-D-Glutamic Acid Antigenemia during Inhalation Anthrax in Rhesus
Macaques. Infect. Immun. 77: 3432-3441 [Abstract] [Full Text]

Detection of anthrax vaccine virulence factors by

polymerase chain reaction
A Fasanella, S Losito, T Trotta, R Adone, S Massa, F Ciuchini, D Chiocco

Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Via Manfredonia 20,
71100, Foggia, Italy.

Vaccine. 08/2001; 19(30):4214-8.

Abstract

In Italy, an attenuated Bacillus anthracis strain, named 'Carbosap', is used for

immunization against ovine and bovine anthrax. Analysis on 'Carbosap', Sterne vaccine
strain F34 and Pasteur vaccine strain SS104, were performed using primers specific for
the sequences, encoding the toxic factors, located on plasmids pXO1 and pXO2 and
primers specific for the chromosome. The results obtained from polymerase chain
reaction (PCR) assay revealed the presence of both plasmids pXO1 and pXO2 in
'Carbosap' strain. This study showed that the 'Carbosap' vaccine strain has a different
plasmid pattern in comparison to Pasteur vaccine strain SS104 and Sterne vaccine strain
F34.

Disease produced by anthrax toxin:

Antibody alters anthrax toxin

The University of Auckland
Wednesday, 22 September 2010
Anthrax disease is caused by the bacteria
Bacillus anthracis.
Image: dra_schwartz/iStockphoto

University of Auckland scientists have made an important discovery about how an

antibody against the anthrax toxin works, and their findings may have implications for
vaccine development for anthrax and for other similar infectious diseases.

Anthrax is an often-fatal disease caused by the bacteria Bacillus anthracis that can spread
from grazing animals to humans. It is now rare in developed countries but there are
periodic outbreaks in poorer nations, and concerns about its use in bioterrorism have
renewed global attempts to develop a vaccine. Anthrax toxin is responsible for much of
the harm caused by the disease.

“The anthrax toxin is made up of multiple proteins and only becomes active – and starts
doing damage – when those proteins cluster together in a very specific way,” explains
Associate Professor Alok Mitra who led the research.

“We have found that the neutralising antibody destroys the configuration of the toxin
cluster, adding more proteins to the mix and altering their arrangement so that they can no
longer act as they normally would.”

“It has been known for some time that this antibody is very effective at neutralising
anthrax toxin in the laboratory, but it is not a simple case of turning the antibody into an
immunisation device for humans. We need to know how the antibody works in order to
develop an effective vaccine and our research is an important step in this direction.”

The work also has much broader implications, demonstrating that a simple new method
(using transmission electron microscopy) can be used to identify other antibodies with
neutralising potential.

“Our research was the first to show that an antibody can structurally alter its target in this
way, but it is unlikely to be the only example in nature and may in fact prove to be an
important biological process,” explains Dr Mitra.

“We have shown that by mixing antibodies and their targets in the laboratory and looking
under a high-powered microscope for the kinds of abnormal clusters we have seen,
scientists can quickly and easily search for neutralising antibodies for many diseases.”

Researchers around the world are continuing in the attempt to create a traditional anthrax
vaccine using the neutralising antibody and related antibodies.

Dr Mitra says that the next steps for his team will be to study in greater detail how the
abnormal complexes are formed and exactly where the antibody binds to the toxin. This
may allow the development of small synthetic proteins to mimic the antibody’s activity
that could be given prior to exposure to the toxin, as an alternative method of vaccination.

The current research, published in the Proceedings of the National Academy of Sciences
was undertaken by scientists in the School of Biological Sciences at The University of
Auckland, using a neutralising antibody provided by collaborators at the National
Institutes of Health in the United States.
Ref: http://www.sciencealert.com.au/news/20102309-21350.html

by John Dudley Miller

NEWS ANALYSIS Be cereus about anthrax toxin

Email: John Dudley Miller - johnmiller@nasw.org

News from The Scientist 2004, 5(1):20040521-02

Published 21 May 2004

A plasmid coding for the anthrax toxin has been found in a sample of Bacillus
cereus taken from a person who survived a life-threatening pneumonia
symptomatic of inhaled anthrax. The finding, reported in the advanced online
edition of PNAS, is the first time the complete plasmid has been found in any
naturally occurring microbe other than Bacillus anthracis.

The study grew out of a Centers for Disease Control and Prevention (CDC) review
of reported cases of unusually severe illnesses caused by these or other Bacillus
species. The researchers did not study cases of anthrax of the skin. They note that 2
years ago, other scientists found one of the genes for the anthrax toxin in two fatal
cases of pneumonia caused by B. cereus. But those samples were not further
characterized, so if the plasmid was present, it was not found.

B. cereus is known to cause food poisoning, but rarely causes pneumonia.

However, when scientists at the CDC and at the Institute for Genomic Research
(TIGR) quickly sequenced the DNA of their pneumonia-causing sample, they
found a plasmid outside the bacterium's single chromosome that is 99.6%
genetically similar to the plasmid in anthracis, pX01. In anthrax, that codes for the
three-part toxin.

Although the researchers did not find a homologue to pX02—the anthracis

plasmid–containing genes that create an impenetrable capsule surrounding and
protecting the bacterium—they found a different plasmid coding for a similar
capsule, which may serve the same function.

Researchers at the US Naval Medical Research Center showed that when they
injected mice known to be sensitive to B. anthracis peritoneally with the plasmid-
containing B. cereus, all the mice died within 14 days. “It may not be appropriate
to consider B. anthracis, as currently defined, as the only species capable of
causing inhalation anthrax-like disease,” the study concludes.

The study's authors say their research proves that scientists could respond rapidly
to newly emerging diseases, both those caused naturally or others manufactured by
bioterrorists by altering existing diseases. “These findings show that genomics can
rapidly assist public health experts in responding to novel pathogens,” Claire
Fraser, one of the authors and the president of the Institute for Genetic Research,
said in a statement.

But for public health agencies to be able to respond quickly and reliably, the
authors point out, the agencies must revise the way they monitor for anthrax cases,
shifting from analysis of isolates to evaluation of the similarity of the patient's
symptoms to anthrax. Now, when a Bacillus sample is found to be able to move
and to destroy red blood cells, it is assumed not to cause inhalation anthrax
because anthrax cannot do that.

“Normally, when we look for anthrax, we say that it has to be non-hemolytic and
non-motile, whereas this one [B. cereus] is motile and hemolytic,” said
Nammalwar Sriranganathan, an associate professor of biomedical sciences and
pathobiology at Virginia Tech who is developing an anthrax vaccine, but who was
not involved in the present study.

No one knows how this particular isolate of B. cereus acquired its plasmids, but the
feat doesn't surprise Sriranganathan or Arthur Aronson, a professor of biological
sciences at Purdue University who studies the B. cereus variant strain ATCC 4342.
They said the bacillus family of bacteria is known to be capable of “gene
jumping”—transferring genes laterally from one species to another.

However, Aronson said he is not convinced that the B. cereus isolate actually
caused anthrax in the human patient or in the mice that were injected with it. He
said the descriptions of their symptoms do not uniquely describe inhalation
anthrax.

Aronson also said that mice are not a good animal model for anthrax and that
rabbits are better. Moreover, injecting mice in their peritoneal cavities may not
produce the same results as making them inhale the isolate.

“So what they really needed to do,” Aronson said, “is get rid of that plasmid from
that B. cereus isolate to see if it was still pathogenic or disrupt the gene that
encodes one of the principal toxins. That would have been definitive. In the
absence of that, I don't think it's fully convincing evidence.”

“We totally agree,” said Alex Hoffmaster, the lead author of the paper. “It's very
intriguing that this organism caused the very severe disease and it had these
plasmids. But we have not shown that these plasmids contributed to the disease, so
we don't know that this organism produced anthrax toxin and the patient suffered
from anthrax toxin.”

Editor's Note: See The Scientist's special issue on biosecurity here.

Correction (posted May 25): When originally posted, this story incorrectly stated
that the capsule surrounding B. cereus protects it even while the organism is
dormant in soil. The Scientist regrets the error.

Return to top

References

1 [http://www.pnas.org/cgi/content/abstract/0402414101v1]
.
A.R. Hoffmaster et al., “Discovery of anthrax toxin genes in a Bacillus cereus
associated with an illness resembling inhalation anthrax: Public health
challenges in the age of bioterrorism,” PNAS, May 21, 2004.
Return to citation in text: [1]

2 [http://www.tigr.org/faculty/Claire_Fraser.shtml]
 .
Claire Fraser
Return to citation in text: [1]

3 [http://www.vetmed.vt.edu/Organization/Departments/DBSP/faculty/nathan.asp]
.
Nammalwar Sriranganathan
Return to citation in text: [1]

4 [http://www.gradschool.purdue.edu/gradschool/PULSe/faculty/aronson.html]
.
Arthur Aronson
Return to citation in text: [1]

5 [http://www.the-scientist.com/yr2004/may/edit_040524.html]
.
R. Gallagher, "Choices on biosecurity," The Scientist, May 24, 2004.
Return to citation in text: [1]

Read more: Be cereus about anthrax toxin - The Scientist - Magazine of the Life Sciences
http://www.the-scientist.com/article/display/22192/#ixzz1O0nTN53v

Ref: http://www.the-scientist.com/news/20040521/02/

Hyaline Fibromatosis Syndrome inducing mutations in

the ectodomain of anthrax toxin receptor 2 can be
rescued by proteasome inhibitors

Authors
Julie Deuquet, Ekkehart Lausch, Nicolas Guex, Laurence Abrami, Suzanne Salvi, Asvin
Lakkaraju, Maria Celeste M. Ramirez, John A. Martignetti, Dariusz Rokicki, Luisa
Bonafe, Andrea Superti‐Furga, Françoise G. van der Goot

Abstract

Hyaline Fibromatosis Syndrome (HFS) is a human genetic disease caused by mutations

in the anthrax toxin receptor 2 (or cmg2) gene, which encodes a membrane protein
thought to be involved in the homeostasis of the extracellular matrix. Little is known
about the structure and function of the protein or the genotype–phenotype relationship of
the disease. Through the analysis of four patients, we identify three novel mutants and
determine their effects at the cellular level. Altogether, we show that missense mutations
that map to the extracellular von Willebrand domain or the here characterized Ig‐like
domain of CMG2 lead to folding defects and thereby to retention of the mutated protein
in the endoplasmic reticulum (ER). Mutations in the Ig‐like domain prevent proper
disulphide bond formation and are more efficiently targeted to ER‐associated
degradation. Finally, we show that mutant CMG2 can be rescued in fibroblasts of some
patients by treatment with proteasome inhibitors and that CMG2 is then properly
transported to the plasma membrane and signalling competent, identifying the ER folding
and degradation pathway components as promising drug targets for HFS.

Ref:
http://www.embomolmed.org/details/journalArticle/1048271/Hyaline_Fibro
matosis_Syndrome_inducing_mutations_in_the_ectodomain_of_anthrax_to.
html

Anthrax: modern exposure science combats a deadly,

ancient disease
John R Barra, Anne E Boyera and Conrad P Quinna
a
US Centers for Disease Control and Prevention, Atlanta, Georgia, USA

Correspondence: John R Barr, jbarr@cdc.gov

We now have the technology to detect and quantify anthrax toxin levels. This technology
has tremendous promise for diagnosis at very early stages of disease and monitoring the
efficacy of treatments, including those for disease arising from anthrax-related terrorist
events.

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BACKGROUND

Exposure to the spores of Bacillus anthracis causes the disease anthrax. Spores gain entry
into the host through dermal abrasions, gastrointestinal lesions, injection (usually in
intravenous drug use), and inhalation, which cause cutaneous, gastrointestinal, injection,
and inhalation anthrax, respectively.

Anthrax is a naturally occurring disease in many parts of the world, and it remains a
bioterrorism threat. Inhalation anthrax has a mortality rate higher than 90% if untreated
and is often deadly even with antibiotic therapy. During the anthrax-letter attacks of
2001, mortality was 45%, even with antibiotics and aggressive supportive care (Jernigan
et al., 2002). Additionally, there is a “point of no return” during anthrax infection after
which all traditional treatments may fail.

The lethal consequences of this disease are caused by the anthrax toxins. “Lethal toxin”
inactivates cell components central to the victims’ defense against infection. ”Edema
toxin” causes massive fluid retention and swelling (edema), as well as depressing some
parts of immune defenses. Both of these toxins also cause bleeding late in the course of
infection.

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IMPACT AND IMPLICATIONS FOR EXPOSURE SCIENCE

Diagnosis of anthrax in the early stages of disease is critical for effective treatment and
thus to saving lives. Mass spectrometric detection and quantification of anthrax toxins are
highly selective and rapid diagnostic tools for early disease. The ability to quantify the
toxins provides an additional benefit in understanding the course of disease and the
efficacy of treatments and therapies. For the anthrax toxins to be detected early in the
disease, they must be quantified at exquisitely low levels. Exposure science techniques
have been developed to achieve this (Boyer et al., 2007).

Detection and quantification of the anthrax toxin lethal factor represent a major
advancement in the diagnosis of anthrax. The anthrax exposure science technology has
the following important features:

Detects infection earlier. Monitoring lethal factor detects B. anthracis infection as early
as 12 hours after exposure to anthrax spores and as much as 24 hours earlier than other
technologies (Boyer et al., 2009). Anthrax can be detected even before symptoms such as
fever are present.

Shorter time to the first result. Quantitative lethal factor measurements and analyses are
completed within 4 hours. By comparison, traditional biochemical approaches to
diagnosis anthrax may take several days to produce definitive results.

Analyzes many more samples per day. The new method to measure lethal factor has been
automated and can analyze hundreds to thousands of samples per day. This throughput
represents a significant advancement over previous techniques.

Not subject to antibiotic interference. Quantitative lethal factor monitoring can be used to
identify and track infection even if antibiotic treatment has started, unlike with historical
methods, which cannot consistently detect infection in people undergoing treatment.

Quantifies toxin to track course of infection and responses to treatment. Quantification of

lethal factor is an excellent measure of treatment effectiveness by tracking changes in
toxin levels. This capability could be helpful in the clinical management of patients who
are not progressing well. For example, such information could help doctors determine
whether additional or alternative therapeutics are necessary.

Monitoring anthrax lethal factor levels in animal and clinical studies has already yielded
insights into the course of infection and responses to treatment. The animal study data
show that the progression of infection can occur in three stages, during which traditional
diagnostic tests can be at first positive but then transiently revert to negative, only to
become positive again later in infection. In contrast to these traditional tests,
quantification of anthrax lethal factor is detected throughout all stages of infection (Boyer
et al., 2009). In addition, the animal studies have shown that lethal factor levels may be
predictive of the stage or severity of illness, and they have been used to establish a
potential toxin-related point of no return, when mortality is very likely. This would
indicate that additional therapeutics are required to remove high toxin levels from the
blood. Quantification of lethal factor levels on a daily basis during a clinical case would
allow physicians to monitor changes and adjust treatment accordingly.

The application of exposure science to detect and quantify anthrax lethal factor levels has
recently been used to respond to clinical cases of inhalation (Walsh et al., 2007),
cutaneous, injection, and gastrointestinal anthrax. A great deal has been learned about the
course of B. anthracis infection in humans and the efficacy of treatments. The
information gained can be used to evaluate therapeutics and help guide medical
intervention. The use of exposure science to detect and quantify toxins produced by
human pathogens can have a major impact on the diagnosis and treatment of an even
wider range of infectious diseases.

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References

1. Boyer A.E., Quinn C.P., Woolfitt A.R., Pirkle J.L., McWilliams L.G., Stamey
K.L. et al. Detection and quantification of anthrax lethal factor in serum by mass
spectrometry. Anal Chem 2007: 79: 8463–8470.
2. Boyer A.E., Quinn C.P., Hoffmaster A.R., Kozel T.R., Saile E., Marston C.K. et
al. Kinetics of lethal factor and poly-D-glutamic acid antigenemia during
inhalation anthrax in rhesus macaques. Infect Immun 2009: 77: 3432–3241.
3. Jernigan D.B., Raghunathan P.L., Bell B.P., Brechner R., Bresnitz E.A., Butler
J.C. et al. 2002. Investigation of bioterrorism-related anthrax, United States, 2001:
epidemiologic findings. Emerg Infect Dis 2002: 8: 1019-1028
4. Walsh, J.J., Pesik N., Quinn C.P., Urdaneta V., Dykewicz C.A., Boyer A.E. et al.
A case of naturally acquired inhalation anthrax: clinical care and analyses of anti-
protective antigen immunoglobulin G and lethal factor. Clin Infect Dis 2007: 44:
968–971.

Ref: http://www.nature.com/jes/journal/v20/n7/full/jes201049a.html