REVIEW

10.1111/j.1469-0691.2010.03213.x

Pathogenesis and immunity in enterococcal infections

I. G. Sava1, E. Heikens2 and J. Huebner1
1) Division of Infectious Diseases, Department of Medicine, University Medical Center Freiburg, Germany and 2) Department of Medical Microbiology,
University Medical Center Utrecht, The Netherlands

Abstract
Enterococcus faecalis and Enterococcus faecium have emerged as multi-resistant nosocomial pathogens in immunocompromised and critically ill patients. Multi-resistant strains have acquired virulence genes resulting in hospital-adapted clones. The following review summarizes several proteins and carbohydrate- or glycoconjugates that have been identied as putative virulence factors involved in the
pathogenesis of enterococcal infections and may be used as targets for alternative therapies. Several studies describing the host immune
response against enterococci are also summarized.
Keywords: Enterococcus faecalis, Enterococcus faecium, immunity, review, vaccine, virulence factors
Clin Microbiol Infect 2010; 16: 533540
Corresponding author and reprint requests: J. Huebner, Division
of Infectious Diseases, Department of Medicine, University Medical
Center Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany
E-mail: johannes.huebner@uniklinik-freiburg.de

Introduction
Enterococci have until recently been considered as harmless
commensals, especially when compared to other more virulent Gram-positive pathogens. However, there is growing
evidence that these bacteria frequently possess several specic traits that enable them to survive in the hospital environment, colonize patients, and cause infections such as
bacteraemia, peritonitis, endocarditis and urinary tract,
wound, and device-related infections. Several recent studies
have indentied putative virulence factors in E. faecalis, but
also in E. faecium, albeit to a lesser extent (see Table 1). A
better understanding of the factors that enable enterococci
to cause infections and of the host immune response to
these pathogens can contribute to the development of new
therapeutic and prophylactic approaches, which may help to
prevent nosocomial spread of and infections with these
bacteria.

Aggregation Substance
Aggregation substance (AS) is the description used for a
group of surface proteins encoded on pheromone-inducible
conjugative plasmids. AS promotes conjugation by directing

bacterial aggregation, resulting in close cell contact between

donor and recipient [1]. Expression of AS proteins is induced
by a peptide pheromone, which is secreted by plasmid-free
recipient cells [2,3], but can also be induced by a host factor
during in vivo growth [4,5]. Asa1, Asp1, and Acs10 are the
best studied AS proteins and show over 90% amino acid
sequence identity [1,6,7]. AS proteins contain an N-terminal
domain, a variable region, a central domain and two Arg-GlyAsp (RGD) motifs. For Asc10 it has been demonstrated that
both the N-terminal domain and the central domain are
required for aggregation [8]. The N-terminal aggregation
domain promoted binding to cell wall lipoteichoic acid (LTA)
whereas the central aggregation domain did not. Besides its
role in conjugation, AS has also that of a virulence factor in
E. faecalis. Asa1 increased adherence to renal tubular cells
[9] and adherence to and survival in human macrophages
[10]. Asc10 increased internalization [11] and intracellular
survival [12] in polymorphonuclear leukocytes (PMNs). The
RGD motifs probably mediate the interactions with eukaryotic cells [10,11]. Both Asc10 and Asa1 are involved in
adherence to several extracellular matrix proteins [13] and
both increased virulence of E. faecalis in a rabbit endocarditis
model [5,14,15]. For Asc10 it has been demonstrated that
the N-terminal aggregation domain and the RGD motif contribute to virulence during experimental endocarditis [16].
AS did not contribute to virulence in a mouse model of

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TABLE 1. Putative virulence factors in E. faecalis and E. faecium

E. faecium (Efm)
Esp

Acm

Pili
PilA
PilB
Scm
EcbA
E. faecalis (Efa)
AS
Asa1
Asp1
Asc10

Esp
Gelatinase/FSR

Pili
ebp locus

bee locus
Ace
cps locus
epa locus

LTA
WTA
Glycolipids

Pathophysiology/Virulence

Epidemiology

References

Biolm formation
Pathogenesis of rat endocarditis
Pathogenesis of mouse UTIa
Antigenic in humans during endocarditis and bacteraemia
Binding to collagen type I, IV
Pathogenesis of rat endocarditis
Antigenic in humans during endocarditis

Specically linked to HAb Efm

Wide spread among Efm

[34,35], [109th General

Meeting of the American
Society of Microbiology,
Posters B-167 and B-211]
[66,67,70]

Not known

Wide spread among Efm

[53]

Binding to collagen type V

Binding to collagen type V
Binding to brinogen

Widespread among Efm

Specically linked to HA Efm

[59]
[68,69]

Promotes conjugation by directing bacterial aggregation

Internalization into intestinal epithetial cells
Binding to renal tubular cells
Binding to and survival in macrophages
Binding to ECM
Pathogenesis of rabbit endocarditis
Internalization into and survival in PMNsc
Binding to ECM
Pathogenesis of rabbit endocarditis
Biolm formation
Pathogenesis of mouse UTI
Biolm formation
Pathogenesis of mouse peritonitis
Caenorhabditis elegans infection
Pathogenesis of rabbit endophthalmitis

Widespread among Efa

[1,1820]

Biolm formation
Pathogenesis of endocarditis
Pathogenesis of UTI
Antigenic in humans during endocarditis
Biolm formation
Binding to collagen type I, IV
Binding to laminin and dentin
Resistance to complement and PMNs-mediated killing
Biolm formation
Enterocyte translocation
Resistance to killing by PMNs
Resistance to infection by phages
Pathogenesis of mouse peritonitis
Pathogenesis of mouse UTI
Target of opsonic antibodies
Not known
Biolm formation
Binding to colonic epithelial cells
Pathogenesis of mouse sepsis

[9,10]
[1315]
[5,1115]

Widespread among Efa

[2427]

Widespread among Efa

[40,4245]

Widespread among Efa

[52,56,58]

Among 5% of Efa
Widespread among Efa

[54]
[6063]

Efa serotype C and D strains

Present in Efa cell wall

[73,8587]
[79,8284]

Present in enterococcal cell wall

Present in enterococcal cell wall
Present in enterococcal cell membrane

[74]
[72]
[89,90]

UTI, urinary tract infection.

HA, hospital-associated.
PMNs, polymorphonuclear leukocytes.

b
c

urinary tract infection [17]. However, AS proteins did facilitate bacterial internalization by different cultured intestinal
epithelial cells [1820], indicating that they might be involved
in the translocation of E. faecalis through the intestinal epithelia, leading to systemic infection.

Enterococcal Surface Protein

One putative virulence determinant is the enterococcal surface protein gene esp located on a pathogenicity island (PAI)
in both E. faecalis and E. faecium [21,22]. Esp proteins are
expressed in both species at the surface of the bacterium.
They contain an N-terminal signal sequence and an N-termi-

nal domain followed by three repeat domains designated A,

B, and C, with the number of repeats varying among strains
[21,23]. The C-terminal end of Esp consists of a (Y/F)PxTG
motif, which is presumably responsible for cell-wall anchoring.
In E. faecalis, Esp has been identied as a putative virulence factor involved in colonization of the urinary tract
[24]. Conicting results have been reported about the role
of E. faecalis Esp in biolm formation, ranging from signicant loss of biolm formation in E. faecalis Esp-decient
mutants [25,26] to no apparent effect [27]. Also, no correlation was found in different studies between the presence
or absence of esp in clinical isolates and biolm formation
[2831].

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Sava et al. Pathogenesis and immunity in enterococcal infections

Recently, more information has been presented about the

function of E. faecium Esp. Interestingly, in contrast to E. faecalis esp, which is widely spread among strains, E. faecium esp
is predominantly present in hospital-acquired isolates [21,32],
suggesting a role in virulence. It has been shown that esp
expression is affected by changes in environmental conditions, being highest at 37C and under anaerobiosis [33].
Furthermore, expression of esp was correlated with initial
adherence to polystyrene and biolm formation [33]. By constructing an esp-decient mutant and by using different animal models, Heikens et al. [34] demonstrated that Esp of
E. faecium is involved in biolm formation and contributes to
the pathogenesis of experimental endocarditis [109th General Meeting of the American Society of Microbiology, Poster
B-167], urinary tract infection (UTI) [35], and bacteraemia
[109th General Meeting of the American Society of Microbiology, Poster B-211]. No specic role for Esp was found in
peritonitis [35] and colonization of the gastro-intestinal tract
[36]. The importance of E. faecium Esp in UTI and endocarditis probably results from the fact that these models represent typical biolm-associated infection models indicating a
specic role of Esp in the pathogenesis of E. faecium infections.

Gelatinase and fsr Two-Component System

One well-studied virulence factor of E. faecalis is a secreted
bacterial protease called gelatinase which is controlled by the
fsr two-component system comprised of four genes (fsrA,
fsrB, fsrD, and fsrC) [37]. The fsr system responds to the
extracellular accumulation of a peptide lactone, [38] which is
encoded by frsD. It is believed that this peptide is processed
from the FsrD protein and is exported to the extracellular
space by the gene product of fsrB [37]. Extracellular accumulation of the peptide is probably sensed by the FsrC histidine
kinase, subsequently leading to activation of the response
regulator and transcription factor FsrA by a phosphoryl
group. Phosphorylated FsrA activates expression of two
cotranscribed protease genes, gelE and sprE, located downstream of the fsr locus and encoding gelatinase and serine
protease [39]. Microarray results demonstrated that the fsr
locus activates expression of a variety of other genes potentially involved in virulence and metabolic pathways [40].
Mutations in the fsr operon and inactivation of the fsr-controlled gene gelE demonstrated that gelatinase has an important role in biolm formation by E. faecalis, and that the fsr
system controls biolm formation through production of
gelatinase [29]. It was also demonstrated that gelatinase
enzymatic activity is required for biolm formation [29]

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which is probably established by C-terminal processing of

the gelatinase protein [41]. Besides their role in biolm formation, gelatinase and the fsr system have been implicated in
virulence in different animal infection models, including
mouse peritonitis [42,43], Caenorhabditis elegans infection
[44] and rabbit endophthalmitis [45]. Epidemiological data
indicate involvement of gelatinase and the fsr locus in virulence [4648]. Together these data suggest an important role
of the fsr system in the virulence of E. faecalis.

Pili
Pili of Gram-positive bacteria have been implicated in adhesion to multiple types of human cells and in biolm formation, two processes critical in the pathogenesis of many
bacterial diseases [4952]. E. faecalis and E. faecium harbour
pilin gene clusters (PGCs), comprising genes encoding surface proteins with LPxTG-like motifs and sortases, which are
required for pilus assembly [52,53]. E. faecalis harbors two
PGCs, designated as the ebp locus (endocarditis and biolmassociated pili) [52] and the bee locus (biolm enhancer in
enterococci) [54].
Ebp pili seem to be involved in initial adherence and biolm formation [52]. They contribute to the pathogenesis of
experimental endocarditis [52] and urinary tract infection
[55,56] which are both biolm-associated infections. In addition, it was found that Ebp pili are antigenic in humans during
endocarditis [57]. The bee locus is located on a conjugative
plasmid and was only detected in 5% of the E. faecalis isolates, whereas the ebp operon was found in almost all of the
isolates [58]. E. faecium harbours four PGCs, designated
PGC-1 to -4 [53]. Expression of PilA and PilB was demonstrated in a hospital-acquired E. faecium isolate, proving that
E. faecium can express two different types of pili at the surface of a single cell [53]. It remains to be determined
whether these pili are implicated in virulence of E. faecium;
however, their genes display high similarity with genes of the
ebp operon of E. faecalis, suggesting a possible role of these
pili in interactions with the mammalian host.

Enterococcal MSCRAMMs
Colonization of human tissues is assumed to occur via interactions between specic proteins in the extracellular matrix
and the so-called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). The publicly
available genome sequences of E. faecalis V583 and E. faecium
TX0016 revealed the presence of 17 and 15 MSCRAMMs,

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respectively [57,59]. So far, seven enterococcal MSCRAMMs

have been characterized in detail: Ace (adhesion of collagen
of E. faecalis), Fss1, Fss2, and Fss3 (E. faecalis surface protein)
of E. faecalis and Acm (adhesin of collagen of E. faecium),
Scm (second collagen adhesin of E. faecium) and EcbA (E. faecium collagen binding protein A) of E. faecium. Ace was
shown to bind to collagen types I and IV, laminin and dentin
[6062]. Furthermore, Ace was shown to be involved in the
pathogenesis of experimental endocarditis. It was demonstrated that rats immunized against recombinant Ace (rAce)
or given specic anti-rAce antibodies were less likely to
develop E. faecalis endocarditis [63]. Fss1, Fss2, and Fss3 bind
to brinogen [64,65], targeting however different brinogen
polypeptide chains.
Acm binds to collagen type I and to a lesser extent to collagen type IV [66], contributing to the pathogenesis of experimental endocarditis and being antigenic in humans during
endocarditis [67]. Both Scm and EcbA bind to collagen type
V [59,68] and EcbA also binds to brinogen [68]. The presence of ecbA is associated with hospital-acquired E. faecium
isolates [69]. The other MSCRAMMs are widely found
among clinical and non-clinical isolates. Interestingly, a functional acm gene is predominantly present in clinical isolates,
whereas a non-functional acm gene, containing an insertion
element, is mainly present in non-clinical isolates [70].

Enterococcal Cell Wall and Capsular

Polysaccharides
The bacterial cell wall represents a complex structure allowing bacteria to interact with their environment and providing
the factors needed for growth in their ecological niche. In
the infected host it separates the interior of the bacteria
from harmful inuences exerted by the surrounding milieu
[71]. The enterococcal cell wall contains peptidoglycan, proteins, polysaccharides, lipids, lipoproteins and several glycoconjugates. The most abundant glycopolymers are wall
teichoic acid (WTA) and lipoteichoic acid (LTA). WTA is a
polymer usually consisting of a glycosylated and D-alaninesubstituted alditol phosphate repeating unit covalently
attached to the peptidoglycan via a linkage unit [72]. So far,
the contribution of this molecule to enterococcal pathogenesis has not been investigated.
Hufnagel et al. [73] grouped E. faecalis strains into four
capsular serotypes (AD). It was recently shown that the
antibodies used to classify serotype A strains actually recognize LTA [74]. Unpublished observations (Sava, personal
observations) based on cross-reactivity in ELISA and opsonophagocytosis assays showed that the predominant antigen of

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serotype B strains is also LTA. Investigations of the role of

LTA in enterococcal pathogenicity were hampered by the
fact that mutants lacking LTA are lethal [75].
However, information obtained from mutants decient in
alanination of LTA indicated that esterication of LTA with
D-alanine changes the net charge of this polymer, the defect
in D-alanination being associated in E. faecalis with higher
susceptibility to antimicrobial peptides and with reduced ability to form biolm and to adhere to eukaryotic cells [76].
Several studies have demonstrated the presence of an
enterococcal polysaccharide antigen, designated Epa, in the
E. faecalis cell wall that seems to be recognized by sera from
patients with systemic enterococcal infections [7780]. The
epa locus consists of 16 open reading frames, and the polysaccharide produced by the enzymes encoded by this locus
is thought to be a rhamnose-containing polysaccharide that is
widespread among E. faecalis strains. The structure of the
Epa polysaccharide has not yet been elucidated and preliminary information indicates that its localization might be inside
the bacterial cell wall [80]. This carbohydrate seems to play
an important role in enterococcal pathogenicity, since disruption of different genes of the epa locus results in decreased
biolm formation [81] and enterocyte translocation [82],
lower resistance to killing by PMNs [83], higher bacterial
susceptibility to infection by phages [84] and reduced virulence in mouse peritonitis [79] and urinary tract infection
[84] models.
In the serotyping system described by Hufnagel et al. [73]
it was shown that serotype C and D strains are resistant to
opsonophagocytosis by antiserum directed against the serotype A strains (i.e. anti-LTA serum), suggesting the presence
of a capsule that masks LTA and thereby prevents opsonisation by anti-LTA antibodies. Bacterial capsules are a major
determinant of virulence for many pathogens, contributing to
evasion of the hosts immune system by preventing complement activation and/or killing by phagocytes. Several studies
[8587] conrmed the ndings of Hufnagel et al. regarding
the synthesis of a capsule by E. faecalis serotype C and D
strains, but not by serotypes A and B strains. A capsule locus
in E. faecalis, designated cps, contains nine genes (cpsC
cpsK). CpsF encodes a putative glucosyltransferase which is
probably responsible for the serodiversity between serotype
C and D strains, modifying the ratio between glucose and
galactose in the two types of capsules [86]. It was shown in
a recent study that the polysaccharides produced by the
enzymes encoded by the cps locus contribute to enterococcal virulence by conferring resistance to complementmediated opsonophagocytosis of serotype C and D strains [87].
However, to date, the chemical structures of these capsular
polysaccharides have not been published.

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Regarding E. faecium, published information about capsular

polysaccharides is completely lacking. In a study by Huebner
et al. [88] about one-third of the clinical isolates tested were
efciently killed by anti-LTA antibodies, suggesting that these
strains do not express a capsule.

Glycolipids
Recent studies demonstrated an important role of enterococcal membrane glycolipids in bacterial virulence [89,90]. These
molecules are involved in the formation of a permeable layer
between the cytoplasm and the environment. In many
Gram-positive bacteria they also function as anchor of LTA,
representing the hydrophobic part of this amphiphilic polymer
[91,92]. A specic glycolipid, a-diglycosyl-diacylglycerol (DGlcDAG), is the most abundant in E. faecalis, accounting for 8
37% of all polar lipids of the cell membrane. Other glycolipids
are mostly a-monoglycosyl-diacylglycerol (MGlcDAG) and
small traces of a-triglycosyl-diacylglycerol (TGlcDAG) [91,92].
DGlcDAG, either by itself or as part of the LTA molecule, has
recently been demonstrated to specically recognize and bind
the glycosaminoglycans heparin and heparan sulfate present
on the surface and/or in the extracellular matrix of human
colonic cells, representing an important molecule involved in
bacterial adhesion to host tissues [89].
An E. faecalis mutant unable to produce DGlcDAG,
through a deletion in the biolm-associated glycolipid synthesis A gene (bgsA) encoding a putative glucosyltransferase,
showed reduced ability to adhere to enterocytes and to
form biolm on plastic surfaces [90].
The DGIcDAG-decient mutant was cleared more rapidly
from the bloodstream of mice compared to wild-type bacteria
in a bacteraemia model. Since both strains are complementresistant, differ very little in their sensitivity to opsonophagcytic killing and are equally susceptible to antimicrobial peptides,
this suggests that persistence of E. faecalis in the blood stream
might be related to its ability to form biolm [90], conrming
previous studies using a mouse bacteraemia model. Deletion
mutants of the sugar-binding transcriptional regulator bopD
[93] and of the D-alanine-poly(phosphoribitol) ligase dltA
(unpublished observations) genes in E. faecalis, which are also
defective in biolm formation, were also less virulent in the
mouse bacteraemia model.

Host Immune Response Against Enterococci

Several recent studies investigated the host immune
response, in particular to E. faecium. Using a non-lethal

537

peritonitis model in mice it was determined that the normal

immune system reacts to E. faecium infection with a rapid
neutrophil inux into the peritoneal cavity causing a consecutive rapid decline in peritoneal and systemic enterococcal
load. Recognition by TLR2 (Toll-like receptor 2) and subsequent signaling through MyD88 [94], peritoneal macrophages
[95], neutrophil inux [96] and opsonization of bacteria by
complement [97] were all found to be important in the early
containment of E. faecium peritonitis. Depletion of these cells
and molecules caused prolonged high peritoneal loads of
E. faecium with subsequent more serious systemic infection.
Interestingly, eventually all mice were able to clear the bacteria. Apparently, in the healthy host, deciency of one part of
the immune system is counterbalanced by other components
of the immune system. This parallels the clinical situation
where especially severely ill, immunocompromised patients
are vulnerable to infections with enterococci.
To mimic this scenario, mice were rendered immunocompromised by inducing a sterile acute-phase response (APR)
or a sepsis before causing E. faecium peritonitis.
Both APR and sepsis impaired the host defence against
E. faecium suggesting that these conditions may contribute to
the increased vulnerability of critically ill patients to enterococcal infections [98,99].

Conclusions
In this review an overview was given describing cell surface
determinants and structures potentially important in enterococcal pathogenicity and immunity.
Most putative virulence factors found so far in E. faecalis
and E. faecium are involved in adherence to extracellular
structures and biolm formation, important processes in initiating colonization of and infection in the host. In healthy
mice, different components of the normal immune system,
including TLRs, neutrophils, macrophages, and the complement system were found to be important in containing
E. faecium infections. Deciency of one of these components
did not necessarily result in inability to clear the infection,
suggesting compensation of one component by the other.
This parallels the clinical situation demonstrating the moderate virulent nature of E. faecium which is usually not capable
to cause overwhelming infections in the healthy host.
Different surface structures have been identied in E. faecalis and E. faecium that may be promising candidates for the
development of alternative treatment and prevention strategies against enterococcal infections. Only few of them have
been tested in appropriate animal models. Cell-wall carbohydrate antigens (such as LTA and capsular polysaccharides)

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and the enterococcal MSCRAMM Ace have been shown to

be a target of protective antibodies. Several additional protein antigens could also be targets of a protective immune
response, although appropriate protection studies have not
been published so far. Interference with the expression of
specic virulence factors could be exploited for novel treatments, although, to date, no precedent of such a therapeutic
approach has been tested clinically. Some of these new
developments may eventually offer the prospect of replacing
the increasingly obsolete classes of antibiotics against which
enterococci have, so successfully, developed resistances.

11.

12.

13.

14.

Transparency Declaration
The authors have declared that no conict of interest exists.

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Journal Compilation 2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 533540