Cell WallAssociated Virulence Factors

In addition to hemolysins and proteases, E. faecalis contains a number of cell-wall-associated proteins that could potentially be involved in pathogenesis. The ability to adhere to host tissues is a critical step in the onset of most microbial infections. Recent examination of the E.faecalis genome has identified the presence of 41 putative cell-wall-anchored proteins, and 17 of these were predicted to contain structural characteristics of the MSCRAMM (microbial surface component recognizing adhesive matrix molecules) type [95]. In addition to this, the E. faecalis genome contains 134 predicted surface-exposed proteins, of which 65 contain repeat regions and may have a role in antigenic variation via a slippage mechanism [8].

The only characterized adhesion from E.faecalis is Ace (EF1099), a 674-amino-acid protein that has similarities with the central region of the A domain of the collagen-binding protein Cna of S. aureus [96]. Ace is thought to be involved in E. faecalis collagen binding, as mutation of Ace showed reduction in 46 °C growth-elicited binding to immobilized collagen type I, collagen type IV, and mouse lami-nin [97, 98], and Ace-specific antibody has been shown to inhibit this binding in wild-type E. faecalis [96, 97].

Present in the V583 genome is a putative internalin protein family protein (EF2686), which has 29% identity to InlA from L. monocytogenes and contains a premature stop codon. The internalin family of proteins is characterized by the presence of leucine-rich repeats. In L. monocytogenes the receptor for InlA is E-cad-herin, and this protein is required for cell invasion [99, 100]. The interaction of internalin A with E-cadherin on enterocytes is an early critical step for the onset of listeriosis in vivo [101]. In some strains of Listeria, this protein is truncated, but clinical strains express a full-length protein more frequently [102]. Disruption of a homologue of the internalin family of proteins in group A Streptococcus has been shown to result in reduced virulence and increased susceptibility to phagocytosis [103]. Interestingly, EF2686 in E.faecalis was found to be induced 162-fold in stationary phase when cultured in urine compared to culture in 2YT broth, showing an induction of gene expression in an infection-type environment [47].

Aggregation substance (EF0149, EF0485, EFA0047, EFB0011) is expressed on the cell surface of E. faecalis, allowing close contact for conjugation. This protein has been implicated as a virulence trait, due to the fact that cells expressing it have increased adherence and internalization into phagocytes [104, 105], renal cells [106], and epithelia [107-110]. Expression of aggregation substance has been shown to be inducible by human serum in the absence of plasmid-free Enterococcus [106]. Analysis of Asc10, the aggregation substance encoded on pCF10, showed that a domain of this protein was required for aggregation, HT-29 internalization, and maximum levels of lipoteichoic acid binding [107]. Aggregation substance has been shown to be involved in the survival of enterococci within poly-

morphonuclear leukocytes [104], but does not enhance the urinary tract colonization ability of E.faecalis [111].

The E.faecalis genome encodes a protein (EF1249) which shares 44% identity to FbpA from L. monocytogenes. In L. monocytogenes, this protein has been shown to bind to human fibronectin, and a fbpA mutant was found to have a reduced ability to colonize and survive in a listeriosis mouse model [112]. Inactivation of fbpA also resulted in reduction of protein levels of two other Listeria virulence factors, LLO and InlB, suggesting that FbpA also acts to modulate expression of these two proteins [112]. The function of EF1249 in E.faecalis is at present unknown.

Enterococcal surface protein, Esp [113], is found on the pathogenicity island in MMH594, but is absent from V583 due to the previously mentioned 17-kbp deletion in the pathogenicity island of this isolate. The gene, esp, encodes a large cell-wall-associated protein and contains a variable number of highly conserved 82-amino-acid repeats [113]. Within these repeats is a 13-amino-acid stretch that is identical to a sequence found in the repeats of the Rib and Ca proteins from S. agalactiae [113]. Studies using a mouse model of urinary tract infections have shown that this protein enhances colonization and persistence in urinary bladders

The Esp protein has recently been linked to enhanced biofilm formation, and its contribution was most pronounced in the presence of glucose [115]; however, biofilm production has been shown to occur in the absence of this protein, showing that it is not essential for this process [86, 116]. GelE has also been implicated in biofilm formation [86]. Disruption of GelE or the response regulator required for GelE production in V583 affected biofilm formation, while complementation of GelE restored the wild-type biofilm phenotype [86]. The same study showed that of the 17 two-component regulatory systems present in E.faecalis V583, only the fsr locus was involved in biofilm production. Biofilm production in E.faecalis has also been linked to the protein BopD (EF0954). This protein has homology to a sugar-binding transcriptional regulator, and has 50% similarity to CcpA (EF1741). The actual role of this regulatory gene is unknown, but the association of enhanced biofilm formation in the presence of glucose and the possible involvement of a sugar-binding transcriptional regulator suggest a linkage to increased biofilm production in E.faecalis in the presence of specific carbohydrates.

Contributing to immune cell evasion is the E.faecalis variable capsular polysaccharide (EF2495-EF2485). Expression of capsular polysaccharide from the cpsC-K genes has been shown to contribute to host immune evasion in E.faecalis, with mutants being more susceptible to phagocytic killing [117]. Recent work suggests a limited number of E.faecalis capsule serotypes, 60% of the isolates tested being placed into four serotypes [118]. Also present in E.faecalis V583 is a common cell wall polysaccharide, Epa (enterococcal polysaccharide antigen). An epa knockout has been shown to be unable to translocate across a monolayer of polarized human enterocyte-like T84 cells, and complementation was shown to restore its translocation ability [119]. In the same model, 9 out of 15 E.faecalis isolates were able to translocate, showing strain-specific differences in this phenotype [119].

Other genes that may have a role in immune evasion include homologues of the dlt operon (EF2749-EF2746), responsible for D-alanine incorporation of tei-choic acid polymers in cell wall. Formation of D-alanyl-lipoteichoic acid has been shown to be required for host cell adhesion in L. monocytogenes [120]. D-Alanine incorporation into lipoteichoic acid reduces the negative charge, and therefore electrostatic interactions with cationic peptides are altered. The E.faecalis V583 genome also contains a protein, EF0031, which shares identity with MprF. This protein was first described in S. aureus [121], and confers resistance to human defensins. MprF has been demonstrated as modifying phosphatidylglycerol with L-lysine, therefore reducing the negative charge of the membrane [121]. Disruption of the mprF gene in S. aureus led to a 13-fold higher sensitivity to defensin from human neutrophils; however, the action of this protein in E.faecalis has not yet been investigated [121].

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