Hemolysins Proteases and other Enzymes

The genome sequence of E.faecalis V583 shows the presence of three putative hemolysins (EF0700, EF0982, EF1685). EF0700 has its closest identity to L64811 (57.2%) from L. lactis, while EF0982 is most similar to Lm01366 from L. monocytogenes. The third putative hemolysin, EF1685, shares 46% identity to hemolysin III from Bacillus cereus [70]. E.faecalis V583 is nonhemolytic on blood agar; however, it is possible that expression of these genes could be induced in in vivo conditions. It is unknown at present whether these genes do indeed encode functional hemolysins. A putative exfoliative toxin A (EF0645) is likewise present in the chromosome. This protein shares 32% identity to Staphylococcus hyicus exfoliative toxin A and again its function in E.faecalis is unknown.

Also encoded on the chromosome are two putative xanthan lyases, EF0818 and EF3023, which share 36% identity at the protein level. EF0818 and EF3023 have 31-28% identity to the xanthan lyase from Bacillus sp. G11 [71], and 29-27% identity to hyaluronate lyase from S. pneumoniae [72]. Both these enzymes are involved in breaking glycosidic bonds in polysaccharides, although they have different substrate specificities. Hyaluronidases are an important pathogenic bacterial spreading factor, and cleave hyaluronan, which is a constituent of the extracellular matrix of connective tissues [73]. The substrate specificity of these two enzymes in E.fae-calis is uncertain.

GelE (EF1818) encodes a secreted zinc metalloprotease which has been shown to cleave a number of substrates including casein, hemoglobin, collagen, fibrin, and gelatin, as well as degrading pheromone and inhibitor peptides [74, 75]. GelE has been hypothesized to clear the bacterial cell wall of misfolded proteins, and disruption of this gene has been shown to increase the bacterial chain length [76]. The presence of GelE is essential for the degradation of polymerized fibrin, which has important implications in pathogenesis, and the linkage of GelE to decreased chain length may suggest a role in increasing dissemination of bacteria in high-density environments [76]. A gelE knockout has shown reduced virulence in models of mouse peritonitis [77], rabbit endophthalmitis [78], and in a Caenorhabditis elegans virulence model [79].

Located downstream from GelE is an ORF called sprE (EF1817), coding for a serine protease, and the presence of functional GelE has been implicated in SprE maturation [80]. SprE encodes a 26-kDa protein that shares homology with the S. aureus V8 protease, and has also been shown to contribute to pathogenesis in several infection models including C. elegans, mouse peritonitis, and a rabbit endophthalmitis model [78, 79, 81]. Deletion of the serine protease, but not gelatinase has been shown to cause attenuation of bacterial infection in the Arabidopsis thaliana root pathogenicity model with the SprE mutant forming poor bacterial communities [82]. The expression of both gelE and sprE is dependent on the fsr genes that encode a two-component signal transduction system [81, 83].

The fsr locus (EF1822-EF1820) encodes three proteins, FsrA, FsrB, and FsrC, which share sequence similarity with AgrA, AgrB, and AgrC, respectively, of S. aureus. This locus has been identified as a two-component signal transduction system that is dependent upon a secreted peptide lactone, meaning that regulation is controlled in a cell-density-dependent manner [83-85]. Accumulation of the peptide, encoded at the C terminus of the FsrB protein, is thought to be sensed by the FsrC histidine kinase, and leads to activation of the FsrA response regulator [85]. Hancock and Perego recently showed a role for fsr and GelE in biofilm formation [86]. Interestingly, although gelatinase and serine protease were found to contribute to virulence in a rabbit endophthalmitis model, an fsrB mutant showed much greater attenuation, suggesting the effect of this gene on the expression of additional traits that contribute to virulence [78].

E.faecalis V583 also contains a second membrane-associated zinc metallopro-tease (EF2380), Eep. This protein has been shown to play a role in the production of the sex pheromone cAD1, and is thought to be involved in processing the pher-omone precursor structure, or in regulating the expression or secretion [87].

The final secreted virulence factor in E. faecalis is cytolysin, which is a structurally novel toxin. As previously mentioned, this gene locus is present on the patho-genicity island; however, most of this gene locus is absent in E.faecalis V583 due to a 17-kbp deletion. In V583, the genes cylRl, cylR2, clyLL, cylLS, and cylM are present, but the genes cylB, cylA, and cylI, which are essential for mature cytolysin production and self-immunity, are either absent or nonfunctional. Cytolysin is comprised of two nonidentical peptide subunits, both of which are required for lytic activity [88], and is capable of lysing erythrocytes, polymorphonuclear leukocytes, and macrophages [89], as well as a broad range of gram-positive bacteria. Recently cytolysin expression has been shown to be induced in response to target cells present in the environment through a quorum-sensing autoinduction mech anism [90]. In virtually all infection models tested, cytolysin has been shown to contribute to pathogenesis [91-94].

0 0

Post a comment