SummaryThe enterococcal cytolysin, a two-peptide lytic system, is a divergent relative of a large family of toxins and bacteriocins secreted by pathogenic and nonpathogenic Gram-positive bacteria. This family includes the lantibiotics and streptolysin S. The enterococcal cytolysin is of interest because its activities enhance enterococcal virulence in infection models and, in epidemiological studies, it has been associated with patient mortality. The cytolysin is lethal for a broad range of prokaryotic and eukaryotic cells, and this activity requires two non-identical, post-translationally modified peptides. The smaller of the two peptides also plays a role in a quorumsensing autoinduction of the cytolysin operon. As a trait that is present in particularly virulent strains of Enterococcus faecalis , including strains that are resistant to multiple antibiotics, it serves as a model for testing the value of developing new virulencetargeting therapeutics. Further, because of the interest in small membrane active peptides as therapeutics themselves, studies of the molecular structure/ activity relationships for the cytolysin peptides are providing insights into the physical basis for prokaryotic versus eukaryotic cell targeting.

The cytolysin is a novel, two-peptide lytic toxin produced by some strains of Enterococcus faecalis. It is toxic in animal models of enterococcal infection, and associated with acutely terminal outcome in human infection. The cytolysin exerts activity against a broad spectrum of cell types including a wide range of gram positive bacteria, eukaryotic cells such as human, bovine and horse erythrocytes, retinal cells, polymorphonuclear leukocytes, and human intestinal epithelial cells. The cytolysin likely originated as a bacteriocin involved with niche control in the complex microbial ecologies associated with eukaryotic hosts. However, additional anti-eukaryotic activities may have been selected for as enterococci adapted to eukaryotic cell predation in water or soil ecologies. Cytolytic activity requires two unique peptides that possess modifications characteristic of the lantibiotic bacteriocins, and these peptides are broadly similar in size to most cationic eukaryotic defensins. Expression of the cytolysin is tightly controlled by a novel mode of gene regulation in which the smaller peptide signals high-level expression of the cytolysin gene cluster. This complex regulation of cytolysin expression may have evolved to balance defense against eukaryotic predators with stealth.

Many virulent strains of Enterococcus faecalis produce a two-subunit toxin, termed cytolysin. Cytolysin expression is regulated by one of the subunits (CylL(S)'') through a quorum-sensing autoinduction mechanism. We found that when target cells are absent, the other subunit (CylL(L)'') forms a complex with CylL(S)'', blocking it from autoinducing the operon. When target cells are present, however, CylL(L)'' binds preferentially to the target, allowing free CylL(S)'' to accumulate above the induction threshold. Thus, enterococci use CylL(L)'' to actively probe the environment for target cells, and when target cells are detected, allows the organism to express high levels of cytolysin in response.

Enterococcus faecalis is a leading cause of nosocomial infections and is known for its ability to acquire and transfer virulence and antibiotic resistance determinants from other organisms. A 150-kb pathogenicity island (PAI) encoding several genes that contribute to pathogenesis was identified among antibiotic-resistant clinical isolates. In the current study, we examined the structure of the PAI in a collection of isolates from diverse sources in order to gain insight into its genesis and dynamics. Using multilocus sequence typing to assess relatedness at the level of strain background and microarray analysis to identify variations in the PAI, we determined the extent to which structural variations occur within the PAI and also the extent to which these variations occur independently of the chromosome. Our findings provide evidence for a modular gain of defined gene clusters by the PAI. These results support horizontal transfer as the mechanism for accretion of genes into the PAI and highlight a likely role for mobile elements in the evolution of the E. faecalis PAI.Enterococcus faecalis is a core constituent of the intestinal flora of humans and a leading cause of nosocomial infections worldwide (35). Enterococci are associated with a variety of pathologies, including pelvic infections, intra-abdominal abscesses, postsurgical infection, bacteremia, endocarditis, and urinary tract infections (12,20,30). The ability of E. faecalis to cause serious infection is connected to the inherent hardiness of the bacterium, which enables it to tolerate desiccation, persist in the hospital environment, and then endure host defenses (20,22). In addition, enterococci are particularly adept at acquiring resistance to antibiotics and disseminating these elements within and beyond the genus (30, 48).Pathogenicity islands (PAIs) are large, horizontally transmitted elements found in many gram-positive and gram-negative pathogens (14). They are believed to contribute to the rapid evolution of nonpathogenic organisms into pathogenic forms (2, 26). The PAI of E. faecalis is approximately 150 kb and encodes multiple factors that contribute to its virulence, including the cytolysin toxin (3, 18), the enterococcal surface protein Esp (40), and Gls-24-like proteins (44), as well as traits suspected of contributing to pathogenicity or altering its relationship with the host, including a bile acid hydrolase, carbohydrate utilization pathways, and many additional genes of unknown function (38). The PAI, or parts thereof, has been identified in hundreds of E. faecalis isolates, and variation in genetic content has been noted (25,30,32,39). It is enriched among infection-derived isolates (38) and highly clonal lineages containing multiple antibiotic resistance elements (30). Variation has been found in the occurrence of genes within the PAI, even within a genetic (clonal) lineage, suggesting that segments of the island can vary independently of the whole (38). Indeed, movement of genes derived from an internal portion of the PAI has been det...

Inflammation caused by infection with Gram-positive bacteria is typically initiated by interactions with Toll-like receptor 2 (TLR2). Endophthalmitis, an infection and inflammation of the posterior segment of the eye, can lead to vision loss when initiated by a virulent microbial pathogen. Endophthalmitis caused by Bacillus cereus develops as acute inflammation with infiltrating neutrophils, and vision loss is potentially catastrophic. Residual inflammation observed during B. cereus endophthalmitis in TLR2 ؊/؊ mice led us to investigate additional innate pathways that may trigger intraocular inflammation. We first hypothesized that intraocular inflammation during B. cereus endophthalmitis would be controlled by MyD88-and TRIF-mediated signaling, since MyD88 and TRIF are the major adaptor molecules for all bacterial TLRs. In MyD88 ؊/؊ and TRIF ؊/؊ mice, we observed significantly less intraocular inflammation than in eyes from infected C57BL/6J mice, suggesting an important role for these TLR adaptors in B. cereus endophthalmitis. These results led to a second hypothesis, that TLR4, the only TLR that signals through both MyD88 and TRIF signaling pathways, contributed to inflammation during B. cereus endophthalmitis. Surprisingly, B. cereus-infected TLR4 ؊/؊ eyes also had significantly less intraocular inflammation than infected C57BL/6J eyes, indicating an important role for TLR4 in B. cereus endophthalmitis. Taken together, our results suggest that TLR4, TRIF, and MyD88 are important components of the intraocular inflammatory response observed in experimental B. cereus endophthalmitis, identifying a novel innate immune interaction for B. cereus and for this disease. Bacillus cereus is a Gram-positive, spore-forming, and beta-hemolytic soil bacterium (1). Commonly identified as a causative agent of foodborne illnesses, B. cereus is also associated with a multitude of clinical conditions, such as central nervous system infections (2), pneumonia (3), endocarditis (4), and gas-gangrene-like cutaneous infections (5). B. cereus also causes a virulent form of endophthalmitis, an intraocular inflammatory condition resulting from the introduction of microorganisms into the posterior segment of the eye following surgery or injury or from a distant site of infection. This infection causes irreversible damage to the retina, often leading to vision loss within 1 or 2 days (6). Typically, B. cereus endophthalmitis occurs following a penetrating eye injury (posttraumatic) with retained intraocular foreign bodies (7, 8) but has also been reported in postoperative patients (9-11). Fewer than 30% of posttraumatic B. cereus endophthalmitis patients retained useful vision, and out of these, only 9% retained 20/70 vision or better (7, 12). Moreover, 48% of B. cereus and other Bacillus species infections required evisceration or enucleation of the eye despite therapeutic intervention (7, 12). Intraocular inflammation that occurs during B. cereus endophthalmitis interferes with the clarity of the visual axis, contributing to disruption...