Pheromone-responsive conjugative plasmids are unique to the species Enterococcus faecalis. Many pheromone-responsive plasmids, including those frequently isolated from sites of infection, express a novel cytolysin that possesses both hemolytic and bacteriocin activities. Further, this cytolysin has been shown to be a toxin in several disease models. In the present study, nucleotide sequence determination, mutagenesis, and complementation analysis were used to determine the organization of the E. faecalis plasmid pAD1 cytolysin determinant. Four open reading frames are required for expression of the cytolysin precursor (cylL9, cylLS, cyiM, and cyiB). The inferred products of two of these open reading frames, Cyll and CylLs, constitute the cytolysin precursor and bear structural resemblance to posttranslationally modified bacteriocins termed lantibiotics. Similarities between the organization of the E. faecalis cytolysin determinant and expression units for lantibiotics exist, indicating that the E. faecalis cytolysin represents a new branch of this class and is the first known to possess toxin activity.Enterococcusfaecalis isolates derived from infection sites are more frequently hemolytic than isolates from the oral cavity or stools of healthy volunteers (22,26,48). The variable nature of the hemolytic phenotype results from the hemolysin determinant being located on highly transmissible, pheromone-responsive plasmids (e.g., pAD1; recently reviewed in references 8 and 9), although evidence has been obtained recently for its occasional residence on the E. faecalis genome (23). The observation of acute toxicity of hemolytic E. faecalis, when injected intraperitoneally in mice (25), suggested that the hemolysin may contribute to bacterial virulence in models of human disease. Hemolytic E. faecalis strains have been observed to cause a more rapid and fulminant endophthalmitis in a rabbit infection model than isogenic strains rendered nonhemolytic as the result of insertion of a transposon into various areas of the hemolysin determinant (29). Similar observations of an endocarditis model where hemolytic strains were found to be significantly more toxic than isogenic, nonhemolytic mutants have been made (6; for a recent review of enterococcal virulence, see reference 28).Contributing to virulence is a common motif for bacterial hemolysins (3,35,46,47). The E. faecalis hemolysin, however, is unique in that in addition to mediating lysis of erythrocytes, it also possesses antibacterial activity toward a broad range of gram-positive bacteria (4,5,27 Immediately 5' to cylA is cylB, whose nucleotide sequence has been reported (17). cylB is the first member of the HlyB family of ATP-binding transport proteins to have been identified in an operon from a gram-positive bacterium (15,17). CylB was observed to be essential for externalization of the E. faecalis cytolysin precursor activity, component L.Although a substantial amount of information describing (i) the protein that activates the E. faecalis cytolysin precursor extra...

Clinical isolates of Enterococcus faecalis more commonly produce a cytolysin than do commensal isolates. Epidemiologic evidence and animal-model studies have established a role for the cytolysin in the pathogenesis of enterococcal disease. The cytolysin consists of two structural subunits, CylLL and CylLS, that are activated by a third component, CylA. Genetic and biochemical characterization of CylA indicate that it is a serine protease, and that activation putatively results from cleavage of one or both cytolysin subunits. Genetic evidence also suggests that the cytolysin subunits are related to the rapidly growing class of bacteriocins termed lantibiotics. However, unlike lantibiotics, the cytolysin is lytic for eukaryotic as well as prokaryotic cells, and it consists of two structural subunits. This report describes the purification and characterization of the cytolysin subunits and detection of lanthionine-type post-translational modifications within their structures. Furthermore, the cleavage specificity of the CylA activator is reported and it is shown that proteolytic activation of both subunits is essential for activity.

The severity of endophthalmitis has been associated generally with the virulence of the offending pathogen. However, precisely what constitutes the virulence in intraocular infections remains ill defined. We therefore sought to identify the basis for virulence for three common ocular pathogens (Bacillus cereus,Enterococcus faecalis, and Staphylococcus aureus) in terms of intraocular growth rates, bacterial localization patterns, and the contribution of cell walls and secreted products to the pathogenesis of endophthalmitis. Rabbit eyes were injected intravitreally with (i) viable B. cereus, E. faecalis, or S. aureus, (ii) metabolically inactiveB. cereus, E. faecalis, or S. aureus, (iii) sacculus preparations from each strain, or (iv) culture fluid containing products secreted by each strain. Eyes were assessed at various times following injection by slit lamp biomicroscopy, electroretinography (ERG), bacterial and inflammatory cell enumeration, and histology. B. cereus endophthalmitis followed a more rapid and virulent course than E. faecalisor S. aureus endophthalmitis, eliminating retinal responsiveness, as measured by ERG, by 12 h. Analysis of bacterial localization revealed that B. cereus uniquely migrated rapidly from posterior to anterior segment during infection. Although injection of neither metabolically inactive bacteria nor cell wall sacculi greatly affected ERG, significant intraocular inflammation was observed. Injection of B. cereus or S. aureusculture fluids caused both significant reductions in retinal responsiveness and significant intraocular inflammation, paralleling that seen in natural infections. The results demonstrate that toxins, intraocular localization, and, to a lesser extent, the intraocular host response to cell walls all contribute to the pathogenesis of B. cereus, S. aureus, and E. faecalisendophthalmitis in a pathogen-specific manner. The key pathophysiologic differences in these intraocular diseases highlight opportunities for optimizing conventional therapies and deriving new ones.

Previous studies showed that an agr mutant strain of Staphylococcus aureus was partially attenuated in virulence compared to a parental strain in experimental endophthalmitis. The purpose of this study was to determine whether the sar locus, either alone or through interactions with agr, contributes to the regulation of virulence in S. aureus endophthalmitis. Experimental endophthalmitis was established by the midvitreous injection of approximately 30 CFU of S. aureus RN6390 or the isogenic attenuated strains RN6911 (agr mutant), ALC136 (sar mutant), and ALC135 (agr sar double mutant). Unexpectedly, the rate of reduction in electroretinographic B-wave amplitude in eyes infected with strain ALC136 (sar mutant) was not significantly different from the parental strain on postinfection day (PID) 5 (10% retention). In contrast, ALC135 (agr sar double mutant)infected eyes retained 73% of preoperative B-wave amplitude on PID 5. Therefore, unlike agr, a mutation in the sar locus alone does not alter the overall virulence of wild-type S. aureus in experimental endophthalmitis. However, the combined effect of insertional mutations in both the sar and agr global regulators leads to near-complete attenuation of virulence.