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...

Thirty-seven nonhemolytic/nonbacteriocinogenic mutations in Enterococcus (Streptococcus) faecalis plasmid pADi were generated by Tn917 insertion. All were found to belong to one of two complementation classes.Each class of mutants secreted either hemolysin/bacteriocin (Hly/Bac) component A or L into the culture medium. DNA encoding Hly/Bac was cloned in Escherichia coli in which both components of the hemolysin were expressed individually and collectively. The region encoding components A and L was further defined by deletion analysis and physically mapped. A total of approximately 8.4 kilobases of pADl DNA were observed to be required for hemolysin expression. Hly/Bac activity of the wild-type and the inactive L substance was observed to be heat stable. Active Ely/Bac resulting from incubating separately secreted components A and L was also found to be heat stable. The results indicate that component A activates component L and that activated component L possesses the Hly/Bac activity. Component A was also observed to be associated with host immunity to the Hly/Bac.In certain geographical locations as many as 60% of clinical Enterococcus (formerly Streptococcus) faecalis isolates produce a cytolytic toxin which lyses human, rabbit, and horse erythrocytes (20,23). This hemolysin also possesses bacteriolytic activity and mediates the lysis of a broad range of gram-positive bacteria (2, 4, 6). Epidemiological studies have shown that hemolysin/bacteriocin (Hly/Bac) production correlates with the presence of conjugative plasmids which transfer at a frequency of 10-1 to 10' in broth matings (23). A high percentage of the hemolysin plasmids bear structural similarities to the conjugative plasmid pAD1 (20,26). pAD1 is a 59.6-kilobase (kb) plasmid originally identified in E. faecalis DS16 (30) and belongs to the incompatibility class IncHly (9). The pAD1 hlylbac determinant was shown to contribute to pathogenicity in a mouse model (24). In addition to Hly/Bac synthesis, pAD1 has been shown to confer resistance to UV light (7) and a conjugative mating response to the peptide sex pheromone cAD1 secreted by recipient cells (5,11,13).Early studies on the physical and chemical nature of the E. faecalis Hly/Bac found the hemolytic activity to be heat sensitive and inhibited by a teichoic acid produced by hemolytic strains (10). Granato and Jackson (16) 50 ,uM isopropyl-,-D-thiogalactopyranoside (Sigma) and 0.01% 5-bromo-4-chloro-3-indolyl-,3-D-galactopyranoside (Sigma) were included in the media. Unless otherwise indicated, the medium used for other aspects of this study was brain heart infusion (BHI, Difco). Solid media were prepared by the addition of 1.5% (wt/vol) agar (Difco

The nucleotide sequence of a 3,422-bp internal restriction fragment from the Enterococcus faecalis pADI hemolysin/bacteriocin-encoding region was determined. This fragment was associated with expression of hemolysin/bacteriocin component L and contained a 2,142-bp open reading frame. The inferred amino acid sequence revealed a protein which shared extensive similarity with HlyB of the Escherichia coli alphahemolysin operon. The inferred protein, CylB, was observed to be independently expressed in E. coli and capable of complementing an insertion mutation in the cloned hemolysin/bacteriocin operon in trans. Despite the extensive similarity to HlyB, CylB was incapable of complementing an insertion mutation in hlyB. Cytolysin determinants possessing an HlyB-type transport function are widely dispersed throughout gram-negative genera. We believe this to be the first example of an HlyB-type protein encoded within a cytolysin determinant from a gram-positive bacterium.

The gene encoding component A (cylA), the activator protein of the Enterococcusfaecalis cytolysin, has been localized on pADI, and the nucleotide sequence was determined. cylA consists of a 1,236-bp open reading frame encoding a 412-amino-acid polypeptide. A search of the National Biomedical Research Foundation data base revealed significant homology between the inferred amino acid sequence of component A and subtilisin BPN'. Component A activation of the cytolysin'precursor (component L) was observed to be inhibited by the serine protease inhibitor diisopropylfluorophosphate. Mature component A exhibits a molecular weight of approximately 30,000 and an isoelectric point of 4.5. Differences between the size of the primary translation product (45,625 daltons) and the mature enzyme suggest that, as for subtilisin, component A is secreted as a proenzyme. These results provide the basis for a model of component A activation of component L and a role for component A in protecting the cytolysin-producing cell from lysis.