Virulence properties of a peptide hemolysin produced by Enterococcus faecalis

Abstract

The characterization and prevalence of virulence fac- tors associated with enterococcal invasiveness and severity of disease are important areas to be investi- gated. Recently, we described the production of a heat- stable hemolysin by clinical isolates of Enterococcus faecalis cultived in BHI-GA (BHI with glucose and L- arginine). Now, we purified the hemolysin from the cul- ture supernatant by ultrafiltration (PM-10 membrane) and ethanol extraction followed by chromatography in a Bondapak C18 and Superdex Peptide columns. The hemolytic activity was not affected by the proteo- lytic enzymes. Cholesterol, phospholipids, EDTA and also bivalent ions did not inhibit the hemolytic activity. Among the various carbohydrates, only dextran 4 protected the erythrocytes against lyse. Scanning elec- tron microscopy showed that lyse of erythrocytes oc-cured at once after the exposure to the hemolysin. The mitochondrial activity and the cell membrane integrity were significantly affected by the hemolysis, within 20 min of exposure and caused apoptosis after 12 h incubation, 51.92% in HeLa and 68% in HEp-2 cells, analyzed by flow cytometry. These results sug- gest that the heat-stable pore forming hemolysin might be a putative virulence factor in enterococci in- fections.