Enterococcus faecalis produces extracellular superoxide and hydrogen peroxide that damages colonic epithelial cell DNA

Enterococcus faecalis is a commensal microorganism of the human intestinal tract that produces substantial extracellular superoxide (O-2(-)), and derivative reactive oxygen species such as H2O2 and hydroxyl radical, through autoxidation of membrane-associated demethylmenaquinone. Because these oxidants may be important as a cause of chromosomal instability (CIN) associated with sporadic adenomatous polyps and colorectal cancer, the ability of E. faecalis to damage eukaryotic cell DNA was examined using the alkaline lysis single cell gel electrophoresis (comet) assay. Both Chinese hamster ovary and HT-29 intestinal epithelial cells showed increased DNA damage after co-incubation with wild-type E. faecalis strain OG1RF, but not a transposon-inactivated mutant with attenuated extracellular O-2(-) production. E. faecalis-mediated DNA damage was prevented by catalase, but not manganese superoxide dismutase, indicating H2O2 arising from O-2(-) was the genotoxin. In a rat model of intestinal colonization, OG1RF resulted in significantly higher stool concentrations of H2O2 and 5,5-dimethyl-1-pyrroline N-oxide adducts of hydroxyl and thiyl radicals, as identified by electron spin resonance-spin trapping, compared with rats colonized with a mutant strain having attenuated O-2(-) production. Using the comet assay, luminal cells from the colon of rats colonized with O-2(-)-producing E. faecalis showed significantly increased DNA damage compared with control rats colonized with the mutant. These findings suggest a potentially profound role for extracellular free radical production by E. faecalis in promoting CIN associated with sporadic adenomatous polyps and colorectal cancer.