In vivo production of hydroxyl radical by enterococcus faecalis colonizing the intestinal tract using aromatic hydroxylation

Enterococcus faecalis is an intestinal commensal that produces extracellular superoxide (O2•−) through autoxidation of membrane-associated demethylmenaquinone. To assess free radical production by E. faecalis in vivo, intestinal tracts of rats were colonized using wild-type E. faecalis or a mutant strain with attenuated O2•− production. Ex vivo electron paramagnetic resonance spin trapping study of colonic contents (mean ± SD) showed 1.4 ± 1.5 and 0.094 ± 0.24 μM 5,5-dimethyl-1-pyrroline-N-oxide-hydroxyl radical adduct/gm stool for rats colonized with wild-type and mutant strains, respectively (p = .002). In vivo hydroxyl radical production was further assayed by aromatic hydroxylation using phenyl N-tert-butylnitrone (PBN) and image-phenylalanine. Hydroxylated PBN and image-phenylalanine products were recovered from stool (μM/gm colonic contents/109 colony forming units) and urine (μM/h/ml), respectively, and quantified using electrochemical detection. Hydroxylated (OH) PBNs and isomeric tyrosines (hydroxylated phenylalanine) were significantly increased (mean ± SD) for rats colonized with wild-type E. faecalis (2-OH PBN, 63 ± 58; 3-OH PBN, 63 ± 84; ortho-tyrosine, 31 ± 27; meta-tyrosine, 17 ± 14) compared to the mutant strain (2-OH PBN, 2.5 ± 7.3 (p < .001); 3-OH PBN, 3.9 ± 12.3 (p = .01); ortho-tyrosine, 1.9 ± 6.0 (p < .001); meta-tyrosine, 1.5 ± 3.4 (p = .03)). Similar differences were observed following in vitro incubations of these bacteria with aromatic targets. These results confirm in vivo production of hydroxyl radical by E. faecalis colonizing the intestine, and indicate this bacterium may be a potent source of oxidative stress on the intestinal epithelium.