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 (O(2)(*-)) 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 O(2)(*-) production. Ex vivo electron paramagnetic resonance spin trapping study of colonic contents (mean +/- SD) showed 1.4 +/- 1.5 and 0.094 +/- 0.24 microM 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 D-phenylalanine. Hydroxylated PBN and D-phenylalanine products were recovered from stool (microM/gm colonic contents/10(9) colony forming units) and urine (microM/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.
The Muchmore Laboratories for Infectious Diseases Research, Medical Service, Department of Veterans Affairs Medical Center, Oklahoma City, OK 73104, USA. mark-huycke@ouhsc.edu
Mark M
MM
Huycke
Danny R
DR
Moore
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003106:Colon / Q000382:microbiology / Q000473:pathology; D004578:Electron Spin Resonance Spectroscopy; D013293:Enterococcus faecalis / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000494:pharmacology; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017665:Hydroxyl Radical; D007422:Intestines / Q000382:microbiology*; D009578:Nitrobenzenes; D009589:Nitrogen Oxides / Q000494:pharmacology; D018384:Oxidative Stress; D010649:Phenylalanine / Q000494:pharmacology; D051381:Rats; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase / Q000378:metabolism; D014443:Tyrosine / Q000652:urine
D016166:Free Radical Scavengers; D005609:Free Radicals; D009578:Nitrobenzenes; D009589:Nitrogen Oxides; D017382:Reactive Oxygen Species; D017665:Hydroxyl Radical; C070322:N-tert-butyl-alpha-(4-nitrophenyl)nitrone; D014443:Tyrosine; D010649:Phenylalanine; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase