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Effect of Nitric Oxide on the Oxygen Metabolism and Growth of E. faecalis.

Nishikawa T, F Sato E, Choudhury T, Nagata K, Kasahara E, Matsui H, Watanabe K, Inoue M - J Clin Biochem Nutr (2009)

Bottom Line: Kinetic analysis revealed that E. faecalis generated 0.5 micromol O(2) (-)/min/10(8) cells in a glucose-dependent manner as determined using the cytochrome c reduction method.However, the growth rate of NOC12-pretreated E. faecalis in NO-free medium was similar to that of untreated cells.These observations suggested that O(2) (-) generated by E. faecalis reacted with NO to form peroxinitrite (ONOO(-)) that preferentially nitrated tyrosyl residues in cytosolic proteins, thereby reversibly inhibited cellular growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Pathology, Osaka City Medical School, 1-4-3 Asahimachi, Abeno 545-8585, Japan.

ABSTRACT
Gastro-intestinal mucosal cells have a potent mechanism to eliminate a variety of pathogens using enzymes that generate reactive oxygen species and/or nitric oxide (NO). However, a large number of bacteria survive in the intestine of human subjects. Enterococcus faecalis (E. faecalis) is a Gram-positive bacterium that survives not only in the intestinal lumen but also within macrophages generating NO. It has been reported that E. faecalis generated the superoxide radical (O(2) (-)). To elucidate the role of O(2) (-) and NO in the mechanism for the pathogen surviving in the intestine and macrophages, we studied the role and metabolism of O(2) (-) and NO in and around E. faecalis. Kinetic analysis revealed that E. faecalis generated 0.5 micromol O(2) (-)/min/10(8) cells in a glucose-dependent manner as determined using the cytochrome c reduction method. The presence of NOC12, an NO donor, strongly inhibited the growth of E. faecalis without affecting in the oxygen consumption. However, the growth rate of NOC12-pretreated E. faecalis in NO-free medium was similar to that of untreated cells. Western blotting analysis revealed that the NOC12-treated E. faecalis revealed a large amount of nitrotyrosine-posititive proteins; the amounts of the modified proteins were higher in cytosol than in membranes. These observations suggested that O(2) (-) generated by E. faecalis reacted with NO to form peroxinitrite (ONOO(-)) that preferentially nitrated tyrosyl residues in cytosolic proteins, thereby reversibly inhibited cellular growth. Since E. faecalis survives even within macrophages expressing NO synthase, similar metabolism of O(2) (-) and NO may occur in and around phagocytized macrophages.

No MeSH data available.


Related in: MedlinePlus

Effect of NO and ONOO− on the O2 consumption by E. faecalis. Oxygen consumption by E. faecalis (1 × 108 cells/ml) was determined polarographically using a Clark type oxygen electrode fitted to a 2 ml water-jacketed chamber at 37°C in HEPES-KRP medium (50 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM KCl, 1 mM each of MgCl2, NaH2PO4 and CaCl2) containing 1 mM D-glucose. NO (5 µM) or ONOO− (10 µM) were added to the reaction mixture at various oxygen tension.
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Figure 3: Effect of NO and ONOO− on the O2 consumption by E. faecalis. Oxygen consumption by E. faecalis (1 × 108 cells/ml) was determined polarographically using a Clark type oxygen electrode fitted to a 2 ml water-jacketed chamber at 37°C in HEPES-KRP medium (50 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM KCl, 1 mM each of MgCl2, NaH2PO4 and CaCl2) containing 1 mM D-glucose. NO (5 µM) or ONOO− (10 µM) were added to the reaction mixture at various oxygen tension.

Mentions: We previously reported that a low concentration of NO (5 µM) inhibited the respiration of E. coli and rapidly decreased their ATP levels in a reversible manner [4]. However, the presence of NO had no appreciable effect on the oxygen consumption by E. faecalis (Fig. 3). To elucidate the mechanism of the NOC12-induced inhibition of cell growth, we also analyzed the effect of peroxynitrite, a reaction product of O2− and NO, on the oxygen consumption by E. faecalis. The presence of peroxynitrite had no appreciable effect on their oxygen consumption.


Effect of Nitric Oxide on the Oxygen Metabolism and Growth of E. faecalis.

Nishikawa T, F Sato E, Choudhury T, Nagata K, Kasahara E, Matsui H, Watanabe K, Inoue M - J Clin Biochem Nutr (2009)

Effect of NO and ONOO− on the O2 consumption by E. faecalis. Oxygen consumption by E. faecalis (1 × 108 cells/ml) was determined polarographically using a Clark type oxygen electrode fitted to a 2 ml water-jacketed chamber at 37°C in HEPES-KRP medium (50 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM KCl, 1 mM each of MgCl2, NaH2PO4 and CaCl2) containing 1 mM D-glucose. NO (5 µM) or ONOO− (10 µM) were added to the reaction mixture at various oxygen tension.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654474&req=5

Figure 3: Effect of NO and ONOO− on the O2 consumption by E. faecalis. Oxygen consumption by E. faecalis (1 × 108 cells/ml) was determined polarographically using a Clark type oxygen electrode fitted to a 2 ml water-jacketed chamber at 37°C in HEPES-KRP medium (50 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM KCl, 1 mM each of MgCl2, NaH2PO4 and CaCl2) containing 1 mM D-glucose. NO (5 µM) or ONOO− (10 µM) were added to the reaction mixture at various oxygen tension.
Mentions: We previously reported that a low concentration of NO (5 µM) inhibited the respiration of E. coli and rapidly decreased their ATP levels in a reversible manner [4]. However, the presence of NO had no appreciable effect on the oxygen consumption by E. faecalis (Fig. 3). To elucidate the mechanism of the NOC12-induced inhibition of cell growth, we also analyzed the effect of peroxynitrite, a reaction product of O2− and NO, on the oxygen consumption by E. faecalis. The presence of peroxynitrite had no appreciable effect on their oxygen consumption.

Bottom Line: Kinetic analysis revealed that E. faecalis generated 0.5 micromol O(2) (-)/min/10(8) cells in a glucose-dependent manner as determined using the cytochrome c reduction method.However, the growth rate of NOC12-pretreated E. faecalis in NO-free medium was similar to that of untreated cells.These observations suggested that O(2) (-) generated by E. faecalis reacted with NO to form peroxinitrite (ONOO(-)) that preferentially nitrated tyrosyl residues in cytosolic proteins, thereby reversibly inhibited cellular growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Pathology, Osaka City Medical School, 1-4-3 Asahimachi, Abeno 545-8585, Japan.

ABSTRACT
Gastro-intestinal mucosal cells have a potent mechanism to eliminate a variety of pathogens using enzymes that generate reactive oxygen species and/or nitric oxide (NO). However, a large number of bacteria survive in the intestine of human subjects. Enterococcus faecalis (E. faecalis) is a Gram-positive bacterium that survives not only in the intestinal lumen but also within macrophages generating NO. It has been reported that E. faecalis generated the superoxide radical (O(2) (-)). To elucidate the role of O(2) (-) and NO in the mechanism for the pathogen surviving in the intestine and macrophages, we studied the role and metabolism of O(2) (-) and NO in and around E. faecalis. Kinetic analysis revealed that E. faecalis generated 0.5 micromol O(2) (-)/min/10(8) cells in a glucose-dependent manner as determined using the cytochrome c reduction method. The presence of NOC12, an NO donor, strongly inhibited the growth of E. faecalis without affecting in the oxygen consumption. However, the growth rate of NOC12-pretreated E. faecalis in NO-free medium was similar to that of untreated cells. Western blotting analysis revealed that the NOC12-treated E. faecalis revealed a large amount of nitrotyrosine-posititive proteins; the amounts of the modified proteins were higher in cytosol than in membranes. These observations suggested that O(2) (-) generated by E. faecalis reacted with NO to form peroxinitrite (ONOO(-)) that preferentially nitrated tyrosyl residues in cytosolic proteins, thereby reversibly inhibited cellular growth. Since E. faecalis survives even within macrophages expressing NO synthase, similar metabolism of O(2) (-) and NO may occur in and around phagocytized macrophages.

No MeSH data available.


Related in: MedlinePlus