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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 on the growth of E. faecalis. E. faecalis of mid-log-phase incubates (OD = 0.1 at 660 nm) were inoculated into 100 ml BHI medium and cultured with shaking in the presence (open circle) or absence (open square) of 1 mM glucose at 37°C for 4 h. The growth was inhibited by 2 mM NOC12 in the presence (closed circle) and absence (closed square) of glucose.
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Figure 2: Effect of NO on the growth of E. faecalis. E. faecalis of mid-log-phase incubates (OD = 0.1 at 660 nm) were inoculated into 100 ml BHI medium and cultured with shaking in the presence (open circle) or absence (open square) of 1 mM glucose at 37°C for 4 h. The growth was inhibited by 2 mM NOC12 in the presence (closed circle) and absence (closed square) of glucose.

Mentions: NOC12 spontaneously releases 2 mol NO per mol of the compound and, hence, it has been used as a useful NO donor [14]. Under the present experimental culture conditions, E. faecalis grow similarly in the presence or absence of 1 mM glucose. However, the rate of growth slightly decreased thereafter in the absence of glucose. The growth of E. faecalis was inhibited by the presence of NOC12 either in the presence or absence of glucose (Fig. 2). The inhibitory effect of NOC12 depended on its concentration (data not shown).


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 on the growth of E. faecalis. E. faecalis of mid-log-phase incubates (OD = 0.1 at 660 nm) were inoculated into 100 ml BHI medium and cultured with shaking in the presence (open circle) or absence (open square) of 1 mM glucose at 37°C for 4 h. The growth was inhibited by 2 mM NOC12 in the presence (closed circle) and absence (closed square) of glucose.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Effect of NO on the growth of E. faecalis. E. faecalis of mid-log-phase incubates (OD = 0.1 at 660 nm) were inoculated into 100 ml BHI medium and cultured with shaking in the presence (open circle) or absence (open square) of 1 mM glucose at 37°C for 4 h. The growth was inhibited by 2 mM NOC12 in the presence (closed circle) and absence (closed square) of glucose.
Mentions: NOC12 spontaneously releases 2 mol NO per mol of the compound and, hence, it has been used as a useful NO donor [14]. Under the present experimental culture conditions, E. faecalis grow similarly in the presence or absence of 1 mM glucose. However, the rate of growth slightly decreased thereafter in the absence of glucose. The growth of E. faecalis was inhibited by the presence of NOC12 either in the presence or absence of glucose (Fig. 2). The inhibitory effect of NOC12 depended on its concentration (data not shown).

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