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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.


Denitration of NO-treated E. faecalis. After incubation with 2 mM NOC12 and 1 mM glucose for 4 h, E. faecalis was washed with fresh medium and subsequently cultured for ~60 min in NOC12-free medium. The cells were washed and fractionated into cytoplasm and particulate fractions. SDS-PAGE followed by western blotting was performed by the same way as Fig. 7. Nitrated BSA (peroxynitirite-treated BSA) is a positive control for detection of protein tyrosine nitration. MW, molecular weight (kilodalton)
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Figure 8: Denitration of NO-treated E. faecalis. After incubation with 2 mM NOC12 and 1 mM glucose for 4 h, E. faecalis was washed with fresh medium and subsequently cultured for ~60 min in NOC12-free medium. The cells were washed and fractionated into cytoplasm and particulate fractions. SDS-PAGE followed by western blotting was performed by the same way as Fig. 7. Nitrated BSA (peroxynitirite-treated BSA) is a positive control for detection of protein tyrosine nitration. MW, molecular weight (kilodalton)

Mentions: To evaluate the role of protein nitration in the mechanism of reversible inhibition of E. faecalis growth by NO, possible involvement of the denitration of the modified proteins in cells was studied. After incubation with 2 mM NOC12 and 1 mM glucose for 4 h, the cells were washed with fresh medium and subsequently cultured for ~60 min in NOC12-free medium. Western blotting analysis of the cell fractions revealed that the nitrotyrosine-positive protein bands in NOC12-pretreated cells disappeared rapidly after incubation (Fig. 8). As nitration of tyrosyl residues in proteins occurred in an irreversible manner, its disappearance might depend on cell growth. Alternatively, the nitrated proteins might be rapidly eliminated by proteolysis.


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)

Denitration of NO-treated E. faecalis. After incubation with 2 mM NOC12 and 1 mM glucose for 4 h, E. faecalis was washed with fresh medium and subsequently cultured for ~60 min in NOC12-free medium. The cells were washed and fractionated into cytoplasm and particulate fractions. SDS-PAGE followed by western blotting was performed by the same way as Fig. 7. Nitrated BSA (peroxynitirite-treated BSA) is a positive control for detection of protein tyrosine nitration. MW, molecular weight (kilodalton)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Denitration of NO-treated E. faecalis. After incubation with 2 mM NOC12 and 1 mM glucose for 4 h, E. faecalis was washed with fresh medium and subsequently cultured for ~60 min in NOC12-free medium. The cells were washed and fractionated into cytoplasm and particulate fractions. SDS-PAGE followed by western blotting was performed by the same way as Fig. 7. Nitrated BSA (peroxynitirite-treated BSA) is a positive control for detection of protein tyrosine nitration. MW, molecular weight (kilodalton)
Mentions: To evaluate the role of protein nitration in the mechanism of reversible inhibition of E. faecalis growth by NO, possible involvement of the denitration of the modified proteins in cells was studied. After incubation with 2 mM NOC12 and 1 mM glucose for 4 h, the cells were washed with fresh medium and subsequently cultured for ~60 min in NOC12-free medium. Western blotting analysis of the cell fractions revealed that the nitrotyrosine-positive protein bands in NOC12-pretreated cells disappeared rapidly after incubation (Fig. 8). As nitration of tyrosyl residues in proteins occurred in an irreversible manner, its disappearance might depend on cell growth. Alternatively, the nitrated proteins might be rapidly eliminated by proteolysis.

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.