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

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.

Keywords: Enterococcus faecalis; Superoxide; nitric oxide; nitro-tyrosine; peroxynitrite.

Fig. 1

Superoxide generation assessed by cytochrome c reduction E. faecalis used in this study was RIMD 5803 (JCM5803) strain. E. faecalis were incubated in BHI medium (Difco) at 37°C and pH 7.5 for overnight. The incubated cells (OD = 0.1 at 660 nm) were inoculated into 100 ml BHI medium and cultured with shaking at 37°C and pH 7.5 for 4 h. Mid-log-phase incubates (OD = 0.4–5 at 660 nm) were obtained and used. The incubated cells were centrifuged at 3000 × g and 4°C for 10 min, and washed once with PBS. Superoxide generation by E. faecalis was analyzed by the cytochrome c reduction method [1, 11]. Reaction mixtures contained in a total volume of 1 ml PBS, 1 mM cytochrome C, and 1 × 10⁸ cells/ml in the presence (open square) or absence (closed circle, open circle) of 600 units of Cu/Zn-SOD. A) The reaction was started by adding E. faecalis in the presence (closed circle) or absence (open circle) of 1 mM glucose at 37°C. B) The rate of superoxide generation was calculated by cytochrome C reduction.

Fig. 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.

Fig. 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.

Fig. 4

Effect of long-term exposure to NO on E. faecalis. After E. faecalis was incubated in the presence (closed circle) or absence (open circle) of 2 mM NOC12 and 1 mM glucose for 4 h, the cells washed with fresh medium to remove the remaining NO donor, and then cultured in the absence of NOC12.

Fig. 5

Effect of long-term exposure to NO on the O² consumption by E. faecalis. After E. faecalis was incubated in the presence of 2 mM NOC12 and 1 mM glucose for 4 h, the cells washed with fresh medium to remove the remaining NO donor, and O² consumption was measured on the same condition as Fig. 3. NO (5 µM) or ONOO⁻ (10 µM) were added to the reaction mixture at various oxygen tension.

Fig. 6

Detection of nitrotyrosine in E. faecalis proteins. E. faecalis was incubated in the presence or absence of 2 mM NOC12 for 4 h. The cells were washed and fractionated into cytoplasm and particulate fractions. Total and fractionated proteins (10 µg) were subjected to SDS-PAGE followed by western blot analysis using anti-nitrotyrosine antibody. Nitrated BSA (peroxynitirite-treated BSA) is a positive control for detection of protein tyrosine nitration. MW, molecular weight (kilodalton)

Fig. 7

Effect of DIDS on protein nitration in E. faecalis. After E. faecalis was incubated with 300 µM DIDS in PBS included 1 mM glucose and 2 mM NOC12 for 20 min, E. faecalis was incubated in the presence of 2 mM NOC12 for 4 h. 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)

Fig. 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)