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 (O2-). To elucidate the role of O2- and NO in the mechanism for the pathogen surviving in the intestine and macrophages, we studied the role and metabolism of O2- and NO in and around E. faecalis. Kinetic analysis revealed that E. faecalis generated 0.5 μmol O2-/min/108 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 O2- 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 O2- and NO may occur in and around phagocytized macrophages.