Protective Role of Bacillus anthracis Exosporium in Macrophage-Mediated Killing by Nitric Oxide▿

The ability of the endospore-forming, gram-positive bacterium Bacillus anthracis to survive in activated macrophages is key to its germination and survival. In a previous publication, we discovered that exposure of primary murine macrophages to B. anthracis endospores upregulated NOS 2 concomitant with an ·NO-dependent bactericidal response. Since NOS 2 also generates O2·−, experiments were designed to determine whether NOS 2 formed peroxynitrite (ONOO−) from the reaction of ·NO with O2·− and if so, was ONOO− microbicidal toward B. anthracis. Our findings suggest that ONOO− was formed upon macrophage infection by B. anthracis endospores; however, ONOO− does not appear to exhibit microbicidal activity toward this bacterium. In contrast, the exosporium of B. anthracis, which exhibits arginase activity, protected B. anthracis from macrophage-mediated killing by decreasing ·NO levels in the macrophage. Thus, the ability of B. anthracis to subvert ·NO production has important implications in the control of B. anthracis-induced infection

Neuronal nitric oxide synthase-induced S-nitrosylation of H-Ras inhibits calcium ionophore-mediated extracellular-signal-regulated kinase activity

nNOS (neuronal nitric oxide synthase) is a constitutively expressed enzyme responsible for the production of NO(•) from L-arginine and O(2). NO(•) acts as both an intra- and an inter-cellular messenger that mediates a variety of signalling pathways. Previous studies from our laboratory have demonstrated that nNOS production of NO(•) blocks Ca(2+)-ionophore-induced activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) of the mitogen-activated protein kinases through a mechanism involving Ras G-proteins and Raf-1 kinase. Herein we describe a mechanism by which NO(•) blocks Ca(2+)-mediated ERK1/2 activity through direct modification of H-Ras. Ca(2+)-mediated ERK1/2 activation in NO(•)-producing cells could be restored by exogenous expression of constitutively active mitogen-activated protein kinase kinase 1. In contrast, exogenous expression of constitutively active mutants of Raf-1 and H-Ras only partially restored ERK1/2 activity, by 50% and 10% respectively. On the basis of these findings, we focused on NO(•)-mediated mechanisms of H-Ras inhibition. Assays for GTP loading and H-Ras interactions with the Ras-binding domain on Raf-1 demonstrated a decrease in H-Ras activity in the presence of NO(•). We demonstrate that S-nitrosylation of H-Ras occurs in nNOS-expressing cells activated with Ca(2+) ionophore. Mutation of a putative nitrosylation site at Cys(118) inhibited S-nitrosylation and restored ERK1/2 activity by constitutively active H-Ras even in the presence of NO(•). These findings indicate that intracellular generation of NO(•) by nNOS leads to S-nitrosylation of H-Ras, which interferes with Raf-1 activation and propagation of signalling through ERK1/2