Importance of Nitric Oxide Synthase in the Control of Infection by Bacillus anthracis

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Abstract
The spore-forming, gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, has achieved notoriety due to its use as a bioterror agent. In the environment, B. anthracis exists as a dormant endospore. Upon infection, germination of endospores occurs during their internalization within the phagocyte, and the ability to survive exposure to antibacterial killing mechanisms, such as O2·−, NO·, and H2O2, is a key initial event in the infective process. Macrophages generate NO· from the oxidative metabolism of l-arginine, using an isoform of nitric oxide synthase (NOS 2). Exposure of murine macrophages (RAW264.7 cells) to B. anthracis endospores up-regulated the expression of NOS 2 12 h after exposure, and production of NO· was comparable to that achieved following other bacterial infections. Spore-killing assays demonstrated a NO·-dependent bactericidal response that was significantly decreased in the presence of the NOS 2 inhibitor l-N6-(1-iminoethyl)lysine and in l-arginine-depleted media. Interestingly, we also found that B. anthracis bacilli and endospores exhibited arginase activity, possibly competing with host NOS 2 for its substrate, l-arginine. As macrophage-generated NO· is an important pathway in microbial killing, the ability of endospores of B. anthracis to regulate production of this free radical has important implications in the control of B. anthracis-mediated infection.