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

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.