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 O 2 ⅐؊ , NO · , and H 2 O 2 , 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-N 6 -(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. anthracismediated infection.Endospores of the gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, when inhaled and deposited in the lung, are phagocytosed by resident macrophages, where the spore evades host defenses and germinates into the vegetative bacterium (6,13,14). Germination is a prerequisite to replication and dissemination of B. anthracis (2). Unfortunately, mechanisms by which the spore enters the macrophage, germinates, survives therein, and escapes from this phagocytic cell are poorly understood. To study spore-specific interactions, the ⌬gerH mutant of the B. anthracis Sterne 34F2 strain, which is a germination-deficient variant incapable of conversion from spores to bacilli in macrophages, is an appropriate model (34). The use of this strain focuses on distinct spore-macrophage interactions independent of contamination of B. anthracis vegetative bacilli.The virulence of B. anthracis and the efficiency with which it can infect humans via inhalation are reasons the organism and the disease are currently being investigated. B. anthracis spores are comprised of an endospore, containing the infectious portions of the bacterium, and a surrounding exosporium, the outermost layer of the spore, composed of polysaccharides, lipids, and approximately 20 different proteins (27). The exosporium is the primary site of contact for spore interactions with host defenses. The process of sonication has been shown to selectively remove the exosporium while keeping the endospore intact (18). We postulate that spore-macrophage interaction at the exosporium is pivotal to the anthrax disease process.Once inside the...

Bacillus anthracis -derived nitric oxide is essential for pathogen virulence and survival in macrophages

“…Considering that macrophages produce a large amount of NO to control infection (18)(19)(20) our seemingly paradoxical results can be rationalized if NO production by bacteria and macrophages is separated in time, leading to opposite outcomes. Indeed, it is well established that superoxide generation begins immediately after phagocytosis of bacteria.…”

“…Indeed, it is well established that superoxide generation begins immediately after phagocytosis of bacteria. However, inducible NOS is activated only 8-12 h after infection, when the oxidative burst is largely over (4,19). In contrast, B. anthracis germinating in macrophages would be expected to begin producing its own NO immediately from bNOS to antagonize the antimicrobial action of ROS.…”

Bacillus anthracis spores and lethal toxin induce IL‐1β via functionally distinct signaling pathways

“…We previously observed that murine peritoneal macrophages were capable of killing BA in vitro through an NO-mediated bactericidal mechanism that targets the emerging vegetative bacilli [8,9,13,14]. Therefore, we determined whether the susceptibility of caspase-1 -/-mice to lethal infection was due to an inability of its macrophages to kill the BA.…”

“…No germination of DgerH spores was evident either by direct inspection or by susceptibility of spores in culture supernatants to heating. Since macrophages kill the emerging vegetative bacilli within macrophages in an NO-dependent manner [8,13], we measured whether BA could induce NO, a key microbicidal effector mechanism of murine macrophages, in the absence of caspase-1. While DgerH BA spores induced the expression of NO in macrophages of caspase-1 +/+ mice, there was minimal NO generated following the same stimulation in macrophages of caspase-1 -/-mice (Fig.…”

“…Recent data suggest that the spore, rather than being an inert structure, may actually interact with the innate immune system to promote germination and subsequent infection [7,8]. While >10 6 spores/mL are required to elicit TNF-a and other cytokines, levels of BA spores likely to be encountered initially in the environment ( 10 3 spores/mL) [9].…”