Guan-Liang Cao

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.

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

ABSTRACT 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 O2. 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 Ca2+-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 Ca2+-mediated ERK1/2 activity through direct modification of H-Ras. Ca2+-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 Ca2+ ionophore. Mutation of a putative nitrosylation site at Cys118 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.

Effect of trifluoromethyl ketone‐based elastase inhibitors on neutrophil function in vitro

Neutrophils release elastase, which is known secondarily to cause tissue damage. However, it is rapidly inactivated by the endogenous α1‐proteinase inhibitor (α1Pi). Nevertheless, under pathological conditions, α1Pi is inactivated by oxidants released from neutrophils, resulting in an excess of elastase at the site of inflammation. This elastase/α1Pi imbalance has been implicated as a pathogenic factor in cystic fibrosis, acute respiratory distress syndrome, and emphysema. Elastase inhibitors, which do not interfere with the microbicidal activity of neutrophils and are resistant to neutrophil‐released oxidants, would undoubtedly represent an important advance in the management of neutrophil‐mediated tissue injury. We report that a new family of elastase inhibitors ICI200355 and ZD0892 was found to be resistant toward superoxide, hypochlorous acid, hydrogen peroxide, hydroxyl radical, and peroxynitrite mediated degradation as well as having no effect on the formation of these oxidants by activated neutrophils. More importantly, we found that these inhibitors did not interfere with the ability of human neutrophils to phagocytose and to kill Staphylococcus aureus. In conclusion, a new potent class of elastase inhibitors, while blocking the effects of neutrophil elastase, was found not to impede various physiological functions of human neutrophils, in particular the ability of these phagocytic cells to phagocytose and kill bacteria. J. Leukoc. Biol. 64: 322–330; 1998.

Spin labelling of Bacillus anthracis endospores : A model for in vivo tracking by EPR imaging

Anthrax is caused by the gram-negative bacterium, Bacillus anthracis. Infection by this microbe results from delivery of the endospore form of the bacillus through direct contact, either topical or inhalation. With regard to the latter route of administration, it is proposed that endospores of B. anthracis enter the lungs and are phagocytized by host alveolar macrophages. Thereafter, it is unclear as to how endospores travel to distal loci and what tissues are the targets. Herein, this study describes the spin labelling of endospores through two different approaches with various aminoxyls. Indeed, after exposure to RAW 264.7 cells, these aminoxyl-containing endospores were phagocytized, as demonstrated by EPR spectroscopy of the infected macrophage, thus providing a potential tool for EPR imaging in animals.

The 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 O(2).(-), experiments were designed to determine whether NOS 2 formed peroxynitrite (ONOO(-)) from the reaction of .NO with O(2).(-) 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.