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 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.

FIG. 1.

Representative plot depicting spin trapped O₂·⁻ generated by purified NOS 2. The reaction system consisted of NOS 2 (6.5 μg), l-arginine (100 μM), BMPO (50 mM), and NADPH (150 μM) in potassium phosphate buffer (Chelexed [50 mM] containing EGTA [1 mM] and DTPA [1 mM] adjusted to pH 7.4). Each bar on the graph is the peak height of the first low-field peak of EPR spectrum of BMPO-OOH from the reaction of BMPO with NOS 2-generated O₂·⁻. The data are representative of three independent experiments, expressed as the means and standard deviations. The inset shows a typical EPR spectrum for the reaction of BMPO with O₂·⁻ generated by NOS 2 in the presence of l-arginine. EPR spectra were recorded continuously, and data presented in this figure were obtained 10 min after the addition of NADPH. Receiver gain was 10 × 10⁴.

FIG. 2.

Killing of B. anthracis in spore-infected macrophages. Primary murine peritoneal macrophages (10⁶ cells/ml) were infected with spores (10⁵ spores/ml) prepared from sonicated exosporium (exo⁻) B. anthracis strain Sterne 34F2 and treated either with l-nil (1 mM) (▪) or PEG-SOD (100 U/ml) (○) and incubated at 37°C in 5% CO₂ for 30 min to allow phagocytosis. Samples of infected macrophages were obtained at 1, 3, 5, and 24 h after initial infection; washed; and lysed for viable count plating, and the CFU were determined. The data are expressed as log values. Log kill was determined as defined in Materials and Methods. The data are expressed by their differences in log values. *, P < 0.05; **, P < 0.01 (versus the l-nil treatment group). The data are shown as means ± the standard deviation of values obtained from two independent experiments, each conducted in duplicate.

FIG. 3.

Arginase activity from Sterne exosporium samples of various concentrations. Arginase activity assay was used to determine enzyme activity from various concentrations of exosporium isolated from Sterne 34F2 strain B. anthracis (25 to 75 μg/ml). Initiation of enzyme activity began with the addition of l-arginine (25 mM), and the reaction was allowed to proceed for 3 h before being terminated with perchloric acid. Each bar on the graph is the average of three independent experiments, expressed as means and standard deviations.

FIG. 4.

Exosporium-dependent killing of B. anthracis in spore-infected macrophages. Primary murine peritoneal macrophages (10⁶ cells/ml) were infected with either native (exo⁺) or sonicated endospores (exo⁻) (10⁵ spores/ml) or isolated exosporium at indicated concentrations (25, 50, and 75 μg/ml) to sonicated endospores and incubated at 37°C in 5% CO₂ for 30 min to allow phagocytosis. Samples of infected macrophages were obtained at 1, 5, and 24 h after initial infection; washed; and lysed for viable count plating, and CFU were determined. The data are expressed as log values. Log kill was determined as defined in Materials and Methods. The data are expressed by their differences in log values. *, P < 0.05; **, P < 0.01 (versus exo 0 μg/ml, i.e., sonicated). The data are shown as means ± the standard deviation of values obtained from two independent experiments, each conducted in duplicate.

FIG. 5.

Differential nitrite production by primary murine peritoneal macrophages infected with B. anthracis in the presence of exosporium. Primary murine peritoneal macrophages (10⁶ cells/ml) were infected with either native (exo⁺) or sonicated endospores (exo⁻ or exo 0 μg/ml) (10⁵ spores/ml) or isolated exosporium at the indicated concentrations (25, 50, and 75 μg/ml) to sonicated endospores and incubated at 37°C in 5% CO₂ for 30 min to allow phagocytosis. The Griess assay was used to determine the concentration of nitrite, the oxidation product of ·NO, in the extracellular milieu at 24 h after primary murine peritoneal macrophages were infected with sonicated endospores. **, P < 0.01 (versus exo 0 μg/ml). The data are representative of three independent experiments.