Bacillus anthracis phospholipases C facilitate macrophage-associated growth and contribute to virulence in a murine model of inhalation anthrax

Abstract

Several models of anthrax pathogenesis suggest that early in the infectious process Bacillus anthracis endospores germinate and outgrow into vegetative bacilli within phagocytes before being released into the blood. Here, we define the respective contributions of three phospholipases C (PLCs) to the pathogenesis of B. anthracis. Genetic deletions of the PLCs were made in the Sterne 7702 background, resulting in the respective loss of their activities. The PLCs were redundant both in tissue culture and in murine models of anthrax. Deletion of all three PLC genes was required for attenuation of virulence in mice after intratracheal inoculation. This attenuation may be attributed to the inability of the PLC-null strain to grow in association with the macrophage. Complementation of these defects in both models of anthrax was achieved by expression of the PLC genes in trans. The functional redundancy between PLCs in the virulence of B. anthracis implies that their activities are important for anthrax pathogenesis.

FIG. 1.

PLC activity of B. anthracis. PC-PLC activity was assayed on McClung Toabe agar supplemented with 10% egg yolk enrichment (A), SMase activity was assayed on TSA II 5% sheep blood agar supplemented with 1 mM MgCl₂ and 1 mM CaCl₂ (B), and PI-PLC activity was assayed on BHI supplemented with 500 μg of X-PI/ml (C). Plates were inoculated with B. anthracis or B. subtilis strains, incubated at 37 or 40°C for 36 h, and monitored for opacity (PC-PLC), hemolysis (SMase), or blue-white screen (PI-PLC).

FIG. 2.

Functional cooperation between B. anthracis PLCs is important for growth and/or survival in BMM. BMM obtained from BALB/c mice were challenged with B. anthracis endospores at an input MOI of 10:1 as described in Materials and Methods. Cell-associated growth was determined every 2 h for 8 h after gentamicin treatment. Changes in cell-associated growth were normalized to viable counts at the 0-h time point. Experiments were done in triplicate three separate times, and the average of three representative experiments are depicted here with the standard deviation. The differences in the fold increase in growth at 8 h after gentamicin treatment between the ΔplcBsmcA, ΔplcBplcA, and ΔsmcAplcA strains compared to the Sterne 7702 strain were significant (P < 0.02 as determined by an unpaired two-tailed t test). The differences between the ΔplcBsmcAplcA strain compared to the Sterne 7702, ΔplcB, ΔsmcA, ΔplcA, ΔplcBsmcA, ΔplcBplcA, and ΔsmcAplcA strains were significant (P < 0.02 as determined by an unpaired two-tailed t test at 8 h).

FIG. 3.

B. anthracis PLC-null strains are less cytotoxic to macrophages. BMM were challenged with B. anthracis endospores at an input MOI of 10:1 as described in Materials and Methods. Supernatants were collected at 8 h after gentamicin treatment and assayed for cytotoxicity by scoring for the release of the cytoplasmic LDH. Experiments were done in triplicate, and the average of three representative experiments are depicted here with the standard deviation. The differences between ΔplcBsmcA, ΔplcBplcA, and ΔsmcAplcA strains compared to the Sterne 7702 strain were significant (P < 0.02 as determined by an unpaired two-tailed t test). The differences between the ΔplcBsmcAplcA strain compared to the Sterne 7702, ΔplcB, ΔsmcA, ΔplcA, ΔplcBsmcA, ΔplcBplcA, and ΔsmcAplcA strains were significant (P < 0.05 as determined by an unpaired two-tailed t test).

FIG. 4.

PLC activity aids the growth of B. anthracis in association with the macrophage. BMM were challenged with B. anthracis endospores as described in Materials and Methods, except that infections were performed in Lab Tek II chamber slides. Slides were stained at 8 h after gentamicin treatment with Wright-Giemsa-like technique and visualized by light microscopy at ×1,000 magnification. Photomicrographs of representative fields are shown.