Murine macrophages kill the vegetative form of Bacillus anthracis

Abstract

Anti-protective antigen antibody was reported to enhance macrophage killing of ingested Bacillus anthracis spores, but it was unclear whether the antibody-mediated macrophage killing mechanism was directed against the spore itself or the vegetative form emerging from the ingested and germinating spore. To address this question, we compared the killing of germination-proficient (gp) and germination-deficient (DeltagerH) Sterne 34F2 strain spores by murine peritoneal macrophages. While macrophages similarly ingested both spores, only gp Sterne was killed at 5 h (0.37 log kill). Pretreatment of macrophages with gamma interferon (IFN-gamma) or opsonization with immunoglobulin G (IgG) isolated from a subject immunized with an anthrax vaccine enhanced the killing of Sterne to 0.49 and 0.73 log, respectively, but the combination of IFN-gamma and IgG was no better than either treatment alone. Under no condition was there killing of DeltagerH spores. To examine the ability of the exosporium to protect spores from macrophages, we compared the macrophage-mediated killing of nonsonicated (exosporium+) and sonicated (exosporium-) Sterne 34F2 spores. More sonicated spores than nonsonicated spores were killed at 5 h (0.98 versus 0.37 log kill, respectively). Pretreatment with IFN-gamma increased the sonicated spore killing to 1.39 log. However, the opsonization with IgG was no better than no treatment or pretreatment with IFN-gamma. We conclude that macrophages appear unable to kill the spore form of B. anthracis and that the exosporium may play a role in the protection of spores from macrophages.

FIG. 1.

Uptake of germination-proficient Sterne strain 34F2 B. anthracis spore and the germination-deficient Sterne 34F2 mutant (ΔgerH) by murine peritoneal macrophages. Macrophages (10⁶/ml) were infected with spores (10⁶/ml) prepared from B. anthracis strain Sterne 34F2 and ΔgerH. After macrophage uptake of spores for 30 min, the infected macrophages were washed and incubated for another 30 min with gentamicin to kill any extracellular vegetative B. anthracis (i.e., total incubation time of 1 h after addition of spores to the macrophages). The macrophages were then washed further and lysed, and viable CFU were determined. Aliquots from each sample also were incubated at 65°C for 30 min to assess the presence of vegetative cells. The percentage of spore germination was calculated by loss of heat resistance. There was nearly 60% germination of Sterne strain 34F2 spores during the initial 1 hour following addition of spores to the macrophages (*, P < 0.05 compared to nonheated samples). There was no statistically significant decrease in CFU following heating of the ΔgerH spores. Data are shown as means ± standard deviations of values obtained from two independent experiments, each conducted in duplicate.

FIG. 2.

Time course of macrophage bactericidal activity in an MOI-dependent manner. Macrophages (10⁶) were infected with different numbers of spores at ratios of 1:1, 1:2, 1:10, and 1:20 (1 × 10⁶, 5 × 10⁵, 1 × 10⁵, and 5 × 10⁴), respectively, prepared from B. anthracis strain Sterne 34F2 (A) and the ΔgerH strain (B). CFU were determined at 1, 3, 5, and 24 h postinfection. Data are shown as means ± standard deviations of values obtained from two independent experiments, each conducted in duplicate.

FIG. 3.

Comparison of intracellular CFU between Sterne 34F2 and ΔgerH strains in a time-dependent manner. Macrophages (10⁶/ml) were pretreated with IFN-γ (100 U/ml) overnight and infected with spores (10⁶/ml) prepared from B. anthracis strain Sterne 34F2 (A) and the ΔgerH strain (B). Spores (10⁶) were opsonized by IgG (50 μg/ml) or medium alone before phagocytosis by macrophages (10⁶). The infected macrophages were incubated for 1, 3, 5, and 24 h in 5% CO₂ at 37°C, washed, and lysed for viable count plating, and CFU were determined. The data are expressed as log kill, which is defined as log₁₀ CFU at 1 h − log₁₀ CFU at 3, 5, or 24 h, respectively. Data are shown as means ± standard deviations of values obtained from two independent experiments, each conducted in duplicate.

FIG. 4.

Transmission electron micrographs of B. anthracis Sterne 34F2 strain spores with and without exosporium. Sterne 34F2 spores were examined by transmission electron microscopy before (A) and after (B) sonication as described in Materials and Methods. The arrow in panel A indicates the exosporium, which is not evident in panel B.

FIG. 5.

Comparison of intracellular CFU between sonicated and nonsonicated Sterne 34F2 cells in a time-dependent manner. Macrophages (10⁶/ml) were pretreated with IFN-γ (100 U/ml) overnight and infected with spores (10⁶/ml) prepared from B. anthracis nonsonicated (A) and sonicated (B) Sterne 34F2 strain cells. Spores were opsonized by IgG (50 μg/ml) or medium alone before phagocytosis by macrophages. The infected macrophages were incubated for 1, 3, 5, and 24 h in 5% CO₂ at 37°C, washed, and lysed for viable count plating, and CFU were determined. Data were expressed in log values. Data are shown as means ± standard deviations of the values obtained from two independent experiments, conducted in duplicate.