Murine Macrophages Kill the Vegetative Form of Bacillus anthracis

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 (gerH) 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- )o r 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- and IgG was no better than either treatment alone. Under no condition was there killing of gerH 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 IFNincreased the sonicated spore killing to 1.39 log. However, the opsonization with IgG was no better than no treatment or pretreatment with IFN-. 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. Bacillus anthracis, the causative agent of anthrax, is a highly virulent gram-positive and spore-forming bacterium that is typically acquired through contact with anthrax-infected animals or animal products or atypically through intentional exposure as a biological weapon (6, 8). Virulent strains of B. anthracis carry two large plasmids, pXO1 and pXO2, that carry the genes encoding anthrax toxin production and capsule formation, respectively. Dormant spores are highly resistant to adverse environmental conditions but are able to reestablish vegetative growth in the presence of favorable environmental