Factors involved in the germination and inactivation of Bacillus anthracis spores in murine primary macrophages

Since macrophages have been implicated in inhalation anthrax either for defense and/or as enablers for spore trafficking, their function has been further defined. Spores were efficiently taken up by primary mouse bone marrow-derived macrophages even in the absence of serum but a minimal amount was required for spore germination and subsequent inactivation. With 10% fetal bovine serum (FBS) virtually all of the spores germinated but when the concentration of FBS was lowered to 1.0% or less, or when 10% horse serum replaced FBS, only 50% of the spores were inactivated within 1 h with no subsequent loss. Chloramphenicol, which blocks spore outgrowth but not germination, did not inhibit spore killing in macrophages. Based on complete inhibition of germination by D-alanine plus D-histidine, it is likely that only two of the several Bacillus anthracis germination systems are involved within macrophages. D-Histidine inhibits the gerH system previously implicated in germination within macrophages. D-Alanine is likely to block the gerX system since disruption of the gerXA gene resulted in little germination within 4 h in macrophages. Macrophages provide a major line of defense against infection by efficiently sequestering spores and in the presence of minimal nutrients effectively killing those that germinate before outgrowth.