Effects of dipicolinic acid on Bacillus anthracis spore biology and cytotoxicity

Bacillus anthracis is a gram-positive spore-forming bacterium that causes lethal inhalation anthrax. The use of B. anthracis as a bioweapon is predicated in its ability to form dormant and resistant infective spores that can be used as agents. B. anthracis spores contain large concentrations of dipicolinic acid complexed with cations, especially calcium (Ca-DPA). Following phagocytosis by alveolar macrophages, spores germinate inside the phagolysosome and excrete the Ca-DPA depot into the phagosomal space. In this study, we assessed the effects of DPA on B. anthracis spore biology and cytotoxicity. We generated B. anthracis mutants with defects in DPA synthesis ( ΔspoVFA, ΔspoVFB, ΔspoVFAB ) or transport ( ΔspoVV ). To increase the viability of DPA-less spores, we also constructed double mutants ( ΔsleBΔspoVFA, ΔsleBΔspoVFB, ΔsleBΔspoVFAB and ΔsleBΔspoVV ) by deleting the cortex lytic sleB gene. We found that single- and double-mutant DPA-less spores were profoundly compromised in dormancy, viability, germination, and heat resistance. Contrary to expectations, each DPA synthesis mutant exhibited distinct viability and resistance phenotypes. Even with compromised stability, DPA-less B. anthracis spores, with the exception of the Δ sleB Δ spoVV double mutant, were as cytotoxic as wild-type spores. In summary, DPA is required to sustain normal B. anthracis spore biology but is not required for macrophage-targeted virulence. Furthermore, the SpoVV transporter and SleB lytic protein seem to have redundant roles in anthrax spore cytotoxicity beyond DPA accumulation.