Effects of dipicolinic acid on Bacillus anthracis spore biology and cytotoxicity

Abstract

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 ({Delta}spoVFA, {Delta}spoVFB, {Delta}spoVFAB) or transport ({Delta}spoVV). To increase the viability of DPA-less spores, we also constructed double mutants ({Delta}sleB{Delta}spoVFA, {Delta}sleB{Delta}spoVFB, {Delta}sleB{Delta}spoVFAB and{Delta} sleB{Delta}spoVV) 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 {Delta}sleB{Delta}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. ImportanceBacillus anthracis causes deadly pulmonary anthrax and has been used as a weapon for bioterrorism. B. anthracis form spores that germinate and establish infection. During germination, B. anthracis spores release large amounts of calcium complexed with dipicolinic acid (DPA). In this study, we deleted the B. anthracis genes that are required to synthesize and transport DPA into spores. We found that B. anthracis DPA-less mutant spores exhibited differential biological effects that were DPA independent. Furthermore, we found that DPA was not required for anthrax cytotoxicity. Finally, we found that proteins involved in DPA synthesis, transport, and cortex lysis have biological and virulence functions that extend beyond DPA accumulation.