The role of calcium-DPA in the virulence of Bacillus anthracis spores

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DESCRIPTION (provided by applicant): The anthrax attacks of October 2001 caused five fatalities and 17 illnesses, disrupted business and government activities, and caused widespread anxiety. The infective form of B. anthracis is the spore, a dormant and resistant structure formed during periods of stress. Following the inhalation route, B. anthracis spores are phagocytized by alveolar macrophages where they germinate. Interestingly, B. anthracis does not interfere with phagosome maturation and is able to thrive in the phagolysosome. After a delay, the newly germinated cells produce a tripartite toxin that is necessary for survival in the host macrophage. In contrast to their vulnerability to B. anthracis spores, macrophages kill toxin-producing vegetative B. anthracis cells with ease. These results are counterintuitive: Newly germinated cells do not produce toxins early and should be more vulnerable than vegetative cells to macrophage attack. To account for this paradox, we propose that factors unique to Bacillus anthracis spores can account for the ability of germinated cells to survive the macrophage's phagosome. One distinctive candidate is the large concentrations of calcium ions complexed with dipicolinic acid (DPA) that is released upon B. anthracis spore germination. The Ca-DPA depot will be localized inside macrophages when B. anthracis spores germinate in the host. Calcium levels are strictly controlled in mammalian cells and disruptions can lead to apoptosis. Alternatively, DPA release could buffer the acidification of the phagolysosome. As proof-of principle, we have shown that B. anthracis spores containing K+ instead of Ca+2 exhibit reduced cytotoxicity (Fig. 2). Based on these results, we hypothesize that the release of calcium and/or DPA protects germinated B. anthracis cells from macrophage action and is a determining factor in anthrax virulence. To test our hypothesis, we will look into two related issues: (1) is te cytotoxicity of B. anthracis spores dependent on calcium and/or DPA concentrations and (2) what is the fate of calcium and DPA in infected macrophages?