Control of Bacillus Anthracis Spore Formation

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Spore germination represents a pivotal intervention point for controlling anthrax infection. Germination occurs after uptake by alveolar phagocytes, concurrent with their transport to the lymphatics. However, the timing of the initial association of spores with phagocytes varies depending on inoculum's dose and host factors. Spores are phagocytosed soon after exposure or can linger in the lung up to 100 days before initiating a lethal infection. Spores are hardy, not easily cleared from the body and not readily susceptible to antibiotics; this necessitates prolonged antibiotic treatment & real risks of drug intolerances. Abilities to block or delay spore germination could be utilized to block the infectious cycle. Alternatively, efficiently inducing germination, under controlled medical supervision, may render the bacillus vulnerable to antibiotics, shortening the requirement for lengthy antibiotic regimens. Aim 1" Develop 96-well platforms for screening compounds that act to either block or stimulate germination of anthrax spores and screening for stimulators and inhibitors of germination. Development of these assays will be of use to other research groups, and will facilitate the mechanistic studies of the other aims. Aim 2" GerA-like protein structure/function analysis will determine the membrane topology of the 6 gerA family of B. anthracis operons, determine protein-protein interactions between the 3 GerA-like proteins encoded by each tricistronic operon and examine potential physical interactions ("cross-talk") between the proteins of the 6 family members. Aim 3: Determine the spores' repertoire of non-gerA-like proteins that form the complete germination machinery using yeast two-hybrid analysis, phage display, and genetic analysis of hyper germination mutants. Knowledge of the spore proteins responsible for germination, as well as their functions and extracellular domains will directly test the hypothesis that the rapid & dramatic switch from dormancy to growing pathogen is controlled by an intricate membrane sensing/signaling system. It will also facilitate discovery of compounds that can be used to control these germination events.