Spore germination represents a pivotal intervention point for controlling anthrax infection.Germination occurs after uptake by alveolar phagocytes, concurrent with their transport to thelymphatics. However, the timing of the initial association of spores with phagocytes variesdepending on inoculum's dose and host factors. Spores are phagocytosed soon after exposure orcan linger in the lung up to 100 days before initiating a lethal infection. Spores are hardy, not easilycleared from the body and not readily susceptible to antibiotics; this necessitates prolongedantibiotic treatment & real risks of drug intolerances. Abilities to block or delay spore germinationcould be utilized to block the infectious cycle. Alternatively, efficiently inducing germination, undercontrolled medical supervision, may render the bacillus vulnerable to antibiotics, shortening therequirement for lengthy antibiotic regimens.
Aim 1 ' Develop 96-well platforms for screeningcompounds that act to either block or stimulate germination of anthrax spores and screening forstimulators and inhibitors of germination. Development of these assays will be of use to otherresearch groups, and will facilitate the mechanistic studies of the other aims.
Aim 2 ' GerA-likeprotein 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 encodedby each tricistronic operon and examine potential physical interactions ('cross-talk') between theproteins of the 6 family members.
Aim 3 : Determine the spores' repertoire of non-gerA-likeproteins that form the complete germination machinery using yeast two-hybrid analysis, phagedisplay, and genetic analysis of hyper germination mutants. Knowledge of the spore proteinsresponsible for germination, as well as their functions and extracellular domains will directly test thehypothesis that the rapid & dramatic switch from dormancy to growing pathogen is controlled by anintricate membrane sensing/signaling system. It will also facilitate discovery of compounds that canbe used to control these germination events.
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