Bacillus anthracis Spores: Initiation of Germination

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DESCRIPTION (provided by applicant): An essential early step in the initiation of anthrax in the mammalian host is the germination of the Bacillus anthracis (endo)spores that have been engulfed by macrophages. The biochemistry of early steps in Bacillus spore germination is not yet understood. Current therapeutic interventions in the anthrax infectious cycle target much later steps. The proposed studies will extend promising preliminary findings on the three-protein GerB germinant receptor of Bacillus subtilis to the GerX and Gerl homologues of B. anthracis. GerX is encoded by a virulence plasmid and is required for full virulence, and Gerl is one of the chromosomally encoded receptors that may also contribute to germination in the macrophage. Like GerB, GerX and Gerl are homologous members of the GerA-type germinant receptors required for germination of Bacillus spores in response to specific nutrient germinants. Mechanisms for these important complexes will be sought. Preliminary evidence indicates that the GerB is an ion channel that is regulated by its nutrient germinants. The hypothesis underlying the application is: (i) that other GerA-type receptors also are either potassium or sodium channels; and (ii) when triggered by a germinant, ion flux through the receptor channel activates downstream ion transporters and other germination-related proteins leading to a cascade of germination events. Specific Aim 1 is to further characterize the biophysical, inhibition and regulatory properties of the GerB channel in the heterologous systems (Escherichia coli membrane vesicles and human embryo kidney cells) used in preliminary studies. The roles of the three GerB proteins will be further defined. Specific Aim 2 is to apply the same approaches to define the GerX and Gerl ion channel properties, germinants, inhibitors, and response to signals of the phagolysosomal environment. Single protein components of these complexes that exhibit channel activity will also be characterized. Specific Aim 3 is to identify candidates for the most proximal, "downstream" transporter(s) of GerX and Gerl and to characterize their activities and the basis whereby specific germinant receptors may activate specific downstream transporters. Such transporters may be new virulence factors, i.e. additional potential therapeutic targets.