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The specific aims are: 1) To define functional domains within InvC. This will involve the isolation of loss-of-function InvC mutants that retain the ability to hydrolyze ATP. It is expected that these mutants will define protein-protein interaction domains. 2) To identify protein(s) with which InvC associates. This will involve two-hybrid analysis, screening for suppressors of InvC mutants, and biochemical assays. 3) To elucidate the role(s) of the identified interaction(s) in the pathogenesis of S. typhimurium by examining the phenotypes of InvC and/or interacting partner mutants in mouse models of infection.
Contact-dependent type III secretion has evolved as an intricate system of biochemical communication between infecting bacteria and eukaryotic hosts. This method of inter-kingdom signaling has been identified only in human and plant pathogenic bacteria, including the human pathogen S. typhimuriuin. Type III secretion has proven elemental to the pathogenesis of these bacteria. Through this pathway, effector proteins can be translocated from the bacterial cytoplasm directly into the host cells. Of the at least 20 proteins required for the construction and function of this apparatus, one of the most highly conserved factors is a cytoplasmic ATPase, which may provide energy for translocation. While this ATPase, called InvC in S. typhimurium, hydrolyzes ATP in vitro, little is known about the relationship between InvC (or any type III system or related flagellar ATPase) and the export apparatus. Study of InvC will clarify the architecture and molecular mechanisms of this bacterial virulence system.