Purification of a Modified Flagellar Export Apparatus

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DESCRIPTION (provided by applicant): The bacterial flagellum is a complex, proton-driven rotary nanomachine responsible for motility in many species. It contains a specialized Type III secretion ("T3S") apparatus that allows the flagellum to self-assemble. T3S is a membrane transport process that is of clinical interest as a primary mechanism of bacterial pathogenesis. Many human pathogens such as Salmonella and Yersinia use T3S apparatuses known as needle complexes to deliver bacterial proteins into host cells to affect virulence. The flagellar T3S apparatus consists of soluble and integral membrane proteins housed within the basal body of the flagellum. Much remains to be learned about how the apparatus functions. Using genetic and biochemical means, we will construct, purify and reconstitute a modified export apparatus as a prelude to development of an in vitro export assay. Coincident saturation and competition binding studies will also be developed and undertaken to analyze interactions between apparatus components, exported proteins and chaperones. Other efforts will involve examination of the organization and function of the membrane protein components of the apparatus. The flagellum is an excellent model system for T3S in that question of fundamental importance with broad impact can be investigated in a comprehensive manner without the coincident technical complications of working directly with virulence secretion systems. The similarities between needle complexes and flagella are such that understanding gained from one system is usually applicable to the other. Moreover, the flagellum is molecular machine. This work will lead to an enhanced understanding of how the flagellum is assembled, transmembrane transport processes in general and greater specific knowledge of T3S, which may contribute to more effective treatments for bacterial infections. Many human pathogens such as Salmonella and Yersinia use Type III secretion apparatuses known as needle complexes to deliver bacterial proteins into host cells to affect virulence. We will examine the dynamics of Type III secretion by investigating the organization and function of the homologous bacterial flagellar export apparatus. Construction and purification of a modified export apparatus and studies of the dynamics of apparatus and substrate interactions will lead to an enhanced understanding of this primary mechanism of bacterial pathogenesis.