A Mechanism for Shigella Type III Secretion Activation

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DESCRIPTION (provided by applicant): Shigella flexneri, the causative agent of bacillary dysentery, uses its type III secretion system (TTSS) to deliver proteins into host cells to promote bacterial entry. From its position at the tip of the TTSS needle, invasion plasmid antigen D (IpaD) serves to control Shigella type III secretion. By incubating Shigella with deoxycholate (DOC), we have shown that the first secreted translocator protein, IpaB, can be recruited to the needle tip complex without further induction of type III secretion. Recruitment of the second translocator (IpaC) to the needle tip complex occurs upon incubating the bacteria with liposomes rich in cholesterol and sphingomyelin, which also results in full induction of type III secretion. Thus, the Shigella TTSS provides a novel model system for exploring the individual steps of type III secretion induction. Based on the preliminary findings presented here, we hypothesize that IpaD senses environmental signals to trigger the controlled recruitment of IpaB to the needle tip complex. Because DOC mimics the environmental signal(s) needed for this distinct step on the pathway to type III secretion induction, we plan to determine the physical and molecular mechanism responsible for IpaB recruitment to the TTSA needle tip complex. To do this, the specific aims of this investigation are to: 1) solve the crystal structure of IpaD with DOC bound to identify the conformational changes responsible for IpaB recruitment to the Shigella surface; 2) target key sites on IpaD for mutational analysis and phenotypic characterization; and 3) determine the influence of targeted mutations on solution properties of IpaD IpaD and generate new crystals and co-crystals of IpaD 1 DOC to determine how specific mutations that alter IpaD function affect its structure and dynamics. TTSSs are essential virulence determinants for many significant human pathogens. Using the Shigella system, we are the first to describe the distinct steps of type III secretion induction. This unprecedented dissection of the steps of type III secretion now provides a tool for revealing the structural basis for type III secretion induction. In this investigation, we have embarked on a collaborative effort to explore type III secretion at a level not currently possible in any other bacterial system. The information obtained here will contribute to the future design of practical methods for blocking type III secretion. PUBLIC HEALTH RELEVANCE: Shigella flexneri is global public health problem as the causative agent of bacillary dysentery (shigellosis). To initiate infection, S. flexneri uses a complex type III secretion system to deliver proteins into human intestinal epithelial cells to promote bacterial entry into these cells. In this investigation, we will identify the mechanism by which this secretion system becomes activated so that it may be possible to identify compounds that can neutralize Shigella's ability to cause human disease.