IpaD triggers type III secretion in Shigella

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DESCRIPTION (provided by applicant): The ability for Shigella flexneri to cause bacillary dysentery requires the use of a type III secretion system (TTSS) that delivers the translocator proteins IpaB and IpaC to host cell membranes to create a pore for the transport of effector proteins into the host cytoplasm. The net result is pathogen invasion of the target cell. IpaD controls the secretion and delivery of the translocator proteins to the host membrane from a position at the tip of the TTSS needle. ipaD null mutants constitutively secrete IpaB and IpaC and cannot invade mammalian cells. In the same respect, deletions within IpaD that prevent its stable association with the TTSS needle tip result in a loss of secretion control and invasiveness, demonstrating the importance of IpaD localization at the needle tip. The addition of bile salts to the Shigella growth medium promotes the recruitment of IpaB to the needle tip in an IpaD dependent manner. This is the first demonstration of a translocator protein stably associating with the TTSS needle. We thus hypothesize that IpaD senses environmental signals to trigger the mobilization of IpaB to the needle tip as the final step in secreton maturation. To test this hypothesis, the specific aims of this investigation are: 1) determine the molecular basis for environmental sensing by IpaD; and 2) determine the biochemical and structural basis for the effects of bile salts. The first aim of this investigation will center around a mutagenesis study of bile salt effects based on the crystal structure of IpaD to determine the roles of specific domains in sensing bile salts and triggering IpaB mobilization. The second aim will require the chemical synthesis of fluorescent bile salts that will be used in fluorescence spectroscopy studies to assess bile salt interactions with IpaD and mutant IpaD proteins. The presence of TTSS needle tip proteins appears to be a common feature among TTSSs. Because these secretions systems are key virulence factors for many gram-negative pathogens, defining the mechanism used by the tip proteins to control the first steps in translocon formation should help in designing new compounds for preventing infections by these pathogens and identifying new targets for vaccine development. This investigation explores the interaction of bile salts with sensory proteins on the surface of Shigella flexneri that control the delivery of virulence proteins to human intestinal cells. Because the type III secretion proteins targeted here are conserved among a broad range of gram-negative pathogens, completion of this work will have long term implications in understanding the pathogenesis of diverse gram-negative bacterial pathogens and in identifying steps at which the pathogenesis of these bacteria can be blocked.