IpaD triggers type III secretion in Shigella

描述（由申请人提供）：福氏志贺氏菌引起细菌性痢疾的能力需要使用III型分泌系统（TTSS），该系统将转运蛋白IpaB和IpaC传递到宿主细胞膜，以创建一个孔，将效应蛋白运输到宿主细胞质中。最终的结果是病原体侵入目标细胞。IpaD控制转运蛋白从TTSS针尖的位置分泌和传递到宿主膜。ipad零突变体组成性地分泌IpaB和IpaC，不能侵入哺乳动物细胞。同样，IpaD内部的缺失会阻止其与TTSS针尖的稳定联系，从而导致分泌控制和侵袭性的丧失，这表明IpaD定位在针尖的重要性。在志贺氏菌生长培养基中添加胆汁盐可促进IpaB以IpaD依赖的方式向针尖募集。这是首次证明转运蛋白与TTSS针稳定相关。因此，我们假设IpaD感知环境信号触发IpaB到针尖的动员，这是分泌成熟的最后一步。为了验证这一假设，本研究的具体目的是：1)确定IpaD环境感知的分子基础；2)确定胆盐作用的生化和结构基础。本研究的第一个目标将围绕基于IpaD晶体结构的胆汁盐效应的诱变研究，以确定特定结构域在感知胆汁盐和触发IpaB动员中的作用。第二个目标将需要荧光胆盐的化学合成，将用于荧光光谱研究，以评估胆盐与IpaD和突变IpaD蛋白的相互作用。

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.