Organization and function of Shigella IpaB within plasma membrane translocon pores.

志贺氏菌是与腹泻疾病有关的主要死亡原因，是一种细胞内病原体，可 需要3型分泌系统(T3SS)才能入侵宿主细胞。T3SS在肠道中高度保守 T3SS是细菌的病原体，在发病过程中是必不可少的；T3SS的丢失会导致毒力的减弱。 在福氏志贺氏菌感染过程中，T3SS分泌细菌蛋白IPAB和IPAC，这两种蛋白在细菌的 质膜(转位体孔)，被预测为异源低聚物。福氏假单胞菌附着体(“船坞”) 并通过毛孔分泌毒力蛋白(效应物)到宿主细胞。互动 在IPAB和IPAC之间的天然传递的转位孔已被假设，但从未得到证实。 IPAB和IPAC单体已被证明在特定条件下在体外可以齐聚，这表明类似的 在感染的情况下，转运子的孔功能可能需要相互作用。我的初步数据 建立研究转位孔蛋白在S。 哺乳动物细胞的福氏杆菌感染。这些方法已经证明，分子的构象 转位体孔是动态的，并识别了特定的ipac残基，这些残基在孔中有不同的位置。 不适合对接的毛孔和适合对接的毛孔。IPAB如何参与这一转变 从对接无能状态到对接能力状态以及IPAB参与的影响 毛孔组织尚不清楚。我的总体假设是，当IPAB形成功能转位孔时 和IPAC通过其跨膜跨膜的相互作用进行齐聚，使得IPAB在 这些相互作用在孔隙构象的变化过程中发生变化。我提议 测试这一假设的各个方面，目的如下： (I)在天然传递的转位孔的背景下确定IPAB的拓扑结构。 (2)界定IPAB单体之间以及IPAB和IPAC之间的组织和相互作用 在自然传递的易位毛孔的背景下。 使用S.Flexneri T3SS作为模型，这一提议很有可能产生关于 转位子孔参与了T3SS的功能。由于T3SS及其附近的高度保守的性质 对于革兰氏阴性肠道病原菌的毒力的普遍要求，本文提出的工作具有很高的 可能会对我们对细菌发病机制的理解产生广泛的影响。这些研究将为我提供 分子生物学和蛋白质生物化学方面的新专业知识，使我处于一个理想的位置 成为一名独立研究人员的职业目标。

Shigella spp., the leading cause of death associated with diarrheal illness, are intracellular pathogen that require a type 3 secretion system (T3SS) to invade host cells. The T3SS is highly conserved among enteric bacterial pathogens and is essential for pathogenesis; the loss of T3SS leads to an attenuation of virulence. During S. flexneri infection, the T3SS secretes the bacterial proteins IpaB and IpaC, which form a pore in the plasma membrane (the translocon pore) that is predicted to be hetero-oligomeric. S. flexneri attaches (“docks”) onto the pore and secretes virulence proteins (effectors) through the pore into the host cell. Interactions between IpaB and IpaC in a natively delivered translocon pore have been postulated but never demonstrated. IpaB and IpaC monomers have been shown to oligomerize in vitro under specific conditions, suggesting similar interactions may be required for translocon pore function in the context of infection. My preliminary data establish approaches to investigate the translocon pore proteins when they are natively delivered during S. flexneri infection of mammalian cells. These approaches have demonstrated that the conformation of the translocon pore is dynamic and have identified specific IpaC residues that are differentially positioned in pores that are incompetent for docking and pores that are competent for docking. How IpaB is involved in the shift from a docking incompetent state to a docking competent state and the implications of IpaB involvement on pore organization are unknown. My overall hypothesis is that a functional translocon pore is formed when IpaB and IpaC oligomerize through interactions in their transmembrane spans in such a way that IpaB lines the majority of the pore channel, and that these interactions change during shifts in pore conformation. I propose to test aspects of this hypothesis with the following aims: (I) Determine the topology of IpaB in the context of natively-delivered translocon pores. (II) Define the organization of and interactions among IpaB monomers and between IpaB and IpaC in the context of natively delivered translocon pores. Using S. flexneri T3SS as a model, this proposal is highly likely to generate mechanistic insights into how the translocon pore contributes to T3SS function. Due to the highly conserved nature of the T3SS and its near ubiquitous requirement for the virulence of gram-negative enteric pathogens, the work proposed here is highly likely to have a broad impact on our understanding of bacterial pathogenesis. These studies will provide me with new expertise in molecular biology and protein biochemistry and place me in an ideal position for reaching my career goal of becoming an independent researcher.