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

志贺氏菌属，与呼吸道疾病相关死亡的主要原因是细胞内病原体， 需要3型分泌系统（T3 SS）来侵入宿主细胞。T3 SS在肠道中高度保守， T3 SS是细菌性病原体的一种重要组成部分，并且是致病所必需的; T3 SS的丧失导致毒力的减弱。 在S.在福氏杆菌感染时，T3 SS分泌细菌蛋白IpaB和IpaC，其在细胞中形成孔。 质膜（translocon孔）被预测为异源寡聚体。S. flexneri附件（“码头”） 并通过孔分泌毒力蛋白（效应物）进入宿主细胞。相互作用 在天然传递的易位子孔中IpaB和IpaC之间的关系已经被假定，但从未被证实。 IpaB和IpaC单体已显示在特定条件下在体外寡聚化，表明类似的 相互作用可能是在感染的情况下易位孔功能所必需的。我的初步数据 建立方法来研究转座孔蛋白，当它们在S. 哺乳动物细胞的弗氏杆菌感染。这些方法已经证明， 易位孔是动态的，并且已经鉴定了在孔中差异定位的特异性IpaC残基 不能对接的孔和能对接的孔。IpaB如何参与转变 从一个对接不合格的国家对接合格的国家和IpaB参与的影响， 孔隙结构未知。我的总体假设是，当IpaB 和IpaC通过在其跨膜跨度中的相互作用而寡聚化，使得IpaB排列在 大多数的孔通道，这些相互作用的变化在孔构象的转变。我建议 检验这一假设的各个方面，目的如下： (I)在天然递送的易位子孔的背景下确定IpaB的拓扑结构。 (II)定义IpaB单体之间以及IpaB和IpaC之间的组织和相互作用 在天然递送的易位子孔的情况下。 利用S.作为一个模型，这个建议很有可能产生机械的见解，如何 易位子孔有助于T3 SS功能。由于T3 SS的高度保守性及其近 革兰氏阴性肠道病原体的毒力普遍存在的要求，这里提出的工作是高度 可能对我们理解细菌致病机理产生广泛影响。这些研究将为我提供 新的专业知识，分子生物学和蛋白质生物化学，并把我放在一个理想的位置，达到我的 成为独立研究员的职业目标。

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.