Role of host factors during effector translocation by type III secretion

 描述（由申请方提供）：革兰氏阴性肠道病原体引起显著的疾病负担。为了建立疾病，许多这些生物体将细菌效应蛋白转运到宿主细胞胞质溶胶中。福氏志贺菌是该提案的重点，是导致腹泻病的主要原因，每年在全球造成1.65亿例病例和110万例死亡。S.福氏杆菌使用称为III型分泌系统（T3 SS）的装置易位效应子，所述III型分泌系统在细菌病原体中高度保守。T3 SS的缺失可防止志贺氏菌、沙门氏菌、耶尔森氏菌、弧菌和大肠杆菌的毒力。大肠杆菌物种，这证明了其对细菌致病的重要性。细菌与宿主细胞表面的接触激活T3 SS以递送两种不同的细菌蛋白，从而在宿主细胞膜中形成易位子孔。随后T3 SS介导的效应子易位到宿主细胞胞质溶胶中需要易位子孔。细菌与宿主细胞表面的接触激活T3 SS，但负责T3 SS激活的分子机制是未知的。为了确定宿主因素是否有助于T3 SS活性，进行了全基因组选择。这种选择确定了波形蛋白（一种中间丝）的作用。中间丝是三种主要类型的真核细胞骨架丝中研究最少的，它们在细菌致病过程中的作用知之甚少。我的初步数据表明，中间丝与S的C-末端相互作用。弗氏菌转位孔蛋白IpaC。中间丝对于易位孔的形成不是必需的，但是对于有效的S.福氏杆菌与宿主细胞的对接以及通过T3 SS的有效细菌效应子易位。在缺乏中间丝的细胞中没有随后的细菌效应子易位的情况下发生易位子孔形成的观察结果表明，这些过程可以解离，这为研究细菌对接如何影响T3 SS介导的效应子递送提供了独特的机会。我的总体假设是，中间丝和IpaC之间的相互作用改变了S。福氏杆菌与宿主细胞的结合以促进效应子的易位。我建议测试这一假设的各个方面，其目的如下：I）评估IpaC与中间丝相互作用对T3 SS效应子易位下游过程的影响。 (II)为了表征中间丝与IpaC的相互作用对S. flexneri对接。这一建议将提供机制的见解，如何中间丝有助于对接的S。福氏杆菌通过其T3 SS转运至宿主细胞并转运效应子。由于T3 SS的高度保守性及其对革兰氏阴性肠道病原体毒力的几乎无处不在的要求，这里提出的工作很可能对我们理解细菌发病机制产生广泛影响。

 DESCRIPTION (provided by applicant):Gram-negative enteric pathogens cause significant disease burden. To establish disease, many of these organisms translocate bacterial effector proteins into the host cell cytosol. Shigella flexneri, the focus of this proposal, is a major caus of diarrheal illness, resulting in 165 million cases and 1.1 million deaths globally each year. S. flexneri translocates effectors using an apparatus known as the type III secretion system (T3SS), which is highly conserved among bacterial pathogens. Loss of the T3SS prevents virulence of Shigella, Salmonella, Yersinia, Vibrio, and E. coli species, which demonstrates its importance for bacterial pathogenesis. Bacterial contact with the host cell surface activates the T3SS to deliver two different bacterial proteins to form the translocon pore in the host cell membrane. The translocon pore is required for subsequent T3SS-mediated translocation of effectors into the host cell cytosol. Bacterial contact with the host cell surface activates the T3SS, but the molecular mechanisms responsible for T3SS activation are unknown. To identify whether host factors contribute to T3SS activity, a genome-wide selection was performed. This selection identified a role for vimentin, an intermediate filament. Intermediate filaments are the least studied of the three major types of eukaryotic cytoskeletal filaments, and their role during bacterial pathogenesis is poorly understood. My preliminary data demonstrate that intermediate filaments interact with the C-terminus of the S. flexneri translocon pore protein IpaC. Intermediate filaments are not necessary for translocon pore formation, but are required for both efficient S. flexneri docking to host cells and for efficient bacterial effector translocation by T3SSs. The observation that translocon pore formation occurs without subsequent bacterial effector translocation in cells lacking intermediate filaments shows these processes can be dissociated, which allows a unique opportunity to study how bacterial docking impacts T3SS-mediated effector delivery. My overall hypothesis is that the interaction between intermediate filaments and IpaC alters the association of S. flexneri with the host cell so as to promote translocation of effectors. I propose to test aspects of this hypothesis with the following aims: I) To assess the impact of IpaC interaction with intermediate filaments on downstream processes in T3SS effector translocation. (II) To characterize the effect of intermediate filament interactions with IpaC on processes associated with S. flexneri docking. This proposal will provide mechanistic insights into how intermediate filaments contribute both to docking by S. flexneri to host cells and translocation of effectors by its T3SS. 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.