Role of host factors during effector translocation by type III secretion

 描述(申请人提供)：革兰氏阴性肠道病原菌造成重大疾病负担。为了确定疾病，这些生物中的许多将细菌效应蛋白转移到宿主细胞胞浆中。福氏志贺氏菌是这项提案的重点，是腹泻疾病的主要原因，每年导致全球1.65亿例病例和110万人死亡。福氏志贺氏菌使用一种被称为III型分泌系统(T3SS)的装置来转移效应器，这种装置在细菌病原体中高度保守。T3SS的缺失可以预防志贺氏菌、沙门氏菌、耶尔森氏菌、弧菌和大肠杆菌的毒力，这表明T3SS在细菌致病中的重要性。细菌与宿主细胞表面的接触激活T3SS递送两种不同的细菌蛋白，从而在宿主细胞膜上形成转位孔洞。T3SS介导的效应子转位到宿主细胞胞浆是需要转位孔道的。细菌与宿主细胞表面的接触可激活T3SS，但激活T3SS的分子机制尚不清楚。为了确定宿主因素是否对T3SS活性有贡献，进行了全基因组选择。这一选择确定了波形蛋白的作用，波形蛋白是一种中间纤维。在真核细胞骨架的三种主要类型中，中间丝是研究最少的，它们在细菌发病中的作用也鲜为人知。我的初步数据表明，中间丝与福氏志贺氏菌转位孔蛋白IPAC的C末端相互作用。中间丝对转位孔的形成不是必需的，但对于福氏志贺氏菌与宿主细胞的有效对接和T3SS对细菌效应器的有效转位都是必需的。在没有中间丝的细胞中，转位子孔的形成没有随后的细菌效应器移位，这表明这些过程可以被解离，这为研究细菌对接如何影响T3SS介导的效应器输送提供了一个独特的机会。我的总体假设是，中间丝和IPAC之间的相互作用改变了福氏志贺氏菌与宿主细胞的联系，从而促进了效应器的易位。我建议检验这一假设的各个方面，目的如下：i)评估IPAC与中间丝相互作用对T3SS效应器易位下游过程的影响。(Ii)表征中间纤维与IPAC相互作用对与福氏志贺氏菌对接相关的过程的影响。这一提议将为中间细丝如何促进福氏志贺氏菌与宿主细胞的对接和其T3SS对效应器的移位做出贡献提供机械性的见解。由于T3SS的高度保守性，以及它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对细菌发病机制的理解产生广泛的影响。

 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.