Identifying the formate-sensory mechanism in Shigella flexneri

摘要 肠道细菌和细胞内人类病原体志贺氏菌导致数亿例 全世界每年都有腹泻病、志贺氏菌病(痢疾)和多达100万人死亡。目前， 目前还没有被批准的预防志贺氏菌病的疫苗，以及抗菌素耐药性的发生率 在志贺氏菌属中。正在上升。为了引起疾病，福氏志贺菌被摄取到人类宿主中， 然后通过消化道迁移，侵入结肠上皮细胞，在那里复制并导致 急性炎症；这一发病机制需要一个协调的毒力基因表达程序。 Flexneri以适应不同的寄主环境和状态。即使在宿主上皮细胞内，福氏链球菌也是如此 毒力基因的表达是动态的，福氏志贺氏菌用来调节这种表达的一个信号是它自己 甲酸盐的生产。当福氏志贺氏菌在宿主细胞内复制时，它会产生并分泌副产品甲酸盐。 混合酸发酵，这种甲酸在宿主细胞中积累。福氏杆菌然后感觉到这种甲酸盐 积累以正向调节与细胞间传播相关的毒力基因的表达和 抑制宿主免疫反应；然而，福氏志贺氏菌识别甲酸盐的机制 宿主细胞内的累积是未知的。本研究的目的是确定福氏志贺氏菌用于 在致病过程中感觉细胞外甲酸在宿主细胞胞浆中的积累。这样做的第一个目的是 建议用差示配基径向毛细管作用试验(DRaCALA)鉴定福氏志贺氏菌 甲酸盐结合蛋白，然后表征它们在促进福氏志贺氏菌空斑形成中的作用。第二 这项建议的目的是对福氏志贺氏菌转座子突变文库进行遗传筛选，以确定基因 与福氏志贺氏菌对甲酸盐的反应有关。确定这一重要的毒力调控系统将 使未来的研究能够表征受感染宿主细胞中甲酸盐信号的动态，并有可能 为抗菌治疗和疫苗开发提供新的靶点。

Abstract The enteric bacteria and intracellular human pathogen Shigella causes hundreds of millions of cases of the diarrheal disease shigellosis (dysentery) per year worldwide and as many as one million deaths. Currently, there is no approved vaccine for the prevention of shigellosis, and the incidence of antimicrobial resistance amongst Shigella spp. is on the rise. To cause disease, Shigella flexneri is ingested into a human host, and then migrates through the digestive tract and invades colonic epithelial cells where it replicates and causes acute inflammation; this pathogenesis requires a coordinated program of virulence gene expression for S. flexneri to adapt to different host environments and states. Even within a host epithelial cell, S. flexneri virulence gene expression is dynamic, and one signal that S. flexneri uses to regulate this expression is its own production of formate. As S. flexneri replicates within a host cell, it produces and secretes formate, a byproduct of mixed acid fermentation, and this formate accumulates in the host cell. S. flexneri then senses this formate accumulation to positively regulate the expression of virulence genes associated with intercellular spread and dampening the host immune response; however, the mechanism by which S. flexneri recognizes formate accumulation within a host cell is unknown. The goal of this study is to identify the S. flexneri system used to sense extracellular formate accumulation in the host cell cytoplasm during pathogenesis. The first aim of this proposal is to use the differential radial capillary action of ligand assay (DRaCALA) to identify S. flexneri formate binding proteins, and then characterize their role in promoting S. flexneri plaque formation. The second aim of this proposal is to perform a genetic screen of a S. flexneri transposon mutant library to identify genes associated with the S. flexneri response to formate. Identifying this important virulence regulatory system will enable future studies to characterize the dynamics of formate signaling in infected host cells, and potentially provide novel targets for antimicrobial therapeutics and vaccine development.