Identifying the formate-sensory mechanism in Shigella flexneri

摘要 肠道细菌和细胞内的人类病原体志贺氏菌导致数亿例 志贺氏菌病（痢疾）每年在全世界范围内流行，死亡人数多达100万。目前， 没有批准的疫苗用于预防志贺氏菌病， 志贺氏菌属正在上升为了引起疾病，福氏志贺菌被摄入人类宿主， 然后通过消化道迁移并侵入结肠上皮细胞， 急性炎症;这种发病机制需要一个协调的计划，毒力基因表达的S。 flexneri以适应不同的主机环境和状态。即使在宿主上皮细胞内，S.福氏 毒力基因的表达是动态的，是S. flexneri用来调节这种表达的是它自己的 甲酸盐的生产。作为S.弗氏杆菌在宿主细胞内复制，它产生并分泌甲酸盐， 在混合酸发酵过程中，这种甲酸盐在宿主细胞中积累。S.然后，弗莱克斯纳里感觉到这种甲酸盐， 积累以积极调节与细胞间传播相关的毒力基因的表达， 抑制宿主免疫反应;然而，S.弗氏识别甲酸盐 在宿主细胞内的积累是未知的。本研究的目的是确定S。Flexneri系统用于 有义细胞外甲酸积累在宿主细胞质中的发病过程中。第一个目标是 建议采用配体示差径向毛细管作用试验（DRaCALA）鉴定S.福氏 甲酸结合蛋白，然后表征它们在促进S.弗氏菌斑形成。第二 该建议的目的是进行S.弗氏转座子突变体文库以鉴定基因 与S。对甲酸盐的弗氏反应。确定这一重要的毒力调节系统将 使未来的研究能够表征受感染宿主细胞中甲酸信号的动态，并可能 为抗微生物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.