Coordinated stationary phase control of Campylobacter motility and biofilm

描述(申请人提供)：空肠弯曲杆菌是美国胃肠炎的主要细菌来源，每年在美国至少有200万例空肠弯曲菌胃肠炎(发病率等于或超过沙门氏菌和志贺氏菌的总和)。空肠弯曲菌可引起零星疾病以及由受污染的食物和接触娱乐水域引起的食源性/水源性暴发。一些空肠弯曲菌感染导致格林-巴利综合征的发展，这是世界上导致急性瘫痪的主要原因。尽管空肠弯曲菌病的发病率很高，经过20多年的研究，但空肠弯曲菌致病的机制仍不完全清楚，研究严重不足。在许多细菌中，CSRA调节许多重要的表型，包括毒力、碳代谢、运动性、群体感应、生物膜产生和动物定植。虽然到目前为止所研究的所有革兰氏阴性细菌中，CSRA的活性都是由可诱导的小调节RNA(SRNAs)控制的，但新的数据首次表明，空肠弯曲菌CSRA是由新的蛋白质-蛋白质与鞭毛生物合成途径的成员FliW相互作用调节的，从而将关键毒力因子鞭毛的合成与其他固定相细胞过程的调节联系在一起，如生物膜的形成。我们构建了一个缺乏CSRA的空肠弯曲菌突变株，并表明该突变株表现出与毒力相关的多效性表型，包括运动性降低、上皮细胞黏附、生物膜形成、对氧化应激的抵抗力和小鼠的定植。相反，CSRA突变体显示出对人类上皮细胞的侵袭增加。这强调了CSRA调节子在空肠弯曲菌致病过程中的重要性。我们已经确定了CSRA调节子，它包含一些与鞭毛运动/趋化以及与生物膜形成和醋酸酯代谢等静止相过程有明确联系的蛋白质。最后，我们证明了FliW与CSRA的直接相互作用，支持鞭毛和CSRA调控之间的联系。总体假设：空肠弯曲菌CSRA通过稳定期、转录后调控毒力/生存特性(包括运动和生物膜形成)在空肠弯曲菌的发病机制中发挥重要作用。我们建议对CSRA介导的空肠弯曲菌转录后稳定期基因调控进行详细的研究，重点研究CSRA控制运动表达和生物膜形成的机制。我们将使用遗传学、蛋白质组学和生化方法来实现这两个特定目标中概述的目标：目的1)确定CSRA、FliW和FlaA在协调运动和趋化作用的静止阶段调节中的作用，以及目的2)确定CSRA在调节生物膜生产和醋酸酯代谢中的作用。

DESCRIPTION (provided by applicant): Campylobacter jejuni is a primary bacterial cause of gastroenteritis in the United States, with at least 2 million cases of C. jejuni gastroenteritis eah year in the U.S. (an incidence equal or greater to that of Salmonella and Shigella combined). C. jejuni is responsible for sporadic disease as well as food-borne / water- borne outbreaks, which result from contaminated food and exposure to recreational waters. Some C. jejuni infections lead to the development of Guillain-Barré Syndrome, the leading cause of acute paralysis in the world. Despite the high prevalence of Campylobacter disease and more than 20 years of study, the mechanisms by which C. jejuni causes disease remain incompletely understood and severely understudied. In many bacteria, CsrA regulates numerous important phenotypes, including virulence, carbon metabolism, motility, quorum sensing, biofilm production, and animal colonization. While in all Gram-negative bacteria studied to date CsrA activity is controlled by inducible small regulatory RNAs (sRNAs), new data suggests for the first time that C. jejuni CsrA is regulated by novel protein-protein interactions with FliW, a member of the flagellar biosynthesis pathway, thus linking the synthesis of the critical virulence factors flagella to the regulation of other stationary phase cell processes such as biofilm formation. We constructed a C. jejuni mutant lacking csrA, and have shown that the csrA mutant exhibits pleiotropic virulence-related phenotypes including decreased motility, epithelial cell adherence, biofilm formation, resistance to oxidative stress, and colonization of mice. Conversely, the csrA mutant shows increased invasion of human epithelial cells. This underscores the importance of the CsrA regulon in C. jejuni pathogenesis. We have determined the presumptive CsrA regulon, and it contains a number of proteins with clear links to flagellar motility / chemotaxis and to stationary phase processes such as biofilm formation and acetate metabolism. Finally, we have demonstrated direct interaction of FliW with CsrA, supporting the link between flagella and CsrA regulation. Overall hypothesis: C. jejuni CsrA plays an important role in the pathogenesis of C. jejuni via stationary phase, post-transcriptional regulation of virulence / survival properties including motility and biofilm formation. We propose a detailed study of CsrA-mediated post-transcriptional stationary phase gene regulation in C. jejuni, focusing on the mechanism by which CsrA controls the expression of motility and biofilm formation. We will use genetic, proteomic and biochemical approaches to achieve the goals outlined in these two specific aims: Aim 1) Define the roles of CsrA, FliW, and FlaA in coordinating stationary phase regulation of motility and chemotaxis, and Aim 2) Define the role of CsrA in regulating biofilm production and acetate metabolism.