Coordinated stationary phase control of Campylobacter motility and biofilm

摘要 空肠弯曲杆菌是美国胃肠炎的主要细菌原因，至少有2 万例C.空肠胃肠炎（发病率等于或大于 沙门氏菌和志贺氏菌合并）。C.空肠是散发性疾病以及食源性/水源性疾病的原因， 由污染的食物和接触娱乐沃茨引起的传染性疾病暴发。一些C。空肠 感染导致格林-巴利综合征的发展，格林-巴利综合征是急性瘫痪的主要原因， 世界尽管弯曲杆菌病的高患病率和20多年的研究， C.空肠引起的疾病仍然不完全了解和严重不足的研究。 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殖。而在所有革兰氏阴性菌中 迄今为止的研究表明，CsrA活性是由诱导型小调控RNA（sRNA）控制的，新的数据表明， C.空肠CsrA是由新的蛋白质-蛋白质相互作用与FliW，一个成员的调控， 鞭毛生物合成途径，从而将关键毒力因子鞭毛的合成与 调节其他稳定期细胞过程，如生物膜形成。我们构建了一个C。空肠 缺乏csrA的突变体，并已显示csrA突变体表现出多效性毒力相关表型 包括运动性降低、上皮细胞粘附、生物膜形成、对氧化应激的抗性，以及 老鼠的殖民地。相反，csrA突变体显示出增加的人上皮细胞侵袭。这 强调了CsrA调节子在C.空肠发病机制吾等已厘定 假定的CsrA调节子，它包含许多与鞭毛运动/趋化性有明确联系的蛋白质 以及固定相过程如生物膜形成和乙酸盐代谢。我们终于有 证明了FliW与CsrA的直接相互作用，支持了鞭毛与CsrA调节之间的联系。 总体假设：C.空肠CsrA在C.空肠固定 阶段，转录后调节毒力/存活特性，包括运动性和生物膜形成。 我们提出了一个详细的研究环孢素A介导的转录后稳定期基因调控在C。 空肠，侧重于CsrA控制运动和生物膜形成的表达的机制。我们 将使用遗传学，蛋白质组学和生物化学方法来实现这两个具体目标， 目的：目的1）确定CsrA、FliW和FlaA在协调运动的稳定期调节中的作用， 2）确定CsrA在调节生物膜产生和乙酸盐代谢中的作用。

ABSTRACT Campylobacter jejuni is a primary bacterial cause of gastroenteritis in the United States, with at least 2 million cases of C. jejuni gastroenteritis each 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-Barr¿ 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.