Global regulation in Clostridium difficile via phase variation of cyclic diguanylate signaling

项目摘要 艰难梭菌是一个主要的公共卫生威胁，引起的疾病从轻度腹泻到潜在的 致死性假膜性结肠炎C.艰难疾病症状主要由分泌的细胞毒素介导， TcdA和TcdB。这种细菌致病性的许多方面仍然知之甚少，包括如何 C.艰难梭菌适应宿主肠道环境。细菌细胞表面的组成部分起着关键作用 在生理学和毒力中的作用，并且通常是免疫原性抗原和潜在的抗生素靶标。在 C.艰难梭菌的信号分子环二鸟苷酸（c-di-GMP）控制鞭毛，IV型皮利， 和多种额外的细胞表面蛋白，表明c-di-GMP在重组C.艰难 细胞表面对宿主肠道环境的反应。相位变化是许多细菌 物种将表型异质性引入种群，作为确保物种生存的策略。 面对不断变化的选择压力。我们最近发现鞭毛和毒素阶段不同， in C.和测序分析确定了几个其他推定的相位可变基因座，包括两个， 编码c-di-GMP水解酶。我们的中心假设是C。difficile结合了c-di-GMP信号传导和 相位变化，以协调细胞表面的整体变化，从而能够适应细胞外压力 在肠道定植过程中遇到的。本提案的目的是界定 控制c-di-GMP信号传导的时相变化机制，对c-di-GMP变化的表型反应， GMP，以及这些调节机制对C。生理学和毒力都很困难。完成 为此，我们采用分子遗传学、生物化学技术和C.艰难病 在群体和单细胞水平上检查相位变化和c-di-GMP信号传导。我们发现C. difficile将相位变化与c-di-GMP信号传导联系起来，揭示了一种以前未知的协调机制， 修饰细菌细胞表面。这些目标的完成可能会为减毒C暴露新的目标。 宿主的适应性很差，有助于对抗这种日益严重的病原体。

PROJECT SUMMARY Clostridium difficile is a major public health threat, causing disease ranging from mild diarrhea to potentially fatal pseudomembranous colitis. C. difficile disease symptoms are largely mediated by the secreted cytotoxins, TcdA and TcdB. Many aspects of the pathogenicity of this bacterium remains poorly understood, including how C. difficile adapts to the host intestinal environment. The components of the bacterial cell surface play critical roles in physiology and virulence and are commonly immunogenic antigens and potential antibiotic targets. In C. difficile the signaling molecule cyclic diguanylate (c-di-GMP) controls the production of flagella, type IV pili, and multiple additional cell surface proteins, indicating a key role for c-di-GMP in reorganizing the C. difficile cell surface in response to the host intestinal environment. Phase variation is a means by which many bacterial species introduce phenotypic heterogeneity into the population as a strategy to ensure survival of the population in the face of changing selective pressures. We recently showed that flagella and toxins phase vary in C. difficile, and sequencing analyses identified several other putative phase variable loci including two that encode c-di-GMP hydrolases. Our central hypothesis is that C. difficile combines c-di-GMP signaling and phase variation to coordinate global changes to the cell surface, enabling adaptation to extracellular pressures encountered during colonization of the intestinal tract. The objective of this proposal is to define the mechanisms of phase variation that control c-di-GMP signaling, the phenotypic responses to changes in c-di- GMP, and the impact of these regulatory mechanisms on C. difficile physiology and virulence. To accomplish this goal, we employ molecular genetics, biochemical techniques, and animal models of C. difficile disease to examine phase variation and c-di-GMP signaling at the population and single cell levels. Our discovery that C. difficile links phase variation with c-di-GMP signaling reveals a previously unknown mechanism for coordinated modification of the bacterial cell surface. Completion of these aims may expose new targets for attenuating C. difficile fitness in the host, facilitating efforts to combat this increasingly problematic pathogen.