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

项目总结 艰难梭菌是一种主要的公共卫生威胁，可引起从轻度腹泻到潜在的各种疾病 致命性伪膜性结肠炎。艰难梭菌的症状在很大程度上是由分泌的细胞毒素介导的， 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 艰难梭菌适应宿主肠道环境。细菌细胞表面的成分起着至关重要的作用 在生理和毒力中的作用，通常是免疫原性抗原和潜在的抗生素靶标。在……里面 艰难梭菌信号分子环二鸟苷(c-di-GMP)控制鞭毛的产生，IV型菌毛， 和多个额外的细胞表面蛋白，表明c-di-GMP在艰难梭菌重组中起关键作用 细胞表面对宿主肠道环境的反应。相变是一种方式，许多细菌通过这种方式 物种将表型异质性引入种群作为一种策略，以确保 面对不断变化的选择压力的人口。我们最近发现鞭毛和毒素的阶段不同 在艰难梭菌中，和测序分析确定了其他几个假定的阶段可变基因座，包括两个 编码c-di-GMP水解酶。我们的中心假设是艰难梭菌结合了c-di-GMP信号和 相位变化协调细胞表面的全局变化，使其能够适应细胞外压力 在肠道定植期间遇到的。这项建议的目标是界定 控制c-di-GMP信号的时相变化机制，对c-di-GMP信号变化的表型反应 GMP，以及这些调控机制对艰难梭菌生理和毒力的影响。要完成 为了实现这一目标，我们利用分子遗传学、生化技术和艰难梭菌病的动物模型 在群体和单细胞水平上检测细胞周期变化和c-di-GMP信号。我们的发现是C. 艰难菌与c-di-GMP信号通路的时相变化揭示了一种以前未知的协调机制 细菌细胞表面的修饰。这些目标的完成可能会暴露出削弱C. 宿主的艰难适应，促进了抗击这种日益成问题的病原体的努力。

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.