Molecular Mechanisms of Clostridium difficile Resistance to Innate Host Defenses

描述（由申请人提供）：艰难梭菌引起一种潜在致命的肠道疾病，其发病率和严重程度正在增加。这些感染通常是慢性的，难以根除。尽管这种微生物带来了巨大的公共卫生负担，但人们对艰难梭菌如何避开宿主的防御而在人体肠道中定植知之甚少。为了生存，细菌必须应对宿主防御系统的持续攻击。该项目的长期目标是揭示艰难梭菌如何逃避宿主先天免疫，以开辟新的治疗途径。本应用的具体目的是确定艰难梭菌抵抗宿主产生的阳离子抗菌肽（camp）的机制。这些肽在宿主先天防御中起关键作用，阻止细菌的生长和传播。迄今为止获得的初步数据表明，艰难梭菌可以对camp的存在做出反应和适应。我假设艰难梭菌已经进化出多种抗性机制，允许细菌绕过宿主免疫反应，例如camp，允许它在人体肠道中定植和复制。本课题将明确艰难梭菌CAMP耐药的遗传机制，确定CAMP耐药的调控机制，并确定这些机制在体内毒力和定殖中的作用。CAMP耐药性的研究将采用传统的遗传和生化方法，以及快速全基因组测序等新技术。成功完成这些目标将阐明艰难梭菌感染的基本方面，并确定治疗疾病的潜在治疗靶点。此外，这一知识将使我们更好地理解肠道中宿主-细菌的相互作用如何导致消化系统疾病。在这个proposal中概述的实验将帮助我学习到很多我以前没有使用过的分子遗传学，生物化学和动物研究技术，并掌握这种重要的医院病原体的使用方法。最终，这项工作将为我未来对艰难梭菌发病机制的研究奠定基础，并为我过渡到独立研究者的职位提供框架。

DESCRIPTION (provided by applicant): Clostridium difficile causes a potentially fatal intestinal disease that is increasing in incidence and severity. These infections are often chronic and incredibly difficult to eradicate. Though this organism presents an enormous public health burden, little is understood about how C. difficile evades host defenses to colonize the human intestine. In order to persist, the bacteria must cope with a continuous onslaught by host defenses. The long-term goal of this project is to reveal how C. difficile evades host innate immunity with the intent of opening new avenues for treatment. The specific objective of this application is to define the mechanisms C. difficile employs to resist cationic antimicrobial peptides (CAMPs) produced by the host. These peptides play a critical role in host innate defenses, preventing the growth and spread of bacteria. Preliminary data obtained thus far indicate that C. difficile can respond and adapt to the presence of CAMPs. I hypothesize that C. difficile has evolved multiple resistance mechanisms that allow the bacterium to bypass host immune responses, such as CAMPs, allowing it to colonize and replicate in the human intestine. This proposal will identify the genetic mechanisms of CAMP resistance in C. difficile, identify and characterize the regulation of CAMP resistance mechanisms, and determine the role of these mechanisms in the virulence and colonization in vivo. CAMP resistance will be studied using traditional genetic and biochemical methods, along with new technologies, such as rapid, whole-genome sequencing. Successful completion of these aims will illuminate fundamental aspects of C. difficile infection and identify potential therapeutic targets for treatment of disease. Furthermore, this knowledge will better our understanding of how host-bacterial interactions in the intestine can lead to digestive diseases. The experiments outlined in this proposal will help me learn a wide range of molecular genetics, biochemistry, and animal research techniques I have not previously used, and also to master methods for use with this important hospital pathogen. Ultimately, this work will set the stage for my future research on C. difficile pathogenesis and provide the framework for my transition into an independent investigator position.