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的致命性肠道疾病，其发病率和严重性正在增加。这些感染往往是慢性的，难以根除。虽然这种微生物给公共卫生带来了巨大的负担，但人们对艰难梭菌如何逃避宿主防御以定植人类肠道的了解很少。为了坚持下去，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是如何逃避宿主的先天性免疫的，目的是开辟新的治疗途径。本申请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。这些多肽在宿主的先天防御中起着关键作用，防止细菌的生长和传播。到目前为止获得的初步数据表明，艰难梭菌可以对营地的存在作出反应和适应。我推测艰难梭菌已经进化出多种耐药机制，使其能够绕过宿主的免疫反应，比如cAMP，从而使其能够在人体肠道内定植和复制。这一建议将确定艰难梭菌cAMP耐药的遗传机制，识别和表征cAMP耐药机制的调节，并确定这些机制在体内毒力和定植中的作用。将使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来研究cAMP耐药性。这些目标的成功完成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目标。此外，这些知识将更好地理解肠道中宿主和细菌的相互作用如何导致消化系统疾病。这项提案中概述的实验将帮助我学习广泛的分子遗传学、生物化学和动物研究技术，我以前没有使用过，也掌握了使用这种重要的医院病原体的方法。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，并为我过渡到独立的研究人员职位提供框架。

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.