Structure & Function of Clostridium difficile Type IV Pili

结构

• 批准号: 10087197
• 负责人: ERIC JOHN SUNDBERG
• 金额: $ 37.9万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-05 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10087197
• 关键词: Structure; Function; Clostridium; difficile; Type

 DESCRIPTION (provided by applicant): Clostridium difficile is a spore-forming anaerobic Gram-positive bacillus that causes gastrointestinal illnesses in humans ranging from mild diarrhea to pseudomembranous colitis, which in severe cases can lead to toxic megacolon, an acute and sometimes lethal form of colonic distension. The incidence, severity and mortality of C. difficile colitis have increased significantly over the last two decades. However, knowledge concerning interactions between C. difficile bacteria and the human host is virtually nonexistent, severely impeding the development of new approaches to the prevention, control, and treatment of C. difficile disease. Type IV pili are fimbrial surface appendages produced by many bacteria that play critical roles in cellular adhesion, colonization, twitching motility, biofilm formation,and horizontal gene transfer. They are often essential for virulence and some have been successfully developed as vaccines. Type IV pili have been characterized extensively in Gram-negative bacteria, but nearly nothing is known about these pili from Gram-positive bacteria. Type IV pilin genes, though, are present in the genomes of all members of the genus Clostridium and all C. difficile strains encode complete sets of T4P biogenesis machinery components and a variable number of Type IV pilin proteins. We hypothesize that C. difficile Type IV pili, through structural features distinct from those of Gram-negative Type IV pili, directy mediate human cell adherence that is important for both colonization and virulence. Accordingly, we seek to define C. difficile Type IV pilin structures, both as individual protein components and assembled supramolecular appendages, and to identify their human host cell receptors, by pursuing the following Specific Aims: (1) to determine the atomic structures of individual C. difficile Type IV pilin proteins; (2) to define the composition and architecture of C. difficile Tye IV pili; and (3) to identify host molecules engaged specifically by C. difficile Type IV pili. The comprehensive approach that we propose to investigate the structure and function of C. difficile Type IV pili will utilize protein structure determination methods throughout a broad range of resolutions, including X-ray crystallography, NMR spectroscopy, small-angle X-ray scattering and cryo-electron microscopy, as well as state-of-the-art mass spectrometry-based approaches to define the composition of multi-component protein assemblies, map protein-protein interfaces and identify novel receptor molecules. To assure the success of our proposed studies, we have assembled a team of outstanding investigators with extensive expertise in these experimental techniques, C. difficile biology and Type IV pilus structure and function.

 描述（由适用提供）：艰难梭状芽胞杆菌是一种散射的厌氧革兰氏阳性芽孢杆菌，可导致人类胃肠道疾病，从轻度腹泻到假膜性结肠炎，在严重的情况下，这可能导致有毒的巨型巨型巨型巨型巨型，有时会导致剧烈的巨型巨型，有时是巨大的，有时是杀死的。在过去的二十年中，艰难梭菌结肠炎的事件，严重程度和死亡率显着增加。但是，关于艰难梭菌细菌与人类宿主之间相互作用的知识实际上是不存在的，严重阻碍了艰难梭菌疾病的预防，控制和治疗的新方法的发展。 IV型Pili是许多细菌产生的纤维化表面附属物，在细胞粘附，定殖，抽搐运动，生物膜形成和水平基因转移中起着关键作用。它们通常对于病毒至关重要，有些是成功开发为疫苗的。但是，IV型PILIN基因在梭状芽胞杆菌的所有成员的基因组中呈现，所有艰难梭菌菌株编码T4P生物发生机械组件的完整集和IV型PILIN蛋白的可变数量。我们假设艰难梭菌IV型菌毛菌通过与革兰氏阴性型IV型菌毛的结构特征不同，直接介导对定殖和病毒都很重要的人类细胞附着。根据，我们试图通过追求以下特定目的来定义艰难梭菌型pilin结构，包括单个蛋白质成分和组装的超分子附属物，并识别其人类宿主细胞受体：（1）确定个体艰难型杆菌类型IV pili pili蛋白的原子结构； （2）定义艰难梭菌IV型pili的组成和结构； （3）确定特异性艰难梭菌IV型菌毛的宿主分子。我们建议研究艰难梭菌IV型PILI的结构和功能的全面方法将利用蛋白质结构的确定方法，包括各种分辨率，包括X射线晶体学，NMR光谱，NMR光谱，小角生角X射线散射和小冷冻电子显微镜，以及基于整体质谱的方法的构图群体的组合群体，蛋白质蛋白界面并鉴定新的受体分子。为了确保我们提出的研究的成功，我们组建了一个在这些实验技术，艰难梭菌生物学和IV型Pyrus结构和功能方面拥有广泛专业知识的杰出研究人员团队。