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（由申请方提供）：艰难梭菌是一种形成芽孢的厌氧革兰氏阳性杆菌，可引起人类胃肠道疾病，从轻度腹泻到伪膜性结肠炎，严重时可导致毒性巨结肠，这是一种急性且有时致命的结肠扩张形式。本文报道了C.在过去的二十年中，艰难性结肠炎的发病率显著增加。然而，关于C.艰难梭菌与人类宿主之间的相互作用几乎不存在，这严重阻碍了预防、控制和治疗艰难梭菌的新方法的开发。艰难病IV型皮利是由许多细菌产生的菌毛表面附属物，其在细胞粘附、定殖、抽搐运动、生物膜形成和水平基因转移中起关键作用。它们通常对毒力至关重要，有些已成功开发为疫苗。IV型皮利在革兰氏阴性菌中已被广泛表征，但对来自革兰氏阳性菌的这些皮利几乎一无所知。然而，IV型菌毛蛋白基因存在于梭菌属和所有C.艰难梭菌菌株编码完整的T4 P生物发生机制组分和可变数量的IV型菌毛蛋白。我们假设C.艰难梭菌IV型皮利通过与革兰氏阴性IV型皮利不同的结构特征直接介导人细胞粘附，这对于定殖和毒力都是重要的。因此，我们试图定义C。艰难梭菌IV型菌毛蛋白结构，作为单独的蛋白质组分和组装的超分子附件，并鉴定它们的人宿主细胞受体，通过追求以下具体目的：（1）确定单个C.艰难梭菌IV型菌毛蛋白;（2）确定C.艰难梭菌Tye IV皮利;和（3）鉴定由C.艰难梭菌IV型皮利。我们提出的研究C.艰难梭菌IV型皮利将利用蛋白质结构测定方法，包括X射线晶体学、NMR光谱学、小角X射线散射和低温电子显微镜，以及最先进的基于质谱的方法，以确定多组分蛋白质组装体的组成，绘制蛋白质-蛋白质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，我们已经组建了一个优秀的研究人员团队，他们在这些实验技术方面具有广泛的专业知识，C。difficile生物学和IV型菌毛结构和功能。