Structural Basis of Type IV Pilus-Induced Clostridium difficile Microcolony Formation

IV型菌毛诱导的艰难梭菌微菌落形成的结构基础

基本信息

批准号：
9088787
负责人：
Kurt Henry Piepenbrink
金额：
$ 15.34万
依托单位：
UNIVERSITY OF NEBRASKA LINCOLN
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-06-01 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9088787
关键词：
Acute Adherence Affinity Alcohols Anaerobic Bacteria Antitoxins Architecture Bacillus (bacterium)Bacteria Binding Biogenesis Biological Assay Bundling Caco-2 Cells Cell Adhesion Cells Clostridium Clostridium difficile Clostridium perfringens Colitis Colon Cryoelectron Microscopy Crystallization Data Defect Deuterium Development Diarrhea Disease Epithelial Cells Eukaryotic Cell Evolution Fiber Fimbriae Proteins Gene Cluster Genes Genome Goals Gram-Negative Bacteria Gram-Positive Bacteria Hair Hela Cells Horizontal Gene Transfer Human Hydrogen Hydrophobicity In Vitro Incidence Individual Infection Interruption Investigation Isotope Labeling Knowledge Lead Learning Mass Spectrum Analysis Measures Mediating Microbial Biofilms Minor Modeling Mutagenesis NMR Spectroscopy Nosocomial Infections Pathogenicity Pathologic Phase Physiological Pilum Play Prevention approach Process Protein Subunits Proteins Pseudomembranous Colitis Refractory Relapse Reproduction spores Research Research Personnel Resistance Resolution Roentgen Rays Role Sampling Severities Source Stomach Structure Surface Symbiosis Technetium Tc 99m ciprofloxacin Testing Toxic Megacolon Toxic effect Vaccines Vibrio cholerae Virulence Wit X-Ray Crystallography alpha helix antimicrobial appendage base cell motility commensal microbes design effective therapy expectation fecal transplantation gastrointestinal improved in vivo insight member mortality mutant novel strategies pilates exercise protein protein interaction protein structure public health relevance reconstitution response three dimensional structure

项目摘要

DESCRIPTION (provided by applicant): Clostridium difficile, a spore-forming anaerobic Gram-positive bacillus, is the cause of a spectrum of gastrointestinal illness ranging from mild diarrhe though pseudomembranous colitis, and toxic megacolon. Alarmingly, the incidence, severity and mortality of C. difficile colitis have all increased significantly in the past twenty years. Th mechanism of C. difficile toxicity is well-characterized but no vaccine against C. difficile infecton exists and our knowledge about the interactions of C. difficile with its host during colonization i limited. We have shown that protein subunits of the Type IV pili of C. difficile are associated wit cellular adherence and the initiation of biofilm formation through the association of bacteria into microcolonies. Type IV Pili (T4P) are hair-like surface appendages produced by many species of pathogenic Gram negative bacteria which play a role in diverse processes such as cellular adhesion, colonization, twitching motility, biofilm formation, horizontal gene transfer and in numerous instances are essential for virulence. T4Ps are composed exclusively or primarily of many copies of pilin protein, tightly packed in a helix so that the highly hydrophobic amino-terminus of the pilin is buried in the pilus core. Although better characterized in Gram-negative bacteria, several Gram-positive bacteria, including C. difficile and C. perfringens, have now been shown to produce T4P and T4P genes have been discovered in the genomes of all members of the Clostridium genus. This project aims to characterize the structure, supramolecular assembly and role in biofilm formation of the Type IV pili of C. difficile. Current Research: I have established structural investigations into six putative pilin genes identified in the C. difficile genome and have solved the structure of two, the major pilin subunit PilA1 and a minor pilin, PilJ, by x-ray crystallography. As both of these proteins have been shown to be incorporated into the pilus fiber, I have used these two structures, in combination with data from other sources, to model the structure of a C. difficile Type IV pilus. This model is supported by mutagenesis studies of in vivo pilus assembly performed by a collaborator, Glen Armstrong. Additionally, I have grown isotopically-labeled samples of another pilin, PilA2, and have collected data using NMR spectroscopy allowing me to assign the resonances and calculate the necessary distance constraints to determine the structure by NMR. Initial studies of biofilm formation by C. difficile R202091 and strains with gene-interruptions in pilin genes show clearly that Type IV pili promote biofilm formation in vitro by increasing bacterial self-association. Assays measuring the direct binding of soluble pilin proteins to eukaryotic cells show that a minor pilin, PilJ, directly associates with HeLa, Caco-2 and gastric epithelial cells. The major pilin has a weak affinity for Caco-2 cells while other pilin proteins, including PilW, show no binding. These studies are designed to probe the hypothesis that C. difficile Type IV pili mediate the attachment of in vivo microcolonies to host cells. Independent Phase: My efforts as an independent investigator will be directed towards elucidating the role of C. difficile minor pilins in pilus assembly and biofilm formation. Due to te extreme variability of the major pilin PilA1, when compared to the minor pilins, I hypothesize that any specific protein-protein interactions mediated by Type IV pili involve minor pilins. I propose to test the ability of C. difficile mutants deficient in minor pilins to produce Type IV pili and t form biofilms in vitro. My expectation is that some of these minor pilins will produce fewer or no Type IV pili because they are required for pilus initiation but any that are pilated to wild-type levels but form biofilm at reduced levels are likely involved in specific interactions which mediat bacterial self-association. Investigations into these mechanisms will benefit from continuing investigations into the three-dimensional structures of these proteins, particularly in improving our understanding of how minor pilins are incorporated into the pilus fiber.

描述（由申请人提供）：艰难梭菌是一种产芽孢厌氧革兰氏阳性杆菌，是一系列胃肠道疾病的病因，从轻度腹泻到伪膜性结肠炎，再到中毒性巨结肠，令人震惊的是，艰难梭菌的发病率、严重程度和死亡率。过去二十年来，艰难梭菌结肠炎均显着增加。艰难梭菌毒性机制已得到充分表征。但不存在针对艰难梭菌感染的疫苗，并且我们对艰难梭菌在定植过程中与其宿主相互作用的了解有限。我们已经证明，艰难梭菌 IV 型菌毛的蛋白质亚基与细胞粘附和起始相关。通过细菌结合形成生物膜 IV 型菌毛 (T4P) 是由多种致病性革兰氏阴性菌产生的毛发状表面附属物，在细胞粘附、定植、抽搐运动、生物膜形成、水平基因转移等多种过程中发挥作用，并在许多情况下发挥作用。 T4Ps 完全或主要由许多菌毛蛋白拷贝组成，紧密地堆积在螺旋中，因此菌毛蛋白的疏水性氨基末端被埋在螺旋中。虽然在革兰氏阴性细菌中得到了更好的表征，但现在已证明包括艰难梭菌和产气荚膜梭菌在内的几种革兰氏阳性细菌可以产生 T4P，并且在梭菌属所有成员的基因组中都发现了 T4P 基因。该项目旨在表征艰难梭菌 IV 型菌毛的结构、超分子组装及其在生物膜形成中的作用。目前的研究：我对艰难梭菌基因组中确定的六个假定菌毛蛋白基因进行了结构研究，并通过 X 射线晶体学解析了两个主要菌毛蛋白亚基 PilA1 和次要菌毛蛋白 PilJ 的结构。蛋白质已被证明融入菌毛纤维中，我使用这两种结构，结合其他来源的数据，对艰难梭菌 IV 型菌毛的结构进行建模。得到了合作者 Glen Armstrong 进行的体内菌毛组装诱变研究的支持。此外，我还培养了另一种菌毛 PilA2 的同位素标记样品，并使用 NMR 光谱收集了数据，使我能够分配共振并计算必要的距离。对艰难梭菌 R202091 和菌毛基因中存在基因中断的菌株形成生物膜的初步研究清楚地表明，IV 型菌毛促进生物膜。测量可溶性菌毛蛋白与真核细胞的直接结合的体外形成表明，次要菌毛蛋白 PilJ 与 HeLa、Caco-2 和胃上皮细胞直接结合。主要菌毛蛋白具有弱亲和力。对于 Caco-2 细胞，而其他菌毛蛋白（包括 PilW）没有显示出结合。这些研究旨在探讨艰难梭菌 IV 型菌毛介导体内附着的假设。小集落到宿主细胞。独立阶段：作为一名独立研究者，我的努力将致力于阐明艰难梭菌次要菌毛蛋白在菌毛组装和生物膜形成中的作用，由于主要菌毛蛋白 PilA1 与次要菌毛蛋白相比具有极大的变异性，我意识到这一点。 IV 型菌毛介导的任何特定蛋白质-蛋白质相互作用都涉及次要菌毛蛋白，我建议测试缺乏次要菌毛蛋白的艰难梭菌突变体在体外产生 IV 型菌毛和形成生物膜的能力。菌毛蛋白将产生较少或不产生 IV 型菌毛，因为它们是菌毛启动所必需的，但任何菌毛达到野生型水平但以降低水平形成生物膜的菌毛都可能涉及介导细菌的特定相互作用对这些机制的研究将受益于对这些蛋白质的三维结构的持续研究，特别是在提高我们对小菌毛蛋白如何融入菌毛纤维的理解方面。