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

 描述(申请人提供)：艰难梭菌，一种形成芽胞的厌氧革兰氏阳性杆菌，是一系列胃肠道疾病的原因，从轻度腹泻到伪膜性结肠炎，以及中毒性巨结肠。令人震惊的是，艰难梭菌结肠炎的发病率、严重程度和死亡率在过去20年里都有显著的上升。艰难梭菌的毒性机制已有很好的描述，但目前还没有针对艰难梭菌感染的疫苗，而且我们对艰难梭菌在定植过程中与宿主的相互作用的了解也很有限。我们已经证明艰难梭菌IV型菌毛的蛋白亚基与细胞黏附和通过与细菌结合而启动生物膜的形成有关。 微集落。IV型菌毛(T4P)是由多种致病性革兰氏阴性菌产生的毛状表面附属物，在细胞黏附、定植、抽动运动、生物膜形成、水平基因转移等过程中发挥作用，在许多情况下是毒力所必需的。T4P完全或主要由许多拷贝的菌毛蛋白组成，紧密地堆积在一个螺旋中，因此菌毛蛋白高度疏水的氨基末端被埋在菌毛核心中。虽然革兰氏阴性菌具有更好的特性，但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现在已经被证明能产生T4P和T4P基因，在梭状芽孢杆菌属的所有成员的基因组中都发现了T4P基因。本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：我已经对艰难梭菌基因组中发现的六个可能的Pilin基因进行了结构研究，并通过X射线结晶学解决了其中两个的结构，主要的Pilin亚基PilA1和次要的PilJ。由于这两种蛋白质都被证明整合到菌毛纤维中，我使用这两种结构，结合来自其他来源的数据，对艰难梭菌IV型菌毛的结构进行了建模。这个型号是 由合作者格伦·阿姆斯特朗进行的体内毛发组装的突变研究支持。此外，我还培养了另一种Pilin PilA2的同位素标记样品，并使用核磁共振光谱收集了数据，使我能够指定共振并计算必要的距离约束，以通过核磁共振确定结构。艰难梭菌R202091和菌毛蛋白基因突变菌株生物膜形成的初步研究表明，IV型菌毛通过增加细菌的自结合而促进体外生物膜的形成。测定可溶性菌毛蛋白与真核细胞直接结合的分析表明，一种少量的菌毛蛋白PilJ直接与HeLa、Caco-2和胃上皮细胞结合。主要的菌毛蛋白对Caco-2细胞的亲和力较弱，而包括PilW在内的其他菌毛蛋白没有结合。这些研究旨在探索艰难梭菌IV型菌毛介导体内微集落与宿主细胞附着的假设。 独立阶段：作为一名独立研究员，我将致力于阐明艰难梭菌细小菌毛在菌毛组装和生物膜形成中的作用。由于主要的Pilin PilA1与次要的Pilin相比有极大的变异性，我假设 由IV型菌毛介导的任何特定的蛋白质-蛋白质相互作用都涉及到少量的菌毛蛋白。我建议在体外测试缺乏少量菌毛的艰难梭菌突变株产生IV型菌毛和形成生物膜的能力。我的预期是，这些微小的菌毛中的一些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，但任何被毛到野生型水平但在较低水平形成生物膜的菌毛都可能参与介导细菌自我结合的特定相互作用。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微小的菌毛蛋白是如何结合到菌毛纤维中的理解方面。

 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.