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

 描述（由申请方提供）：艰难梭菌是一种芽孢形成厌氧革兰氏阳性杆菌，是一系列胃肠道疾病的原因，包括轻度腹泻、假膜性结肠炎和毒性巨结肠。令人担忧的是，C.在过去的二十年里，艰难性结肠炎的发病率都显著增加。C.艰难梭菌的毒性特征很好，但没有针对C.艰难梭菌感染的存在和我们对C.在定殖过程中难以与宿主接触。我们已经证明，蛋白亚基的IV型皮利的C。艰难梭菌与细胞粘附有关，并通过与细菌结合而启动生物膜形成。 小菌落IV型皮利（T4 P）是由许多种致病性革兰氏阴性细菌产生的毛发样表面附属物，其在多种过程中发挥作用，例如细胞粘附、定殖、抽搐运动、生物膜形成、水平基因转移，并且在许多情况下对于毒力是必需的。T4 Ps完全或主要由菌毛蛋白的许多拷贝组成，紧密地包装在螺旋中，使得菌毛蛋白的高度疏水的氨基末端被掩埋在菌毛核心中。虽然革兰氏阴性菌的特征更好，但几种革兰氏阳性菌，包括C。difficile和C.产气荚膜梭菌，现在已经显示产生T4 P，并且已经在梭菌属的所有成员的基因组中发现了T4 P基因。本项目旨在研究梭菌IV型皮利的结构、超分子组装及其在生物膜形成中的作用。很难 目前的研究：我已经建立了六个假定的菌毛蛋白基因的结构研究，在C。艰难梭菌基因组，并解决了两个结构，主要的菌毛蛋白亚基PilA 1和次要的菌毛蛋白，PilJ，通过X射线晶体学。由于这两种蛋白质已被证明是纳入菌毛纤维，我已经使用这两种结构，结合其他来源的数据，以模拟C的结构。艰难IV型菌毛。该模型 由合作者Glen Armstrong进行的体内菌毛组装的诱变研究支持。此外，我还培养了另一种菌毛蛋白PilA 2的同位素标记样品，并使用NMR光谱法收集了数据，使我能够分配共振并计算必要的距离约束，以通过NMR确定结构。对C.艰难梭菌R202091和菌毛蛋白基因中具有基因中断的菌株清楚地表明IV型皮利通过增加细菌自结合促进体外生物膜形成。测量可溶性菌毛蛋白与真核细胞的直接结合的测定显示，次要菌毛蛋白PilJ与HeLa、Caco-2和胃上皮细胞直接缔合。主要的菌毛蛋白对Caco-2细胞具有弱的亲和力，而其他菌毛蛋白，包括PilW，不显示结合。这些研究旨在探讨C.艰难梭菌IV型皮利介导体内小菌落与宿主细胞的附着。 独立阶段：我作为一名独立调查员的努力将指向阐明C的作用。在菌毛组装和生物膜形成中的艰难小菌毛蛋白。由于主要菌毛蛋白PilA 1的极端变异性，当与次要菌毛蛋白相比时，我假设， 由IV型皮利介导的任何特异性蛋白质-蛋白质相互作用涉及次要菌毛蛋白。我建议测试C的能力。艰难梭菌突变体缺乏次要菌毛蛋白以产生IV型皮利并在体外形成生物膜。我的期望是，这些次要菌毛蛋白中的一些将产生较少或不产生IV型皮利，因为它们是菌毛起始所需的，但任何被菌毛化至野生型水平但以降低的水平形成生物膜的菌毛蛋白可能涉及介导细菌自缔合的特异性相互作用。这些机制的调查将受益于继续调查这些蛋白质的三维结构，特别是在提高我们的理解，如何小菌毛蛋白被纳入菌毛纤维。

 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.