Structure and Function of Type IV Pili in Clostridium difficile

艰难梭菌IV型菌毛的结构和功能

• 批准号: 8647642
• 负责人: Kurt Henry Piepenbrink
• 金额: $ 5.33万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8647642
• 关键词: Adherence; Bacillus (bacterium); Bacteria; Bacteriology; Basic Science; Binding; Biogenesis; Biological Assay; Calorimetry; Cell Adhesion; Cells; Clinical; Clostridium; Clostridium difficile; Clostridium perfringens; Colitis; Collaborations; Colon; Crystallography; Cysteine; Data; Deuterium; Diarrhea; Electrons; Fiber; Fimbriae Proteins; Gene Cluster; Genes; Genome; Goals; Gram-Negative Bacteria; Gram-Positive Bacteria; Hair; Health; Horizontal Gene Transfer; Human; Hydrogen; Immune; Immune response; Immune system; Incidence; Infection; Investigation; Knowledge; Label; Learning; Maps; Mass Spectrum Analysis; Measures; Mediating; Microbial Biofilms; Modeling; Molecular; Molecular Conformation; NMR Spectroscopy; Organism; Peptides; Physiological; Pilum; Play; Prevention approach; Process; Protein Subunits; Proteins; Pseudomembranous Colitis; Publishing; Reagent; Relaxation; Reproduction spores; Resolution; Role; Ruminococcus; Sampling; Severities; Signal Transduction; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; Surface; Surface Plasmon Resonance; Technetium Tc 99m ciprofloxacin; Techniques; Tissues; Titrations; Toxic Megacolon; Toxic effect; Vaccines; Virulence; X-Ray Crystallography; antimicrobial; appendage; cell motility; crosslink; disulfide bond; gastrointestinal; insight; member; monomer; mortality; novel; novel strategies; novel vaccines; prevent; public health relevance; receptor

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 is limited. One possible target for the host immune system are the Type IV pili of C. difficile. 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. This project aims to characterize the structure and functional significance of the proteins that make up the Type IV pili of C. difficile. T4Ps are produced by many species of pathogenic Gram-negative bacteria and are known to play a role in both cellular adhesion and colonization. T4Ps are composed exclusively or primarily of many copies of a pilin protein, tightly packed in a helix. Although better characterized in Gram-negative bacteria, T4P genes have been discovered in the genomes of all members of the Clostridium genus, including C. difficile. In collaboration with Dr. Michael Donnenberg, we have established structural investigations into six putative pilin genes identified in the C. difficile genome and have solved the structure of one PILA4, by x-ray crystallography. Rather than consisting of one ?-sheet surrounded by ?-helices, as is the case with all other known pilins, PILA4 contains a second ?-sheet at a ~70 angle to the helical pilin core. This second sheet and the surrounding helices give it a much more extended conformation from the core and this radically different exposed surface may play a role in cellular adhesion and possibly signaling of innate immune cells. We have used this structure in combination with data from other sources to model the structure of a pilus composed of PilA4. We have also grown isotopically-labeled samples of another pilin, PilA2, and have collected data using NMR spectroscopy to being assigning the resonances to determine the structure using distance and relaxation rates from NMR. Functional studies of PILA4 are currently underway while structural investigations into other pilin proteins continue both by x-ray crystallography and NMR.

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，从轻微腹泻到伪膜性结肠炎，再到中毒性巨结肠。令人震惊的是，在过去二十年中，艰难梭菌结肠炎的发病率、严重程度和死亡率均显着增加。艰难梭菌毒性的机制已得到充分表征，但尚无针对艰难梭菌感染的疫苗，而且我们对艰难梭菌与其宿主相互作用的了解有限。宿主免疫系统的一个可能目标是艰难梭菌的 IV 型菌毛。 IV 型霹雳 (T4P) 是许多种致病性革兰氏阴性菌产生的毛发状表面附属物，它们在细胞粘附、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，并且在许多情况下对于毒力至关重要。 T4P 完全或主要由许多菌毛蛋白拷贝组成，紧密堆积在螺旋中，因此菌毛蛋白的高度疏水性氨基末端埋藏在菌毛中 核。 该项目旨在表征构成艰难梭菌 IV 型菌毛的蛋白质的结构和功能意义。 T4P 由多种致病性革兰氏阴性菌产生，已知在细胞粘附和定植中发挥作用。 T4P 完全或主要由紧密堆积在螺旋中的菌毛蛋白的许多拷贝组成。尽管在革兰氏阴性细菌中得到了更好的表征，但在梭菌属所有成员（包括艰难梭菌）的基因组中也发现了 T4P 基因。我们与 Michael Donnenberg 博士合作，对艰难梭菌基因组中鉴定的六个假定的菌毛蛋白基因进行了结构研究，并通过 X 射线晶体学解析了一个 PILA4 的结构。与所有其他已知菌毛蛋白的情况不同，PILA4 包含一个与螺旋菌毛蛋白核心成约 70 度角的第二个 β 折叠，而不是由一个被 β 螺旋包围的 β 折叠组成。第二片层和周围的螺旋赋予其从核心延伸得多的构象，这种完全不同的暴露表面可能在细胞粘附和可能的先天免疫细胞信号传导中发挥作用。我们将此结构与其他来源的数据结合使用，对由 PilA4 组成的菌毛结构进行建模。我们还培养了另一种菌毛素 PilA2 的同位素标记样品，并使用 NMR 光谱收集数据，以分配共振，从而使用 NMR 的距离和弛豫率确定结构。目前正在进行 PILA4 的功能研究，同时通过 X 射线晶体学和 NMR 继续对其他菌毛蛋白进行结构研究。