CHAPERONE ASSISTED PILI ASSEMBLY IN PATHOGENIC E COLI

伴侣协助致病性大肠杆菌中的菌毛组装

• 批准号: 8693164
• 负责人: SCOTT J. HULTGREN
• 金额: $ 38.09万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-03-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8693164
• 关键词: Adherence; Affect; Antibiotics; Antibodies; Automobile Driving; Bacteria; Bacterial Adhesins; Binding; Biochemical; Biochemistry; Biogenesis; Bioinformatics; Biological Assay; Biophysics; C-terminal; Cell Membrane Permeability; Cellular biology; Collagen Receptors; Complex; Coupling; DNA Sequence Rearrangement; Drug resistance; Economic Burden; Equilibrium; Escherichia coli; Evolution; Fiber; Genetic; Genetic Techniques; Genome; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Habitats; Hemophilus; Image; Infection; Interdisciplinary Study; Klebsiella; Lectin; Ligands; Mannose; Measures; Mechanics; Mediating; Medical Device; Membrane; Microbial Biofilms; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Machines; Movement; Multi-Drug Resistance; N-terminal; Pathogenesis; Pathway interactions; Pilum; Plug-in; Positioning Attribute; Prevention; Prevention strategy; Process; Pseudomonas; Reaction; Recurrence; Resolution; Salmonella; Surface; Techniques; Testing; Therapeutic; Tissues; Urinary tract infection; Uropathogenic E. coli; Variant; Virulence; Virulence Factors; Woman; Work; Yersinia; antimicrobial; bacterial genetics; base; beta barrel; biophysical techniques; combat; conformer; design; drug development; extracellular; genetic analysis; in vivo; innovation; insight; macromolecular assembly; mouse model; novel; pathogen; pathogenic Escherichia coli; pathogenic bacteria; periplasm; polymerization; prophylactic; protein protein interaction; public health relevance; receptor; research study; small molecule; socioeconomics; structural biology; treatment strategy

DESCRIPTION (provided by applicant): Urinary tract infections (UTIs), caused mostly by uropathogenic E. coli (UPEC) affect millions of women each year, with frequent recurrences (rUTI) that do not resolve even with repeated use of antibiotics, causing a significant socio-economic burden. Frequent and long-term prophylactic use of antimicrobials for treatment and prevention of UTIs and other infections has contributed to the evolution of multi-drug-resistance among Gram-negative bacteria. This has become a looming worldwide crisis that has created an urgent need to develop novel treatment and prevention strategies for these infections, including the targeting of bacterial virulence mechanisms. Extracellular fibers termed chaperone-usher pathway (CUP) pili are critical virulence factors in gram negative pathogenic bacteria, with many genomes encoding 10 or more types, all containing dedicated adhesins that promote the recognition of specific receptors and/or drive biofilm formation. FimH, the CUP adhesin joined to the tip of type 1 pili, mediates host-pathogen interactions and bacteria-bacteria interactions critical in biofilm formation and UTI. Chaperone-assisted folding of pilus subunits occurs by a reaction called donor strand complementation (DSC), which templates the folding of subunits into a primed high-energy state. These primed complexes are targeted to the outer membrane usher, a gated channel, which is a molecular machine that catalyzes pilus assembly by driving subunit polymerization in a reaction termed donor strand exchange (DSE). This proposal will pioneer new advances to gain insights into the allostery that governs the interactions needed for CUP pili biogenesis and function. This new direction will provide opportunities for the design of alternative therapeutics to combat the rising problem of multi-drug resistance and Gram-negative bacterial infections in general. This work will elucidate: i) how allosteric modulation of an equilibrium between different conformations of FimH impact host-pathogen interactions (Aim 1); ii) how dynamic allosteric mechanisms of the chaperone and usher facilitate sequential processing of chaperone-subunit complexes and their movement through a molecular machine to promote subunit-subunit polymerization (Aim 2); and; iii) the mechanism of usher activation: how the binding of the initiating chaperone-adhesin complex to the usher results in the reordering of the usher domains to transform chaperone-subunit complexes into a highly stable polymerized fiber extruding across the OM to the bacterial surface (Aim 3). This innovative interdisciplinary research, which is critical for understanding a key step in the virulence of many Gram-negative bacterial pathogens, will use a blend of techniques including bacterial genetics, biochemistry, biophysics, structural biology, cell biology mouse models and high-resolution imaging. Elucidating how a molecular machine converts subunit binding and folding energy into work to assemble highly stable macromolecular CUP fibers and their extrusion to the bacterial surface and the conformational changes governing critical CUP adhesin function will spawn new avenues for drug development.

描述（由申请人提供）：尿路感染（UTI），主要由尿路致病性大肠杆菌引起。大肠杆菌（UPEC）每年影响数百万妇女，经常复发（鲁蒂），即使反复使用抗生素也无法解决，造成重大的社会经济负担。经常和长期预防性使用抗菌药物治疗和预防尿路感染和其他感染，导致革兰氏阴性菌的多重耐药性的演变。这已成为一个迫在眉睫的全球性危机，迫切需要为这些感染开发新的治疗和预防策略，包括靶向细菌毒力机制。被称为伴侣引导途径（chaperone-usher pathway，CUP）皮利的细胞外纤维是革兰氏阴性病原菌中的关键毒力因子，具有编码10种或更多类型的许多基因组，其全部含有促进特异性受体的识别和/或驱动生物膜形成的专用粘附素。FimH是连接到1型皮利尖端的CUP粘附素，介导宿主-病原体相互作用和细菌-细菌相互作用。 在生物膜形成和UTI中至关重要的相互作用。分子伴侣辅助的菌毛亚基折叠是通过一种称为供体链互补（DSC）的反应发生的，这种反应将亚基折叠成一种引发的高能状态。这些引发的复合物靶向外膜引导器，门控通道，这是一种分子机器，通过在称为供体链交换（DSE）的反应中驱动亚基聚合来催化菌毛组装。该提案将开拓新的进展，以深入了解控制CUP皮利生物发生和功能所需的相互作用的变构。这一新方向将为设计替代疗法提供机会，以应对日益严重的多药耐药问题。 耐药性和革兰氏阴性细菌感染。这项工作将阐明：i）FimH的不同构象之间的平衡的变构调节如何影响宿主-病原体相互作用（Aim 1）; ii）分子伴侣和usher的动态变构机制如何促进分子伴侣-亚基复合物的顺序加工及其通过分子机器的运动以促进亚基-亚基聚合（Aim 2）;以及; iii）usher激活的机制：起始分子伴侣-粘附素复合物与引导器的结合如何导致引导器结构域的重新排序，以将分子伴侣-亚基复合物转化为高度稳定的聚合纤维，其穿过OM挤出至细菌表面（Aim 3）。这项创新的跨学科研究对于理解许多革兰氏阴性细菌病原体毒力的关键步骤至关重要，将使用包括细菌遗传学，生物化学，生物物理学，结构生物学，细胞生物学小鼠模型和高分辨率成像在内的技术。阐明分子机器如何将亚基结合和折叠能量转化为工作，以组装高度稳定的大分子CUP纤维，并将其挤出到细菌表面，以及控制关键CUP粘附素功能的构象变化，将为药物开发开辟新的途径。