CHAPERONE ASSISTED PILI ASSEMBLY IN PATHOGENIC E COLI

描述(申请人提供)：尿路感染(UTI)，主要由致尿路大肠杆菌(UPEC)引起，每年影响数百万妇女，经常复发(RUTI)，即使反复使用抗生素也不能缓解，造成巨大的社会经济负担。经常和长期预防性使用抗菌药治疗和预防尿路感染和其他感染，导致了革兰氏阴性细菌的多重耐药性的演变。这已成为一场迫在眉睫的世界性危机，迫切需要为这些感染制定新的治疗和预防战略，包括针对细菌毒力机制。胞外纤维称为伴侣-引导者途径(CUP)菌毛是革兰氏阴性病原菌的关键毒力因子，许多基因组编码10种或更多类型，所有这些都含有促进特定受体识别和/或驱动生物膜形成的专用粘附素。FimH是连接在1型菌毛顶端的杯状粘附素，介导宿主与病原体的相互作用和细菌与细菌的相互作用 在生物膜形成和尿路感染中至关重要的相互作用。伴侣辅助的菌毛亚基折叠通过一种被称为供体链互补(DSC)的反应发生，该反应将亚基的折叠模板化成启动的高能状态。这些启动的复合体针对外膜引导器，这是一个门控通道，是一种通过驱动亚单位聚合来催化菌毛组装的分子机器，被称为供体链交换(DSE)。这项提议将开创新的进展，以获得对变构的洞察，该变构管理着杯毛的生物发生和功能所需的相互作用。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问题 耐药和革兰氏阴性细菌感染。这项工作将阐明：i)FimH不同构象之间平衡的变构调节如何影响宿主-病原体的相互作用(目标1)；ii)伴侣和引导者的动态变构机制如何促进伴侣-亚单位复合体的顺序处理及其通过分子机器的移动促进亚单位-亚单位聚合(目标2)；以及iii)引导者激活的机制：启动伴侣-粘附素复合体与引导者的结合如何导致引导者结构域的重新排序，将伴侣-亚单位复合体转化为高度稳定的聚合纤维，穿过OM挤出到细菌表面(目标3)。这项创新的跨学科研究对于了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多种技术。阐明分子机器如何将亚基结合和折叠能量转化为功，以组装高度稳定的大分子杯状纤维及其向细菌表面的挤出，以及调控关键杯状粘附素功能的构象变化，将为药物开发开辟新的途径。

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.