Chaperone-Assisted Pili Assembly in Pathogenic E. Coli

项目总结/摘要： 细菌病原体中抗生素耐药性的上升突出了了解分子生物学的迫切需要。 细菌引起感染的机制，以开发有效的基于精确度的预防性治疗方法。 治疗革兰氏阴性细菌病原体编码超过100种细胞外纤维，称为伴侣蛋白/引导蛋白 CUP途径（CUP）的皮利能够识别和定殖不同的宿主组织和栖息地，这是感染的先决条件。 每个CUP菌毛编码为基因簇的一部分，该基因簇含有指定的周质伴侣和外部分子。 膜（OM）引导蛋白，促进成千上万的结构亚基组装成每个最终的 菌毛结构此外，大多数CUP皮利被双结构域粘附素所修饰，所述粘附素包括：i）N-末端结构域 其识别具有立体化学特异性的受体;和ii）C-末端菌毛蛋白结构域。伴侣- 亚基/粘附素复合物通过供体链互补（DSC）相互作用形成，其中 分子伴侣提供空间信息以促进菌毛蛋白结构域的折叠，然后被递送到 OM引导器，通过供体链交换（DSE）催化亚基-亚基相互作用。DSE发生时， 存在于每个亚基上的氨基末端延伸（Nte）完成其相邻亚基的IG折叠。菌毛生物发生 由OM引导器催化的是一个非常复杂的过程，涉及引导器的多个域 其作为纳米机器来组装以粘附素为尖端的皮利。在这笔赠款的支持下， 在阐明菌毛生物发生机制方面已经取得了相当大的进展，然而， 该提案旨在填补关键的知识空白：了解引座员的分子工作原理。为此我们 将阐明代表组装中临界点的usher中间体的三维结构 级联：i）菌毛起始;和ii）使用X射线晶体学和单颗粒细胞学的亚基掺入/（DSE） 冷冻电子显微镜（Aim 1）。我们将阐明两个结构域的粘附素和 特化的单域菌毛蛋白激活三种不同菌毛系统中的引导者（Aim 2）。了解 CUP皮利的分子生物学已经导致了一种基于FimH的疫苗，该疫苗被开发用于预防复发性 肾盂肾炎大肠大肠杆菌（UPEC）尿路感染（UTI）。这是基于这样的理解而开发的， 顶端为FimH的1型皮利介导膀胱定植。该疫苗已完成1A/1B期研究， 基于有希望的结果，获得了FDA的同情使用津贴。此外，合理设计 FimH功能的抑制剂，称为甘露糖苷，已经显示在治疗和 在小鼠模型中预防UTI，同时能够选择性地从小鼠中消除UPEC 胃肠道贮器在这里，将开发阻断引导功能的小分子（目标3）， 将潜在地阻断多个CUP皮利的组装并与其它治疗剂协同作用。因此，在本发明中， 将获得结构和功能方面的见解，并将其整合，以开发保留肿瘤的治疗方法， 通过阻断usher和粘附素功能来防止UPEC定植。

PROJECT SUMMARY/ABSTRACT: Rising antibiotic resistance in bacterial pathogens highlights the urgent need to understand the molecular mechanisms by which bacteria cause infections, in order to develop effective precision-based antibiotic-sparing therapies. Gram-negative bacterial pathogens encode over 100 extracellular fibers termed chaperone/usher pathway (CUP) pili able to recognize and colonize different host tissues and habitats, a prerequisite to infection. Each CUP pilus is encoded as part of a gene cluster containing a designated periplasmic chaperone and outer membrane (OM) usher protein that facilitates assembly of tens of hundreds of structural subunits into each final pilus structure. In addition, most CUP pili are tipped by two-domain adhesins comprising: i) an N-terminal domain that recognizes a receptor with stereochemical specificity; and ii) a C-terminal pilin domain. Chaperone- subunit/adhesin complexes are formed through a donor strand complementation (DSC) interaction in which the chaperone donates steric information to promote the folding of the pilin domains and are then delivered to the OM usher which catalyzes subunit-subunit interactions via donor strand exchange (DSE). DSE occurs when an amino-terminal extension (Nte) present on each subunit completes the Ig fold of its neighbor. Pilus biogenesis catalyzed by the OM usher is a remarkably complex process involving the multiple domains of the usher functioning as a nanomachine to assemble pili tipped with an adhesin. With the support from this grant, considerable progress has been made towards elucidating the mechanism of pilus biogenesis, however, this proposal seeks to fill key knowledge gaps: understanding the molecular workings of the usher. To do this, we will elucidate three-dimensional structures of usher intermediates representing critical points in the assembly cascade: i) pilus initiation; and ii) subunit incorporation/(DSE) using X-ray crystallography and single-particle cryo-electron microscopy (Aim 1). We will elucidate the mechanisms by which both two-domain adhesins and specialized single-domain pilins activate ushers in three distinct pilus systems (Aim 2). Understanding the molecular biology of CUP pili has already led to a FimH-based vaccine that was developed to prevent recurrent uropathogenic E. coli (UPEC) urinary tract infections (UTI). This was developed based on understanding that type 1 pili tipped with FimH mediate bladder colonization. The vaccine has completed a Phase 1A/1B study and received FDA allowance for compassionate use based on promising results. In addition, rationally designed inhibitors of FimH function, termed mannosides, have been shown to be highly efficacious in treating and preventing UTI in mouse models, while simultaneously being able to selectively deplete UPEC from the mouse gastrointestinal tract reservoir. Here, small molecules that block usher function will be developed (Aim 3), which would potentially block assembly of multiple CUP pili and work synergistically with other therapeutics. Thus, structural and functional insights will be gained and integrated to develop antibiotic-sparing therapeutics that prevent UPEC colonization by blocking usher and adhesin function.