Chaperone-Assisted Pili Assembly in Pathogenic E. Coli

项目摘要/摘要： 细菌病原体对抗生素耐药性的上升突显了了解这种分子的迫切需要 细菌引起感染的机制，以开发有效的基于精确的抗生素节省 治疗。革兰氏阴性细菌编码100多种胞外纤维，称为伴侣/引导者 途径(CUP)菌毛能够识别和定植不同的宿主组织和栖息地，这是感染的先决条件。 每个杯状菌毛被编码为一个基因簇的一部分，该基因簇包含一个指定的周质伴侣和外部 膜(OM)引入蛋白质，促进数以万计的结构亚基组装成每个最终的 Pilus结构。此外，大多数杯状菌毛尖端有两个结构域粘附素，包括：i)N-末端结构域 它识别具有立体化学特异性的受体；以及ii)C-末端的Pilin结构域。监护人- 亚基/粘附素复合体是通过给体链互补(DSC)相互作用形成的，在DSC中 伴侣捐赠立体信息以促进毛细管结构域的折叠，然后将其传递给 OM引导者，通过供体链交换(DSE)催化亚基-亚基相互作用。DSE在以下情况下发生 存在于每个亚基上的氨基末端延伸(NTE)完成了其相邻亚基的Ig折叠。菌毛生物发生 由OM引座员催化的是一个非常复杂的过程，涉及到引座员的多个域 起到纳米机器的作用，用来组装末端有粘附素的菌毛。在这笔赠款的支持下， 在阐明菌毛的生物发生机制方面已经取得了相当大的进展，然而，这 该提案寻求填补关键知识空白：理解引座员的分子工作原理。为了做到这一点，我们 将阐明代表组装关键点的引座者中间体的三维结构 级联：i)菌毛起始；以及ii)亚基掺入/(DSE)，使用X射线结晶学和单粒子 冷冻电子显微镜(目标1)。我们将阐明两个结构域的粘附素和 专门的单域菌毛激活了三个不同的菌毛系统(目标2)。了解 杯状菌毛的分子生物学已经导致了一种基于FimH的疫苗，该疫苗是为了防止复发而开发的 泌尿系致病性大肠杆菌(UPEC)尿路感染(UTI)。这是在理解以下内容的基础上开发的 FimH尖端的I型菌毛介导了膀胱定植。该疫苗已经完成了1A/1B期研究， 根据有希望的结果，获得了FDA的同情使用津贴。此外，设计合理， FimH功能的抑制剂，称为甘露糖苷，已被证明在治疗和 在小鼠模型中预防UTI，同时能够选择性地从小鼠体内耗尽UPEC 胃肠道储存库。在这里，将开发阻碍引座者功能的小分子(目标3)，其 可能会阻止多个杯状菌毛的组装，并与其他疗法协同工作。因此， 将获得并整合结构和功能方面的见解，以开发节省抗生素的疗法 通过阻断引座器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.