Novel Type 1 Pilus Receptors in Pyelonephritis and Recurrent UTI

肾盂肾炎和复发性尿路感染中的新型 1 型菌毛受体

基本信息

批准号：
10378625
负责人：
DAVID ALAN HUNSTAD
金额：
$ 39.38万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10378625
关键词：
Acute Adhesives Affinity Apoptosis Attention Bacteria Bacterial Adhesins Bacterial Adhesion Behavior Binding Biochemical Biological Bladder CRISPR screen Cadherin Domain Cadherins Cells Clinical Colon Communities Confocal Microscopy Cryoelectron Microscopy Crystallization Cystitis Ductal Epithelium Epithelial Epithelial Cells Escherichia coli Escherichia coli Adhesins Escherichia coli Infections Extracellular Domain Family Fiber Gastrointestinal tract structure Genetic Genome Human In Vitro Inbred C3H Mice Infection Infective cystitis Intercellular Junctions Investigation Kidney Knock-out Knockout Mice Lectin Link Mannose Mannosides Mediating Mediator of activation protein Modeling Molecular Molecular Chaperones Monoclonal Antibodies Mus Mutation Nephrons Pathogenesis Pathway interactions Peptides Pharmacology Pilum Polysaccharides Process Proteins Publishing Pyelonephritis Recurrence Role Seeds Site Specific qualifier value Structure Surface System Testing Therapeutic Tissues To specify Tubular formation Urinary tract Urinary tract infection Urine Uropathogenic E. coli Ursidae Family Vaccination Variant Virulence X-Ray Crystallography bacterial community base cell type desmoglein 2 experimental study extracellular gut colonization human tissue in vitro Model in vivo inhibitor intestinal epithelium kidney infection mouse model novel pathogen receptor renal abscess renal epithelium response small molecule small molecule inhibitor sugar type 1 fimbriae receptor

项目摘要

PROJECT SUMMARY / ABSTRACT Bacterial adhesion to the urinary tract epithelium is a critical step in establishing urinary tract infections. During infection of the mammalian bladder (cystitis), uropathogenic Escherichia coli (UPEC) are well described to employ type 1 pili, bearing the tip adhesin FimH, to bind oligomannose-decorated uroplakins that coat the luminal surfaces of superficial bladder epithelial cells. However, less detail is known about host-pathogen interactions in the kidney that enable initiation of upper-tract UTIs, including pyelonephritis and renal abscess. We have found that type 1 pili, previously thought to be essential only in cystitis, also mediate establishment of pyelonephritis and the initiation of renal abscesses in C3H mice. Furthermore, in an in vitro model of UPEC binding to renal collecting duct epithelium, we identified a candidate renal epithelial receptor for FimH, namely the mannosylated cell-junctional protein desmoglein-2 (Dsg2). This protein is expressed throughout the nephron but most highly in collecting duct epithelium, and bears typical N-linked mannose-containing glycans as well as cadherin family-specific O-linked mannosylation. In this project, we will test the central hypothesis that desmoglein-2 is an epithelial receptor for FimH that mediates establishment of UPEC pyelonephritis and can bind FimH in gut and exfoliated bladder. First, we will use multiple genetic and pharmacologic systems to interrogate the importance of FimH binding to mannosylated Dsg2 in recently published, optimized mouse models of UPEC pyelonephritis. Among these systems will be newly generated C3H mice carrying renal epithelial-specific deletion of Dsg2. Next, we will quantify the binding affinity of the FimH lectin domain to the purified extracellular domain of human DSG2 by SPR, and co-crystallize the relevant FimH and DSG2 domains to reveal the structural basis for the DSG2-FimH interaction. Controls in these experiments will include FimHQ133K, which carries a mutation that abrogates mannose binding; mannosides, high-affinity small-molecule inhibitors of FimH binding; enzymatic pre-treatment of purified protein and kidney tissue sections to eliminate N- or O-linked glycans; and monoclonal antibodies generated against the key DSG2 peptides mediating interaction with FimH. Third, desmoglein-2 is also expressed widely on other epithelial cell types (in both humans and mice), raising the added possibility that it binds FimH in other niches relevant to UTI pathogenesis. These include the bladder after exfoliation (a rapid response to initial UPEC infection that eliminates the primary FimH receptor) and the colon (which serves as a UPEC reservoir to seed recurrent UTI). Therefore, we will use mouse and human tissue sections, an in vivo gut colonization model, and additional new conditional Dsg2 knockout mice to investigate whether Dsg2 can serve as a FimH receptor in these tissues. At the conclusion of these studies, we will have specified a novel receptor for type 1 pili, elucidated the structural basis of FimH interaction with desmoglein-2, and defined the functional roles of this pharmacologically targetable interaction in multiple UTI-relevant niches.

项目摘要 /摘要细菌对尿路上皮的粘附是建立尿路感染的关键步骤。期间哺乳动物膀胱感染（膀胱炎），肝癌大肠杆菌（UPEC）被很好地描述为采用带有尖端粘附素FIMH的1型pili，结合少量的尿布状素，以涂层表面膀胱上皮细胞的腔表面。但是，关于宿主病原体知之甚少在肾脏中的相互作用，可以使上额外的UTI启动，包括肾盂肾炎和肾脓肿。我们发现，以前认为只有在膀胱炎中至关重要的1型pili，也可以调解建立的 C3H小鼠中肾盂肾炎和肾脓肿的启动。此外，在UPEC的体外模型中与肾脏收集管道上皮的结合，我们确定了FIMH的候选肾上皮受体甘露糖基化的细胞 - 功能蛋白Desmoglein-2（DSG2）。该蛋白在整个过程中表达肾单位，但在收集管道上皮方面最高，并且含有典型的含甘露糖的甘露糖的聚糖以及钙粘蛋白家族特异性的O连接甘露糖基化。在这个项目中，我们将检验中心假设 Desmoglein-2是FIMH的上皮受体，可介导建立UPEC pyelonephritis和可以在肠道和去角质膀胱中结合FIMH。首先，我们将使用多个遗传和药理系统进行询问FIMH结合在最近发表的，优化的小鼠中与甘露糖基化DSG2的重要性 UPEC肾盂肾炎的模型。在这些系统中，将是新生成的携带肾脏的C3H小鼠 DSG2的上皮特异性缺失。接下来，我们将量化FIMH凝集素结构域的结合亲和力通过SPR纯化人DSG2的细胞外域，并共结晶相关的FIMH和DSG2域揭示DSG2-FIMH相互作用的结构基础。这些实验中的控件将包括 FIMHQ133K，其突变消除了甘露糖的结合。甘露糖苷，高亲和力小分子 FIMH结合的抑制剂；纯化蛋白质和肾脏组织切片的酶促预处理以消除 N-或O连接的聚糖；和针对介导的键DSG2肽产生的单克隆抗体与FIMH的相互作用。第三，Desmoglein-2在其他上皮细胞类型上也广泛表达（在两者中人类和小鼠），增加了它在与uti相关的其他小境中结合的可能性发病。这些包括去角质后的膀胱（对初始UPEC感染的快速反应消除主要的FIMH受体）和结肠（作为种子复发的UPEC储层 UTI）。因此，我们将使用小鼠和人体组织切片，体内肠道定植模型，以及其他新的条件DSG2敲除小鼠，以研究DSG2是否可以用作FIMH受体这些组织。这些研究结束时，我们将指定一个新型的1型菌毛受体，阐明了FIMH与Desmoglein-2相互作用的结构基础，并定义了该功能的作用在多个与UTI相关的小甲基中，具有药理目标的相互作用。