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

• 批准号: 10180267
• 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
• 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/10180267
• 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/antagonist; intestinal epithelium; kidney infection; mouse model; novel; pathogen; receptor; renal abscess; renal epithelium; response; small molecule; small molecule inhibitor; sugar; type 1 fimbriae receptor

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.

项目总结/摘要 细菌粘附到尿路上皮是建立尿路感染的关键步骤。期间 哺乳动物膀胱的感染（膀胱炎）、尿路致病性大肠杆菌（UPEC）被充分描述， 使用1型皮利，轴承尖端粘附素FimH，结合寡甘露糖修饰的uroplakins，包被 浅表膀胱上皮细胞的腔表面。然而，关于宿主-病原体的详细情况知之甚少 肾脏中的相互作用，使上尿路感染，包括肾盂肾炎和肾脓肿的启动。 我们发现，以前认为仅在膀胱炎中必不可少的1型皮利也介导了膀胱炎的建立。 肾盂肾炎和C3 H小鼠肾水肿的起始。此外，在UPEC的体外模型中， 结合肾集合管上皮，我们确定了FimH的候选肾上皮受体，即 甘露糖基化细胞连接蛋白桥粒芯糖蛋白2（Dsg 2）。这种蛋白质在整个 肾单位，但在集合管上皮细胞中最高，并具有典型的N-连接的含甘露糖聚糖 以及钙粘蛋白家族特异性O-连接的甘露糖基化。在这个项目中，我们将测试中心假设 桥粒芯糖蛋白-2是FimH的上皮受体，其介导UPEC肾盂肾炎的建立， 可结合肠道和脱落膀胱中的FimH。首先，我们将使用多种遗传和药理学系统， 在最近发表的优化的小鼠中，询问FimH与甘露糖基化Dsg 2结合的重要性 UPEC肾盂肾炎模型。在这些系统中，新产生的C3 H小鼠携带肾 上皮特异性Dsg 2缺失。接下来，我们将定量FimH凝集素结构域与 通过SPR纯化人DSG 2的胞外结构域，并共结晶相关的FimH和DSG 2结构域 揭示DSG 2-FimH相互作用的结构基础。这些实验中的对照包括 FimHQ 133 K，其携带废除甘露糖结合的突变;甘露糖苷，高亲和力小分子 FimH结合抑制剂;纯化蛋白和肾组织切片的酶预处理，以消除 N-或O-连接的聚糖;以及针对介导DSG 2的关键肽产生的单克隆抗体。 与FimH的相互作用第三，桥粒芯糖蛋白-2也在其他上皮细胞类型上广泛表达（在两种上皮细胞中均是如此）。 人和小鼠），增加了它在与UTI相关的其他小生境中结合FimH的可能性 发病机制这些包括脱落后的膀胱（对初始UPEC感染的快速反应， 消除主要的FimH受体）和结肠（作为UPEC储存库， 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， 额外的新的条件性Dsg 2敲除小鼠，以研究Dsg 2是否可以作为FimH受体， 这些组织。在这些研究的结论中，我们将确定1型皮利的新受体， 阐明了FimH与桥粒芯糖蛋白-2相互作用的结构基础，并定义了这种相互作用的功能作用。 在多个UTI相关的小生境中进行可扩展的目标交互。