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: 10594971
• 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/10594971
• 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 Cells; Epithelium; 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; Modeling; Molecular; Molecular Chaperones; Monoclonal Antibodies; Mus; Mutation; Nephrons; Pathogenesis; Pathway interactions; Peptides; Pilum; Polysaccharides; Process; Proteins; Publishing; Pyelonephritis; Recurrence; Role; Site; Specific qualifier value; Structure; Surface; System; Testing; Therapeutic; Tissues; Tubular formation; Urinary tract; Urinary tract infection; Urine; Uropathogenic E. coli; Vaccination; Variant; Virulence; X-Ray Crystallography; bacterial community; 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; pharmacologic; protein purification; 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.

项目概要/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 哺乳动物膀胱感染（膀胱炎）、泌尿道致病性大肠杆菌（UPEC）已被充分描述 使用带有尖端粘附素 FimH 的 1 型菌毛来结合寡聚甘露糖修饰的尿斑蛋白，该尿斑蛋白包被 表层膀胱上皮细胞的管腔表面。然而，关于宿主病原体的细节知之甚少。 肾脏中的相互作用导致上尿路感染的发生，包括肾盂肾炎和肾脓肿。 我们发现，以前认为仅在膀胱炎中必不可少的 1 型菌毛也能介导 C3H 小鼠肾盂肾炎和肾脓肿的发生。此外，在 UPEC 的体外模型中 通过与肾集合管上皮结合，我们确定了 FimH 的候选肾上皮受体，即 甘露糖化细胞连接蛋白桥粒芯糖蛋白-2 (Dsg2)。该蛋白在整个组织中表达 肾单位，但在集合管上皮中含量最高，并具有典型的 N 连接含甘露糖聚糖 以及钙粘蛋白家族特异性 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，介导 UPEC 肾盂肾炎的建立 可以结合肠道和脱落膀胱中的FimH。首先，我们将利用多种遗传和药理学系统 在最近发表的优化小鼠中探讨 FimH 与甘露糖化 Dsg2 结合的重要性 UPEC肾盂肾炎模型。在这些系统中，新产生的 C3H 小鼠携带肾 Dsg2 的上皮特异性缺失。接下来，我们将量化 FimH 凝集素结构域与 通过SPR纯化人DSG2的胞外结构域，并共结晶相关的FimH和DSG2结构域 揭示 DSG2-FimH 相互作用的结构基础。这些实验中的控制将包括 FimHQ133K，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；对纯化蛋白和肾组织切片进行酶预处理以消除 N-或O-连接聚糖；以及针对关键 DSG2 肽介导的单克隆抗体 与 FimH 的互动。第三，桥粒芯糖蛋白-2 也在其他上皮细胞类型中广泛表达（在 人类和小鼠），增加了它在与 UTI 相关的其他生态位中结合 FimH 的可能性 发病。其中包括剥脱后的膀胱（对初始 UPEC 感染的快速反应， 消除初级 FimH 受体）和结肠（作为 UPEC 储存库来播种复发性 尿路感染）。因此，我们将使用小鼠和人体组织切片，即体内肠道定植模型，并且 另外一些新的条件性 Dsg2 敲除小鼠，以研究 Dsg2 是否可以作为 FimH 受体 这些组织。在这些研究结束时，我们将确定 1 型菌毛的新受体， 阐明了 FimH 与桥粒芯糖蛋白 2 相互作用的结构基础，并定义了其功能作用 多个尿路感染相关领域的药理学靶向相互作用。