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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：
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
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/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

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