Novel Type 1 Pilus Receptors in Pyelonephritis and Recurrent UTI

项目摘要/摘要 细菌与尿路上皮的黏附是确定尿路感染的关键步骤。在.期间 哺乳动物的膀胱感染(膀胱炎)，尿路致病性大肠杆菌(UPEC)被很好地描述为 使用带有粘附素FimH尖端的1型菌毛来结合覆盖在皮肤上的寡糖装饰的尿板 浅表膀胱上皮细胞的管腔表面。然而，关于寄主病原体的细节知之甚少。 肾脏中的相互作用使上尿路感染得以启动，包括肾盂肾炎和肾脓肿。 我们发现，以前被认为只在膀胱炎中必不可少的1型菌毛也介导了 C3H小鼠肾盂肾炎和肾脓肿的发生。此外，在UPEC的体外模型中 结合肾脏集合管上皮细胞，我们确定了FimH的一个候选肾上皮受体，即 甘露糖结合蛋白Desmoglein-2(Dsg2)。这种蛋白质在整个细胞中表达 肾单位，但在集合管上皮中最高，并含有典型的N-连接甘露糖多糖 以及钙粘蛋白家族特有的O-连锁甘露糖基化。在这个项目中，我们将检验中心假设 桥粒芯糖蛋白-2是FimH的一种上皮受体，介导UPEC肾盂肾炎的建立和 能在肠道和膀胱脱落中结合FimH。首先，我们将使用多种遗传和药物系统来 在最近发表的优化小鼠中询问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，将有新生的C3H小鼠携带肾脏 Dsg2上皮特异性缺失。接下来，我们将量化FimH凝集素结构域与 SPR纯化人DSG2胞外区，并共结晶相应的FimH和DSG2结构域 揭示DSG2-FimH相互作用的结构基础。这些实验中的控制措施将包括 FimHQ133K，携带取消甘露糖结合的突变；甘露糖苷，高亲和力小分子 FimH结合的抑制剂；纯化蛋白和肾组织切片的酶前处理以消除 N-或O-连接的糖链；以及针对关键的DSG2多肽产生的单抗 与FimH的交互。第三，桥粒芯糖蛋白-2在其他类型的上皮细胞上也广泛表达(在两种情况下 人类和小鼠)，增加了它在其他与UTI相关的利基中结合FimH的可能性 发病机制。这些包括膀胱脱落后(对最初的UPEC感染的快速反应， 消除主要的FimH受体)和结肠(作为UPEC的储存库，为复发提供种子 UTI)。因此，我们将使用小鼠和人类组织切片，体内肠道定植模型，以及 新的条件性Dsg2基因敲除小鼠用于研究Dsg2是否可作为FimH受体 这些纸巾。在这些研究的结论中，我们将为1型菌毛指定一种新的受体， 阐明了FimH与Desmoglein-2相互作用的结构基础，并确定了其功能作用 多个与尿路感染相关的利基中的药理学靶向相互作用。

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.