Project 1: Pathogenesis of Enteroaggregative E. coli using human colonoids

摘要 肠聚集性大肠杆菌(EAEC)是引起旅行者腹泻、工业化生产中腹泻的重要病原菌 发展中国家的国家和增长步履蹒跚。在这个应用中，我们建议研究EAEC 利用新建立的人类肠样/结肠样物质在人类肠粘膜中的致病作用 模型，这是一种体外自繁殖的人类肠道原代培养物。此应用程序包含一个 约翰霍普金斯大学(JHU)研究人员组成的联盟，他们开创了 肠样/结肠样病变模型，以及马里兰大学(UMD)和华盛顿大学的研究人员 他们在致泻性大肠杆菌引起的腹泻疾病的发病机制研究中处于领先地位 还有志贺氏菌。该项目将利用新的结肠单分子层模型的存在。我们将重点关注 EAEC与结肠系和类结肠系/白细胞模型的相互作用的特征，特别是剖析 在多种模型中已成为主要EAEC毒力因子的AAFS的作用和功能 对粘蛋白和白细胞糖蛋白表现出广谱活性的Spate，促进 免疫调节作用。该项目将包括两个具体目标。在目标1中，我们将描述 EAEC与肠粘膜的相互作用。我们将延长正在进行的研究，这些研究表明EAEC 与MUC糖蛋白的结合是其发病的基础。我们将扩展对其结构和功能的研究 AAF粘附素及其碱性残基在EAEC与人体相互作用中的作用 肠粘膜。我们将确定AAF粘蛋白结合残基对EAEC与EAEC相互作用的贡献 人类的粘膜。我们将描述Pic和DISPIN在AAF介导的相互作用中的作用 用时间推移显微镜方法对粘膜进行观察。在目标2中，我们将描述免疫调节的特征 2类唾液对肠粘膜的作用。我们将讨论肠道卵裂的潜在作用 糖蛋白在EAEC发病机制中的作用我们将通过结合和切割来剖析炎症反应 O-连接的糖蛋白，如肠粘膜上的MUC信号蛋白。我们还将 Pic通过裂解炎性细胞因子受体的免疫调节作用 结肠/巨噬细胞系统。本文所描述的工作将继续推进EAEC的总体研究 发病机制，产生基本的见解，将阐明与其他相关的发病机制的各个方面 肠道病原体。我们将与其他项目和核心项目的调查人员密切合作 组件。

Abstract Enteroaggregative E. coli (EAEC) is an important pathogen of traveler's diarrhea, diarrhea in industrialized countries and growth faltering in developing countries. In this application, we propose to study EAEC pathogenesis in the human intestinal mucosa through the use of newly established human enteroid/colonoid model, which is an ex vivo self–propagating human intestinal primary culture. This application comprises a consortium among investigators at Johns Hopkins University (JHU), who have pioneered the development of the enteroid/colonoid model, and researchers at the University of Maryland (UMD) and the University of Virginia, who are leaders in the study of the pathogenesis of diarrheal disease cause by diarrhegenic E. coli and Shigella. This project will leverage the existence of the new colonoid monolayer model. We will focus on characterization of EAEC interactions with colonoid and colonoids/leukocyte models, in particular to dissect the roles of AAFs, which have emerged as the principle EAEC virulence factor in multiple models, and the function of SPATEs, which have shown broad spectrum activities on mucin and leukocyte glycoproteins, promoting immunomodulatory effects. This project will comprise two Specific Aims. In Aim 1, we will characterize interactions of EAEC with the intestinal mucosa. We will extend ongoing studies which suggest that EAEC binding to MUC glycoproteins is fundamental to its pathogenesis. We will extend structure-function studies of the AAF adhesin and illuminate the contribution of AAF basic residues to interaction of EAEC with the human intestinal mucosa. We will determine the contribution of AAF mucin-binding residues to EAEC interaction with the human mucosa. We will characterize the contributions of Pic and dispersin to AAF-mediated interaction with the mucosa using time-lapse microscopy methods. In Aim 2 we will characterize the immunomodulatory role of class 2 SPATEs on the intestinal mucosa. We will address the potential roles of cleavage of intestinal glycoproteins in EAEC pathogenesis. We will dissect the inflammatory response through binding and cleavage of O-linked glycoproteins such as the MUC signaling proteins on the intestinal mucosa. We will also characterize the immunomodulatory effect of Pic through cleavage of inflammatory cytokine receptors in the colonoid/macrophage system. The work described here will continue to advance the general study of EAEC pathogenesis, generating fundamental insights that will illuminate aspects of pathogenesis relevant to other enteric pathogens. We will work closely and synergistically with investigators in other projects and the Core components.