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

摘要 肠聚集性大肠大肠埃希菌（EAEC）是引起旅行者腹泻、工业化腹泻的重要病原体 发展中国家的经济增长放缓。在本申请中，我们建议研究EAEC 通过使用新建立的人肠样/结肠样 模型，其是离体自繁殖的人肠道原代培养物。此应用程序包括一个 约翰霍普金斯大学（JHU）的研究人员组成的联盟，他们率先开发了 肠样/结肠样模型，以及马里兰州大学（UMD）和马里兰大学的研究人员， Virginia等在大肠杆菌引起的肠炎发病机制的研究中处于领先地位。杆菌 志贺氏菌该项目将利用新的结肠单层模型的存在。我们将专注于 EAEC与结肠样和结肠样/白细胞模型相互作用的表征，特别是为了解剖 AAF在多种模型中已成为EAEC主要毒力因子，其作用及其功能 SPATE对粘蛋白和白细胞糖蛋白具有广谱活性， 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。我们将扩展正在进行的研究，这些研究表明EAEC 与MUC糖蛋白的结合是其发病机理的基础。我们将扩展结构功能研究， 说明AAF碱性残基在EAEC与人的相互作用中的作用 肠粘膜我们将确定AAF粘蛋白结合残基与EAEC相互作用的贡献。 人体粘膜我们将描述Pic和分散素对AAF介导的相互作用的贡献 用延时显微镜的方法观察粘膜。在目标2中，我们将表征免疫调节剂 2类SPATE对肠粘膜的作用。我们将讨论肠上皮细胞分裂的潜在作用， 糖蛋白在EAEC发病机制中的作用。我们将通过结合和裂解来剖析炎症反应 O-连接的糖蛋白，如MUC信号蛋白在肠粘膜上。我们还将 表征Pic通过切割炎性细胞因子受体的免疫调节作用， 类结肠/巨噬细胞系统。本文的工作将继续推进EAEC的研究 发病机制，产生基本的见解，将阐明发病机制的相关方面， 肠道病原体我们将与其他项目和核心项目的研究人员密切合作， 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.