Immunomodulatory role of Pic in EAEC Pathogenesis

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，我们建议研究高度 EAEC发病机制中SPATE家族的普遍丝氨酸蛋白酶，以及它们与慢性 使用新建立的人白细胞-结肠样模型观察炎症性肠病。本申请 由约翰霍普金斯大学（JHU）的研究人员组成的联盟，他们开创了 肠/结肠模型的发展，以及马里兰州大学（UMB）和 弗吉尼亚大学（UVA），谁是领导者在研究的发病机制，由 嗜热E.大肠杆菌和志贺氏菌。我们已经成功地使用结肠模型来表征相关的 EAEC发病机制的特点，突出了凝集素样丝氨酸蛋白酶Pic在肠道中的重要作用， 定植和炎症。在这个项目中将利用新开发的白细胞的存在- 结肠样单层模型来表征杯状体中Pic和其他丝氨酸蛋白酶的调节活性 细胞、白细胞和上皮细胞在EAEC发病中的作用。该项目将包括三个具体目标。在 目的1，我们将通过获得对Pic的机制见解来表征Pic在杯状细胞功能中的作用， 介导的MUC 2胞吐，并通过建立杯状细胞分泌的丝氨酸蛋白酶抑制剂的作用， WFDC 2在EAEC发病机制中的作用，采用无偏转录组学和药理学方法。在目标2中， 我们将通过获得SPATE在EAEC诱导的上皮炎症中的作用机制， EAEC感染中MUC 1介导的上皮炎症，采用基因沉默和 药理学方法。在目标3中，我们将表征Pic凝集素的免疫调节性质， 使用中性粒细胞和巨噬细胞的EAEC发病机制中对白细胞功能的粘蛋白溶解活性 结肠共培养沿着最先进的延时显微镜、流式细胞术和基于Luminex的 测定。这项工作将继续推进我们对EAEC发病机制的理解， 这些见解将阐明与其他肠道病原体相关的发病机制，并可能开辟新的 治疗干预的途径。我们将与其他项目的调查人员密切合作， 和核心组件。

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 the role of highly prevalent serine proteases from the SPATE family in EAEC pathogenesis, and their relationship with chronic inflammatory enteropathy using a newly established human leukocyte-colonoid model. 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 (UMB) and the University of Virginia (UVA), who are leaders in the study of the pathogenesis of diarrheal disease caused by diarrhegenic E. coli and Shigella. We have successfully used the colonoid model to characterize relevant features of EAEC pathogenesis, highlighting the important role of the lectin-like serine protease Pic in intestinal colonization and inflammation. In this project will leverage the existence of the newly developed leukocyte- colonoid monolayer model to characterize the modulatory activity of Pic and other serine proteases in goblet cells, leukocytes and epithelial cells in EAEC pathogenesis. This project will comprise three Specific Aims. In Aim 1, we will characterize the effect of Pic in goblet cell function by obtaining mechanistic insights into Pic- mediated MUC2 exocytosis and by establishing the role of the goblet-cell secreted serine protease inhibitor WFDC2 in EAEC pathogenesis by using unbiased transcriptomics and pharmacological approaches. In Aim 2, we will characterize the role of SPATEs in EAEC-induced epithelial inflammation by obtaining mechanistic insights into MUC1-mediated epithelial inflammation in EAEC infection, employing gene silencing and pharmacological approaches. In Aim 3, we will characterize the immunomodulatory property of Pic lectin and mucinolytic activity on leukocyte functions in EAEC pathogenesis employing neutrophils- and macrophages- colonoids co-cultures along with state-of-the-art time-lapse microscopy, flow cytometry and Luminex-based assays. This work will continue to advance our understanding of EAEC pathogenesis, generating fundamental insights that will illuminate aspects of pathogenesis relevant to other enteric pathogens, and could open up new avenues for therapeutic intervention. We will work closely and synergistically with investigators in other projects and the Core components.