Immunomodulatory role of Pic in EAEC Pathogenesis

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，我们建议研究高度的角色 Spate家族丝氨酸蛋白酶在EAEC发病机制中的流行及其与慢性疾病的关系 使用新建立的人类白细胞-结肠模型的炎症性肠病。此应用程序 由约翰·霍普金斯大学(JHU)的研究人员组成的财团，他们开创了 肠样/结肠样病变模型的发展，以及马里兰大学(UMB)和 他们是研究由以下原因引起的腹泻疾病发病机制的领先者 致泻性大肠杆菌和志贺氏菌。我们已经成功地使用结肠型模型来表征相关的 EAEC的发病特点，强调凝集素样丝氨酸蛋白酶Pic在肠道中的重要作用 殖民和炎症。这个项目将利用新开发的白细胞的存在- 用类结肠单分子层模型研究Pic等丝氨酸蛋白酶在杯中的调节活性 细胞、白细胞和上皮细胞在EAEC发病机制中的作用该项目将包括三个具体目标。在……里面 目的1，我们将通过了解Pic在杯状细胞功能中的作用机制来表征Pic在杯状细胞功能中的作用。 介导MUC2的胞吐作用及杯状细胞分泌型丝氨酸蛋白酶抑制物的作用 WFDC2在EAEC发病机制中的作用在目标2中， 我们将通过获得机制来表征SPATS在EAEC诱导的上皮炎症中的作用 利用基因沉默和基因转录技术研究肠上皮细胞感染中MUC1介导的上皮炎症 药理学方法。在目标3中，我们将表征Pic凝集素的免疫调节特性和 利用中性粒细胞和巨噬细胞在EAEC发病机制中对白细胞功能的粘蛋白溶解活性 结肠型细胞共培养以及最先进的时间推移显微镜、流式细胞仪和Luminex 化验。这项工作将继续推进我们对EAEC发病机制的理解，产生基本的 将阐明与其他肠道病原体相关的致病机制方面的见解，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，协同工作 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.