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.