ABSTRACT Enteric infections caused by bacterial pathogens are often debilitating and life-threatening. The most common models for studying these pathogens are in vivo rodent models and in vitro intestinal epithelial cell monolayers. However, these models often do not manifest the true outcomes of enteric infections that occurred in the human intestine. Therefore, many aspects of the interactions between these pathogens and the human host remain unknown. This project aims to dissect the intricate host-pathogen interactions for two important intestinal enteric pathogens, Vibrio cholerae and pathogenic Yersinia pseudotuberculosis, using a multicellular 3D in vitro human tissue model that has villi and flow. We will develop and employ a bioengineered model of the human intestine tunica mucosa that mimics the physiological structures and functions of the intestine by introducing primary intestinal cells, flow dynamics, and villus topology to a 3D scaffold. Specifically, we will use silk proteins as scaffolds to develop a 3D multicellular matrix system to support human intestinal epithelium formation for sustained cultivation and for infection by enteric pathogens. This scaffold design is based on our previously developed 3D silk scaffold system seeded with the cultured cell lines Caco-2 and HT-29 cells and primary human myofibroblast cells. Once we incorporate primary epithelial cells, villus-topology and flow dynamics and build this 3D human intestinal model, we will study how V. cholerae and Y. pseudotuberculosis colonize and cause damage to the human intestine. Because these pathogens have very different pathophysiological outcomes on the human intestine they are excellent models to use in exploring the versatility of these novel 3D bio-mimetics of the intestinal system. Our aims are (1) to build and characterize a 3D model human small intestinal tissue with primary cells, flow and villi and (2) investigate the spatial and temporary dynamics of pathogen colonization and damage as well as the pathophysiological responses of the host cells to V. cholerae or Y. pseudotuberculosis in these 3D tissues. The end result will be a robust, tractable, and well-characterized 3D small intestinal tissue model system that can be used by the field for studying the specific mechanistic steps that are important for enteric pathogens to successfully colonize the host intestine. Importantly, these studies will provide a platform upon which to build a larger program in which multiple investigators can use these 3D systems to probe interactions with various enteric pathogens, microbiota, and anti-infectives as well as to further modify these systems to incorporate other cell types and host factors.