Project Summary Infections of the human colon are difficult to study mechanistically because of the absence of in vitro model systems that mimic the limited oxygen environment of the colon. In this project, we propose to use a human intestinal model system based on cylindrical silk scaffolds that serve as the backbone for human epithelial and sub-epithelial tissues. This proposed bioengineered 3D model system will be used to address the details of infection by the major human diarrheal pathogen, Clostridium difficile. Steps in C. difficile pathogenesis, such as spore germination, vegetative cell outgrowth, adherence, toxin production and epithelial cell damage will be studied in the tissue model. In Aim 1, further development of the tissue model will be pursued. The tissue models will be incorporated into perfusion bioreactors capable of passing media of variable chemical compositions and low oxygen tension through the lumens at regulatable rates. The central, hollow core of the tissue model will thus mimic an intestinal lumen in which bacteria and host tissue will interact. In Aim 2, a combination of single cell analysis using fluorescent reporter proteins and genetic analysis of mutants using TnSeq will be pursued to analyze the importance of bacterial factors for infection in the tissue model. The experiments proposed are designed to validate the bioengineered 3D intestinal model system as well as generate new information about the pathogenesis of C. difficile. Interaction between C. difficile and the human gut microbiota will be investigated in Aim 3. Microbiota will be established in the tissue model for these experiments. Aim 4 will focus on testing the efficacy of several anti-infective strategies against C. difficile infection, using the scaffold tissue model. This project will establish the bioengineered intestine tissue model as a novel system for detailed, mechanistic analysis of infection by virtually all colonic pathogens, thereby providing the potential for major breakthroughs in the understanding of multiple aspects of intestinal infection.