ABSTRACT A key feature in intestinal immunity is the dynamic intestinal barrier, which separates the host from resident and pathogenic microbiota through a mucus gel impregnated with antimicrobial peptides. The mechanisms underlying the maintenance and function of this intestinal barrier are not completely understood. Using a mouse forward genetic screen for defects of intestinal homeostasis, we have found a mutation in Tvp23b, which conferred susceptibility to both chemically induced and infectious colitis. Golgi apparatus membrane protein TVP23 homolog B (TVP23B) is a transmembrane protein conserved from yeast to humans. In the intestine, the protein is localized to the epithelium and its deficiency in the hematopoietic extrinsic compartment was essential to the colitis phenotype. We found that TVP23B controls the homeostasis of Paneth cells and function of goblet cells in vivo, leading to a decrease in antimicrobial peptides as well as a more penetrable mucus layer. As a result, Tvp23b-/- mice displayed decreased barrier function and a loss of host-microbe separation. TVP23B- deficient colonocytes have a loss of core-3 O-glycosylation of colonic proteins, which is the major O-glycosylation present on gel forming mucins. TVP23B binds with another Golgi protein, YIPF6, which is similarly critical for intestinal homeostasis. The Golgi proteomes of YIPF6 and TVP23B-deficient colonocytes have a common deficiency of several critical glycosylation enzymes, including those necessary for core-3 glycosylation of mucins. TVP23B is necessary for the formation of the sterile mucin layer of the intestine and its absence disturbs the balance of host and microbe in vivo. In this proposal, we will examine the components that mediate the cellular and molecular dysfunction in TVP23B deficiency and the resulting pathology in three Specific Aims: (1) Determine the role of TVP23B in maintaining barrier function during infectious and chronic colitis (2) To examine the role of TVP23B in intestinal secretory cell differentiation and intestinal regeneration. (3) To understand role of TVP23B on Golgi enzyme trafficking. The aims in this proposal will help elucidate the mechanisms underlying the mechanisms by which epithelial cells regulate host-microbe interactions.