Microbial Invasion of the Intestine at the Cell Extrusion Zone of the Villus Tip

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DESCRIPTION (provided by applicant): Invasive gastroenteritis and enteric fever are major sources of mortality and morbidity worldwide. The initial steps in the pathogenesis of these syndromes involve a breach of the gastrointestinal epithelial barrier by virulent bacteria, colonization and growth in the intestinal tissue, and in some cases subsequent systemic spread to other organs via the lymphatics or blood stream. The receptors for epithelial cell invasion have been defined for several of these pathogens. However, many of these receptors for entry are not found on the luminal side of the intestine, but are instead abundant components of the basolateral membranes of epithelial cells. This implies that pathogens must first breach the epithelial tight junctions, or find alternative mechanisms to cross the epithelial barrier before reaching their receptors for epithelial invasion. We have proposed a novel model of intestinal invasion and colonization by the pathogen Listeria monocytogenes. We found that the tips of intestinal villi are a specialized site where the normal cell extrusion process allows basolateral receptors to be exposed at the luminal surface. Listeria monocytogenes finds E-cadherin, its receptor for entry, at these sites and invades the gastrointestinal epithelium of the villus tip. We propose to establish an oral model of Listeria gastrointestinal infection in Guinea pigs to test this model by characterizing the sites and kinetics of intestinal invasion and colonization. We will test the hypothesis that invasion of the cell extrusion zone results in asymptomatic carriage of Listeria in the gastrointestinal tract and study the relationship between this mode of invasion and systemic disease. Because of technical limitations found in Guinea pigs, we also propose to test whether genetic alterations in the Listeria invasin InlA can improve its interaction with murine E-cadherin, in order to develop a murine model of intestinal Listeriosis. In parallel with in vivo experiments, we will use cell culture experiments to define the mechanisms of endocytosis involved in internalization of Listeria at the cell extrusion zone. We will utilize polarized MDCK cell monolayers to test the hypothesis that Listeria take advantage of junction remodeling during invasion of the cell extrusion zone.Listeria monocytogenes has emerged as a significant cause of mortality due to food-borne illness in the United States, since it was responsible for 27.6% of deaths from enteric infection. Other important pathogens that invade the epithelium include rotavirus, Salmonella, Shigella, Yersinia, enteroinvasive E. coli, and Campylobacter. How these organisms breach the gastrointestinal epithelial barrier is not clearly understood. We propose to study a new paradigm that explains how Listeria monocytogenes, and perhaps also other invasive enteropathogens, breach the intestinal barrier.