The enteric, intracellular human pathogen Shigella causes hundreds of millions of cases of thediarrheal disease shigellosis per year worldwide, which results in as many as one million deaths.Currently, there is no approved vaccine for the prevention of shigellosis, and increasing drugresistance complicates treatment of the disease. The bacteria are acquired by ingestion ofcontaminated food or water, and upon reaching the colon, Shigella invade the colonic epithelialcells, replicate intracellularly, spread to adjacent cells, and provoke an intense inflammatoryresponse. While much is known regarding the mechanism of pathogenesis, there are still gaps inour knowledge. Progress in closing these gaps has been hampered by the lack of a small animalmodel that accurately mimics the human disease. Cell culture has been used to study aspects ofinvasion and replication in the cell, but the use of transformed cells in culture does not provide thesame environment as the normal human epithelium. A potential solution to this problem is thedevelopment of enteroids derived from human intestinal stem cells as a model for Shigella flexneriinfection. In this study, we will use human intestinal enteroid monolayers derived from colon tissue toreplicate aspects of the bacterial-host interaction that currently cannot be emulated in cell culture.We will use enteroids to test the current paradigms of S. flexneri invasion, intracellular replication,and spread, in order to determine how S. flexneri responds to its normal host environment. In thefirst Aim, we will establish the basic parameters for colonoid infection by S. flexneri and test thehypothesis that M-cells are required in the invasion process in human intestine. In Aim 2 we willfocus on the dynamics and kinetics of intercellular spread of the bacteria in the enteroid monolayers.One aspect of Shigella infection that is poorly understood is the basis for its tropism for colonicepithelium; in Aim 3 we will use human intestinal enteroids derived from duodenal, jejunal, ileal andcolonic tissue to determine whether there is tissue specificity for S. flexneri invasion. This will becritical in designing future experiments to determine the host receptors for S. flexneri and theenvironmental factors that influence S. flexneri invasion efficiency. Completion of this project will not only allow us to examine basic assumptions about Shigellapathogenesis but will also result in the development of a model system for future studies of theinteraction between Shigella and the human epithelium.