The intracellular pathogen Shigella flexneri is the causative agent of bacillary dysentery in humans. In low-income countries, poor sanitation is responsible for approximately 270 million cases of shigellosis annually,with at least 250,000 of these resulting in death. In high-income countries, shigellosis is typically associatedwith travel to high-risk regions (Latin America, Asia and Africa). The disease is characterized by severe bloodydiarrhea and dramatic destruction of the colonic mucosa. S. flexneri is transmitted via the fecal-oral route andis extremely contagious, with a rate of attack above 90% with an infectious dose as low as 100-1000 bacteriaper individual, as determined in human volunteer studies. S. flexneri is an intracellular pathogen that invadesepithelial cells in the human colon. The bacteria trigger their own entry by injecting bacterial effector proteinsthrough their type 3 secretion system (T3SS), leading to bacterial uptake into primary cellular vacuoles. Thepathogen promptly escapes from primary vacuoles to gain access to the cytosolic compartment. In the cytosol,bacteria recruit components of the actin cytoskeleton and actin polymerization at the bacterial pole propel thepathogen throughout the cell. As motile bacteria reach cell-cell contacts, they form membrane protrusions thatproject into adjacent cells. The resolution of the formed protrusions leads to formation of double-membranevacuoles (DMVs) in adjacent cells, from which the pathogens escape, thereby achieving cell-to-cell spread. Theimportance of this dissemination process in S. flexneri pathogenesis has been initially established throughstudies conducted in non-human primates or in human volunteers and recently demonstrated by our group inan infant rabbit model of bacillary dysentery. Although actin-based motility is now fairly well understood, themechanisms supporting cell-to-cell spread are unclear. Our group has previously shown the importance of theT3SS in bacterial dissemination. Moreover, we have recently identified the T3SS effector protein IcsB as acritical determinant of DMV escape during cell-to-cell spread. Here we propose to explore how IcsB contributeto DMV escape in epithelial cells (Aim1) and how IcsB-mediated DMV escape contributes to pathogenesis invivo (Aim2).