ABSTRACT Vibrio vulnificus is a Gram-negative bacterial pathogen that causes severe life-threatening infections in humans after eating shellfish or swimming in warm seawater. In the US, there are about 200 cases annually with high rates of mortality and morbidity. While the number of cases is low compared to other food-borne pathogens, the high rates of death among patients exerts a significant burden on human life and the economy. This is expected to increase as incidence of V. vulnificus infections is rising due to the climate crisis. The most prominent virulence factor of V. vulnificus is a 5208 aa MARTX family toxin that delivers cytotoxic effectors to cells and these effectors are essential for virulence in a mouse model. While extensive information is known about the mechanisms of the effectors, very little is known about the remaining over 2800 aa of the protein toxin. These regions are known to be both necessary and sufficient for secretion of the toxin from the bacteria, interaction of the toxin with the eukaryotic cell surface, and translocation of the effectors across the plasma membrane. The project proposes to identify host cell receptors and host factors essential for intoxication using a genetic screen and selection for mutant cells that survive the cytolytic action of the toxin. We will also use a structure-function based approach to identify regions of the MARTX toxin essential for Type I secretion from the bacterium, binding of the toxin to a putative surface receptor, formation of a pore in the plasma membrane, and translocation of effectors to the eukaryotic cell cytosol. This study will advance our understanding of the function of the MARTX toxin repeat regions in detail and is expected to impact our understanding of pathophysiology during V. vulnificus infection.