Project Summary/Abstract The marine pathogen Vibrio vulnificus is a highly lethal Gram-negative bacteria inhabiting coastal waters. It causes intestinal infections via consumption of raw seafood and wound infections via open wounds. The infection progresses rapidly to septicemia with high fatality rates. This bacterium exerts the highest economic cost of any seafood-related disease. As multi-antibiotic resistant isolates have been detected and infection incidence is climbing due to climate change, studies elucidating the pathogenic mechanisms of V. vulnificus are increasingly important to support novel therapeutic development. The Multifunctional-Autoprocessing Repeats-in-Toxin (MARTX) toxin has been shown in animal studies to be the primary virulence factor of V. vulnificus associated with death. The goal of the proposed project is to determine pathogenic mechanisms of MARTX at host cell surfaces that are necessary for intoxication. Addressing this question could provide critical information toward design of antibodies that would function to block MARTX from binding to its targeted cells. This goal is particularly pertinent to the NIAID given the institute's mission to support research to better understand, treat, and prevent infectious diseases. Specifically, the first aim will determine host receptors for the V. vulnificus MARTXVv toxin. Our preliminary data using an unbiased Genome-wide CRISPR-Cas9 Screening approach identified a host cell membrane protein kinase as the most promising receptor candidate. In this aim, we will use a combination of genetic manipulation, in vitro cellular assays and FRET based flow cytometry to validate the role of this kinase as a MARTXVv receptor. The second aim will determine which regions of MARTXVv can bind to the cellular surface based on previous studies on MARTXVv fragments, using techniques including protein purification, cell-based binding assays and fluorescence microscopy. Collective, these data will define the host receptor and receptor binding domain of the toxin revealing critical interactions between the toxin and host at cellular surface, the first step of MARTXVv intoxication. Completion of the proposed work will provide training opportunities required to support the long-term career goal of becoming a principal investigator studying bacterial toxins. Specifically, the training will include 1) developing expertise in studying toxin functions in the host cell system, 2) developing experimental techniques in genetic screens, immunofluorescence, and protein biochemistry and 3) developing general skills required for academic independent researchers such as grant writing and scientific communication. Northwestern University Feinberg School of Medicine provides an outstanding training environment for accomplishing these goals. The sponsor Dr. Karla Satchell is an expert in bacterial toxins and protein structure. The trainee will gain a depth of knowledge in studying toxin biology by having weekly meet...