Abstract Bacillus cereus, a spore-forming, gram-positive bacterium, is a human pathogen commonly associated with hospital infections and foodborne outbreaks. Increasingly, B. cereus has been identified as a cause of acute severe infections and deaths in immunocompromised patients and children. One of B. cereus’ virulence factors is the highly potent pore-forming toxin, hemolysin BL (HBL). However, the mechanisms underlying the interactions between HBL and target cells and the identity of the HBL cellular receptor(s) remains unknown. This knowledge gap presents significant challenges for developing effective therapies in clinical management of potentially devastating B. cereus infections. Therefore, in order to develop effective therapeutics, there is a critical need to identify the toxin cellular receptor(s) and the molecular mechanisms underlying the toxin’s action. Our earlier preliminary data demonstrated that a cellular receptor is required for the cytolytic action of HBL. Based on this observation, we performed an unbiased genome-wide CRISPR screen and have identified Lipopolysaccharide-Induced Tumor Necrosis Factor-α Factor (LITAF) as the major, heretofore elusive, HBL receptor. Building on this strong preliminary data, in Aim 1, we will determine the in vivo role of LITAF in HBL pathogenesis in mouse models. To do so, we will characterize the LITAF knockout mice we have generated in this application, where our preliminary data supports the critical role of LITAF in HBL pathogenesis. We will also generate LITAF transgenic mice allowing the restoration of LITAF expression in a cell type-specific manner in a LITAF KO background. These cell type-specific LITAF-expressing mice will allow us to determine the key tissue targets responsible for HBL-induced lethality. In Aim 2, we will establish whether LITAF is a species-independent HBL receptor, perform comprehensive mutagenesis studies to identify the key residues of LITAF responsible for HBL binding, and demonstrate the utility of decoy receptors as anti-HBL therapy. Our initial CRISPR screen for HBL receptor demonstrated that CRISPR knockout of LITAF in mouse RAW264.7 macrophages resulted in complete resistance to HBL, whereas the same knockout in human HT1080 cells only yielded a 4-fold increase in resistance. This suggests additional receptor(s) may be involved in HBL-mediated cytotoxicity within certain cell types. Therefore, in Aim 3, we will delineate the full complement of host factors required for HBL action via sequential CRISPR screens. Supported by strong preliminary data, we hypothesize that this alternative receptor or additional host factors would become increasingly important when LITAF is absent, and can therefore be identified by a CRISPR screen when LITAF knockout cells are used. Together, this work will open new unbiased strategies for studying interactions between pore-forming toxins and mammalian target cells, potentially elucidating common mechanisms used by other enteropa...