PROJECT SUMMARY Innate immune cells such as macrophages detect pathogens by recognizing pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides (LPS, also known as endotoxin) displayed on the surface of Gram-negative bacteria. Extracellular and endosomal LPS molecules are recognized by Toll-like receptor 4. Cytosolic LPS, on the other hand, is detected by caspase-11 (in mice) and caspase-4/5 (in humans). Interestingly, it was recently found that outer membrane vesicles (OMVs) released by extracellular Gram-negative bacteria can deliver LPS and other bacterial molecules to the cytosol of macrophages. OMVs first enter macrophages through endocytosis, followed by the translocation of bacterial molecules across the endosome membrane into the cytosol to trigger pyroptosis, an inflammatory form of cell death. The molecular basis of OMV-triggered pyroptosis remains poorly understood. In particular, it is unknown how OMVs bind and enter macrophages, and how OMVs crosses the endosome membrane. The goal of this proposed research is to address these key questions by genetically dissecting OMV-induced pyroptosis using an unbiased genome-wide CRISPR genetic screen. In our preliminary studies, we established assays to measure OMV-induced pyroptosis in macrophages. Moreover, we developed tools to genetically dissect complex mammalian pathways using genome-wide CRISPR screens. Here, we will take strategic advantage of these systems to dissect OMV- induced pyroptosis in macrophages using an unbiased genome-wide CRISPR genetic screen. We will then validate the identified genes in pooled and targeted secondary screens. Finally, we will further characterize selected genes identified in the screens in primary human macrophages. These studies will provide a genome-scale view of OMV-induced pyroptosis and will expand our knowledge of inflammatory responses in pathogen-host interactions. Insights gleaned from this pilot study will facilitate the development of safer and more effective strategies to treat pathogen infection and inflammatory diseases. Moreover, we anticipate that these findings will guide the engineering of drug delivery vehicles using OMVs.