Identify new regulators of outer membrane vesicle entry into macrophages

PROJECT SUMMARY Outer membrane vesicles (OMVs) released by extracellular Gram-negative bacteria can deliver lipopolysaccharide (LPS) and other bacterial molecules to the cytosol of macrophages. OMVs enter macrophages through endocytosis and reach the endosome of macrophages. Subsequently, components of OMVs including LPS cross the endosomal membrane and reach the cytosol to trigger pyroptosis, an inflammatory form of cell death. While the downstream pyroptotic signaling steps were previously characterized, the upstream stage of OMV entry into macrophages remains largely unknown. The goal of this project is to bridge this major knowledge gap. In our preliminary studies, we established assays to measure OMV-induced pyroptosis in RAW 264.7 macrophages. Using these assays, we performed an unbiased genome-wide CRISPR genetic screen to dissect OMV-induced pyroptosis. The screen isolated known mediators of pyroptosis and a large number of candidate genes not previously linked to pyroptosis or OMV function. In this proposed research, we will validate and characterize the candidate genes isolated in the CRISPR screen to identify new regulators of OMV entry into macrophages. These experiments are expected to establish mediators of OMV-macrophage interaction, OMV endocytosis, and translocation of OMV components across the endosomal membrane. These exploratory studies will shed light on the molecular basis of OMV entry into macrophages and will broaden our knowledge of inflammatory responses. Insights gleaned from this research will facilitate the development of safer and more effective strategies to combat bacterial infection and inflammatory diseases.