Project Summary Every year in the United States, over two million people are afflicted with bacterial infections resistant to FDA-approved antibiotics. More than 23,000 of these patients die as a result of such infections. The rapid surge in drug-resistant bacteria has now become one of the primary public health crises of the 21st century. The large majority of antibiotics in use today were discovered many decades ago. In order to counter the rapid rise in drug-resistance in bacteria, new drug targets and diagnostic tests are urgently needed. The bacterial cell wall has proven to be a rich source of antibiotic drug discovery. However, there are fundamental aspects of bacterial cell wall assembly and its interaction with the host organism that are yet to be fully elucidated. Our proposed strategies will use synthetic chemistry as a platform to construct cell wall analogs that metabolically label live bacteria and mimic key aspects of cell wall architecture. We anticipate that interrogation of cell wall remodeling and processing in pathogenic bacteria will guide the design of next-generation antibiotics that circumvent resistance mechanisms. Furthermore, the development of probes to systematically characterize cell wall sensing and host distribution will add fundamental knowledge to bacterial pathogenesis and human microbiome maintenance. We will focus on: (1) the contribution of individual enzymes to the overall drug resistant phenotype in response to antibiotics in live bacterial cells, (2) key interactions by bacterial membrane-anchored proteins to Lipid II (the bottle-neck point of cell wall biosynthesis), (3) the molecular recognition of cell wall by cell wall receptors on human immune cells, and (4) the processing of disseminated bacterial-derived membrane vesicles, which contain cell wall fragments, by human immune cells.