PROJECT SUMMARY Over two million people are afflicted with bacterial infections resistant to FDA-approved antibiotics in the United States every year. Of those, more than 20,000 of these patients die as a result of these infections. The surge in drug-resistant bacteria has become a growing pandemic and threatens to undermine medical gains made in the last several decades. It has proven difficult to discover new antibiotics for a myriad of reasons, including under developed concepts related to bacterial targets. To improve our ability to design and discover novel tools to combat bacterial infections, it becomes important to better understand their biology. Bacterial cell walls are unmatched targets for antibiotics, including being the target of many of clinically relevant agents. Yet, foundational aspects of bacterial cell wall assembly and its interaction with the host organisms remain vastly under explored. Our laboratory has extensive experience in designing cell wall analogs that become metabolic processed by bacterial cells during cell wall growth and division. Within these substrate analogs, we introduce non-native tags (e.g., click chemistry handles, biotin, fluorophores) that can be leveraged to elucidate the processes underpinning cell wall biology. For this proposal, we will apply these strategies towards two main areas: the development of probes related to the processing of peptidoglycan, a primary component of bacterial cell wall and to the development of assays that report on the accessibility of components on the cell surface of Gram-positive bacteria. Peptidoglycan is a single large biomacromolecule composed of unique building blocks, including a short peptidic fragment called stem peptide. Our laboratory has extensive experience in labeling cell wall of live bacteria to install unnatural epitopes. The metabolic and site-selective labeling will be the basis for elucidating sites of peptidoglycan modifications and will be the anchor point for the accessibility assay. Through these investigations, we project that we will add insight into cell wall biosynthesis that can empower drug discovery and development.