Project Summary Tuberculosis (TB) killed 1.3 million people in 2017 and is the most deadly infectious disease in the world. Infections caused by drug-susceptible Mycobacterium tuberculosis (Mtb) can be cured. But treatment regimens are long, requiring at least 6 months of therapy with multiple drugs. TB has become increasingly difficult to treat due to the global spread of drug resistant strains. Such strains account for at least 5% of infections, and the cure rate for those patients is low. New drugs are urgently needed to treat drug-resistant strains, but drug development is a slow and costly process. But what if clinically-approved β-lactam antibiotics could be repurposed to treat TB and drug resistant TB? This would significantly speed up patient access to new therapies. Since the 1990s, there have been occasional reports of TB infections responding to β-lactam antibiotics. For example, amoxicillin/clavulanate and meropenem/clavulanate have been used successfully to treat patients with drug resistant TB. β-lactam antibiotics target enzymes in the cells wall. Targets include penicillin-binding proteins (PBPs) and the recently discovered L,D-transpeptidases (LDTs). Both classes of enzyme are critical to survival because they maintain the structure and rigidity of peptidoglycan in the bacterial cell wall. We propose to develop new molecular probes to identify and validate β-lactam drug susceptibility in mycobacteria. Our approach facilitates monitoring multiple PBPs and LDTs at once, enabling a comprehensive examination of these enzymes. We will detect enzymes in protein gel-resolved lysates using activity-based probes (ABPs) derived from the major classes of β-lactam drugs. Identifying drugs with activity against dormant mycobacteria is a high-priority. Therefore, in Aim 1, we will use ABPs to profile the regulation of PBPs and LDTs in dormant, reactivating, and actively-replicating Mtb. In Aim 2, we will determine the drug selectivity and inhibitor profiles for the Mtb PBPs and LDTs following exposure to clinically-approved β-lactam antibiotics. In Aim 3, we will investigate the spatio-temporal regulation of the LDTs. In Aim 4, we will extend our findings to a second high-priority mycobacterial pathogen: M. abscessus. Upon successful completion of this project, we will have an effective approach for assessing the susceptibility of mycobacterial strains to treatment with β-lactams and combination therapies. We anticipate that our findings will have a positive impact on selecting effective therapies for patients infected with Mtb or M. abscessus.