ABSTRACT: Nontuberculous mycobacteria (NTM) infections such as Mycobacterium abscessus (Mabs) are a growing health concern, particularly in older and immunocompromised patient populations, and are among the most difficult bacterial infections to eradicate. Thus, there remains a critical need to understand the mechanisms enabling Mabs to survive prolonged exposure to lethal antibiotics. The central hypothesis of this proposal is that antibiotic tolerance is a regulated process, controlled by pathways that sense discrete stresses, and transduce signals leading to a coordinated cellular response that mitigates antibiotic cytotoxicity. In our preliminary data we present the results of a Tn-Seq screen to identify genes contributing to antibiotic tolerance. We now propose to use cutting-edge proteomics to study several proteases that were identified in the Tn-Seq screen.