Pyrazinamide (PZA) is a cornerstone of modern TB chemotherapy due to its unique ability to shorten the duration of nearly all clinically used regimens. However, resistance is emerging while prospects for a replacement remain lacking, threatening the duration of all future chemotherapies. Understanding the molecular basis for the unique activity of PZA will allow development of additional regimen shortening agents that target the same pathways. However, efforts to develop a functional replacement for PZA remain hindered by fundamental gaps in our understanding of its microbiologic and biochemical targets. Here, we propose to overcome these gaps by identifying physiologic biomarkers of PZA activity and susceptibility. These studies will specifically integrate experimental studies of PZA in vitro activity with in vivo studies of PZA activity as reported by the transcriptional profiles of Mtb recovered from the lungs of infected mice or sputa of culture confirmed patients. Based on our preliminary data, these studies will also test the specific hypothesis that PZA targets Mtb subpopulations whose viability is strictly dependent on NAD salvage pathway activity. By integrating parallel in vitro and in vivo studies with hypothesis-based and unbiased approaches, this project will provide deeper insights into the target(s) and/or mechanism(s)-of-action of PZA, the specific microbiologic subpopulations it targets, and a molecular signature of its activity that can be used to facilitate the development of new agents targeting the same pathways as PZA.