Tuberculosis (TB) is a chronic pulmonary disease caused by Mycobacterium tuberculosis (Mtb), which infects approximately one quarter of the world’s population. A variety of drugs have been identified that rapidly kill Mtb and its relatives in vitro, yet clinical treatment requires at least 6 months of combination therapy and resistance is rampant. Furthermore, the current state-of-the-art for detection of Mtb infection employs cumbersome methods that were developed more than 80 years ago. Herein we propose to develop new methods for detection of Mtb that can be employed in low resource settings, and to develop new screens for potential TB drugs, as well as to perform fundamental studies on the role of mycobacterial lipids in virulence. In this renewal application of R37 AI051622 entitled “Chemical Mycobacteriology”, we propose the following four Aims: (1) to develop probes based on the fluorogenic Nile Red and 3-hydroxychromone dyes, which can be used to detect Mtb with low-power, low-cost microscopes; (2) to establish a magnetic bead- based enrichment platform that can be deployed at the point-of-care to enhance detection of fluorescently labeled Mtb cells; (3) to deploy metabolic labeling as a readout for high-throughput drug screens to decrease time and expense in discovery of new TB drugs; and (4) to employ bioorthogonal labeling and chemical biology approaches to elucidate the role of phthiocerol dimycocerosates (PDIM) lipids in mycobacterial virulence. RELEVANCE (See instructions): Tuberculosis (TB) is a global health crisis that has frustrated efforts to treat and contain, and, unlike other bacterial infections that can be treated with a week-long course of a single antibiotic, TB therapy requires several drugs in combination for at least 6 months and often even this regimen does not work. In this project we will: (1) develop a better detection method for active TB in patient sputum samples through collaboration with a group in South Africa that works with TB-infected and HIV/TB-coinfected patients; (2) develop a better method for quickly and cost-effectively screening potential new TB drugs; and (3) conduct fundamental studies to determine how the outer layer of the pathogen that causes TB allows infection to happen. Our new method for clinical TB diagnosis should make it easier to determine whether TB drugs are working in patients; additionally, if they are not working well, new insight gleaned from this work may help us to better understand why.