ABSTRACT The sulfur-containing amino acid, L-cysteine, is indispensable for pathogen virulence and survival. Sulfur for biosyn- thesis is largely derived from the assimilation of inorganic sulfate by microorganisms. Genes required for sulfate as- similation are essential to pathogen survival but absent in the human genome and, therefore, represent potential new targets for therapeutic intervention. In the previous funding cycle, we defined key features in the catalytic cycle of sulfonucleotide reductases (SRs), enzymes that catalyze the first committed step in sulfate reduction for de novo synthesis of cysteine and other reduced-sulfur containing biomolecules. Our studies provided fundamental insights into how thioredoxins (Trxs)–central antioxidant enzymes that maintain protein thiols in their reduced state– recognize their cellular targets. We learned that the iron-sulfur cluster in APS reductase (APSR) plays an essential role active-site preorganization and substrate activation, expanding knowledge on the catalytic activities of Fe-S pro- teins and on the divergent evolution of PAPS reductase (PAPR), which lacks this cofactor. These insights led to molecules that target APSR in a new way, via interaction with the iron-sulfur metallocenter. We also discovered first- in-class inhibitors of sulfate reduction that exhibit potent bactericidal activity against drug-resistant clinical isolates of M. tuberculosis. Given the importance of SRs in pathogen oxidative stress resistance and virulence, we propose here to address research areas where the biggest open questions and greatest unmet needs remain: well-validated chemical probes that acutely inhibitor essential steps in microbial reductive sulfate assimilation (Aim 1); defining the non-redundant functions of pathogenic Trxs to facilitate sulfur reduction and cope with redox stress (Aim 2); and ad- vancing knowledge in trafficking and delivery of reactive sulfur species (RSS) for microbial cysteine biosynthesis. Collectively, the experiments in this renewal application will provide fundamental information on one of the most under-studied metabolic chemistries–reductive sulfate assimilation–in the context of one of the most devastat- ing pathogens–M. tuberculosis. We have established outstanding collaborations, with a proven track record of productivity, in order to support these efforts and ensure timely completion. Our efforts will address areas cen- tral to role of sulfur metabolism in pathogen oxidative stress resistance and virulence that have not received suf- ficient attention despite their importance, ultimately leading to a new level of understanding and leverage in the battle against infectious disease.