PROJECT SUMMARY Antibiotics are central to modern medicine and rising antibiotic resistance is one of the biggest threats to global health. Identifying new and different drug targets for the development of new antibiotics is crucial to overcome resistance. Adjuvant strategies that either enhance the activity of existing antibiotics or improve clearance by the host immune system provide another mechanism to combat antibiotic resistance. Targeting a combination of essential and non- essential enzymes that play key roles in bacterial metabolism is a promising strategy to develop new antimicrobials and adjuvants, respectively. The enzymatic synthesis of L-cysteine is one such strategy. Cysteine plays a key role in proteins and is vital to the synthesis of biomolecules important for defense against the host immune system. In contrast to mammals, the biosynthesis of cysteine occurs de novo in microbes using sulfide (S2–) as the sulfur source derived from the reductive sulfate assimilation pathway. Inhibition of sulfate assimilation has been proven to interfere with a pathogen’s ability to fight oxidative stress, infect the host and establish long-term infection. Inhibition of sulfate assim- ilation has also been associated with a dysregulated oxidative stress response, enhancing the antimicrobial activity of existing antibiotics. In previous funding cycles, we have defined the mechanism and structure of mycobacterial 5’- adenylylsulfate (APS) reductase an iron-sulfur protein that catalyzes the two-electron reduction of APS to sulfite (SO32– ) using thioredoxin (TrxA) as the preferred electron donor. We subsequently used these insights to discover first-in- class inhibitors of mycobacterial APS reductase (APR), with potent in vivo bactericidal activity against MDR and XDR clinical isolates of Mycobacterium tuberculosis (Mtb) and synergistic activity with known anti-TB drugs (isoniazid, ri- fampicin, clofazimine) in killing H37Rv Mtb. In this renewal, we now seek to expand our early focus on cysteine bio- synthesis and redox metabolism in mycobacteria to pathogens implicated in fatal secondary bacterial infections in influenza infection, specifically in patients with COVID-19: Pseudomonas aeruginosa and Streptococcus pneumoniae. Given the importance of microbial sulfur metabolism in oxidative stress resistance and virulence, in this renewal ap- plication we propose the following Specific Aims: (1) Define the mechanistic and structural basis for inhibition of the Fe-S protein APR from P. aeruginosa; (2) Investigate the effect of P. aeruginosa APR inhibitors on sulfur metabolism and redox metabolism in cells; (3) Determine whether a virus family that infect S. pneumoniae in human environments and encode genes for reductive sulfate assimilation increase the fitness of the bacterial host, which lacks this pathway.