Reductive activation of the disulfide-containing antibiotic thiolutin is mediated by both bacillithiol and FAD-dependent disulfide reductases.

Metal ions are universally essential for life and are required for critical enzymes throughout metabolism. Metalloenzymes rely on metal import and trafficking pathways for loading of the desired metal. Many microbes produce natural products that serve as metal chelators, both for their own nutrition and to serve as antimicrobials. In response to infection, our immune cells restrict bacterial growth by deploying proteins that chelate metal ions as part of nutritional immunity. Cells respond to metal depletion by the activation of pathways that prioritize metal delivery to the most essential enzymes. Dithiolopyrrolone (DTP) class natural products are prodrugs that are reduced in cells to generate a potent, dithiol-containing zinc chelator. Here, we identify the cellular reductants, bacillithiol and the FAD-dependent oxidoreductases TrxB and AhpF, that activate the DTP antibiotic thiolutin in Bacillus subtilis. Genetic studies reveal that loss of the Spx transcription factor also increases thiolutin resistance, consistent with the known role of Spx in transcriptional activation of thioredoxin reductase (trxB) and genes required for bacillithiol synthesis. Collectively, our results support a model in which several parallel pathways all contribute to the reductive activation of DTP class prodrugs in vivo.IMPORTANCEMetal ion chelators (metallophores) are deployed by microbes to obtain nutrient metals, sequester excess metals, and act as antimicrobials to inhibit the growth of other organisms. Dithiolopyrrolones (DTPs) are a class of natural products that inhibit bacterial growth by the intracellular chelation of zinc and iron, two metal ions essential for growth. Thiolutin, a model DTP antibiotic, is activated by reduction inside cells and selectively chelates intracellular metals. Here, we demonstrate that the activation of the thiolutin prodrug is mediated by several parallel pathways, which greatly reduces the ability of cells to evolve antibiotic resistance. Since DTP antibiotics appear to primarily target zinc enzymes, they provide a powerful tool for exploring how cells adapt to zinc deficiency.