CRISPR Screening and Comparative LC‐MS Analysis Identify Genes Mediating Efficacy of Delamanid and Pretomanid against <i>Mycobacterium tuberculosis</i>

Tuberculosis (TB), the leading cause of death from bacterial infections worldwide, results from infection with Mycobacterium tuberculosis (Mtb). The antitubercular agents delamanid (DLM) and pretomanid (PMD) are nitroimidazole prodrugs that require activation by an enzyme intrinsic to Mtb; however, the mechanism(s) of action and the associated metabolic pathways are largely unclear. Profiling of the chemical-genetic interactions of PMD and DLM in Mtb using combined CRISPR screening reveals that the mutation of rv2073c increases susceptibility of Mtb to these nitroimidazole drugs both in vitro and in infected mice, whereas mutation of rv0078 increases drug resistance. Further assays show that Rv2073c might confer intrinsic resistance to DLM/PMD by interfering with inhibition of the drug target, decaprenylphophoryl-2-keto-b-D-erythro-pentose reductase (DprE2), by active nicotinamide adenine dinucleotide (NAD) adducts. Characterization of the metabolic pathways of DLM/PMD in Mtb using a combination of chemical genetics and comparative liquid chromatography-mass spectrometry (LC-MS) analysis of DLM/PMD metabolites reveals that Rv0077c, which is negatively regulated by Rv0078, mediates drug resistance by metabolizing activated DLM/PMD. These results might guide development of new nitroimidazole prodrugs and new regimens for TB treatment.