PrrAB is a Selective Therapeutic Target and Regulator of Respiration in Mycobacterium tuberculosis

The essentiality of the PrrAB two-component system (TCS) in Mycobacterium tuberculosis positions it as a promising therapeutic target, prompting investigation into its potential as a universal drug target across mycobacterial species. To evaluate its essentiality and regulatory influence across mycobacterial species, we leveraged CRISPR interference (CRISPRi) to systematically investigate PrrAB function and its interaction with diarylthiazole-48 (DAT-48), an experimental tuberculocidal agent. Strong CRISPRi-mediated knockdown of M. tuberculosis prrAB reproduced a lethal knockout phenotype, whereas knockdown of Mycobacterium abscessus prrAB1 and/or prrAB2 had no effect on growth, highlighting functional divergence between orthologs. Strikingly, M. tuberculosis prrAB repression universally downregulated respiratory chain genes, cementing its role as a master regulator of mycobacterial respiration. DAT-48 displayed potent, sterilizing effects against laboratory and clinical M. tuberculosis strains (H37Rv 4 μg/mL; Mt103 16 μg/mL) but lacked efficacy against M. abscessus ATCC19977 Smooth morphotype (MIC > 128 μg/mL), further highlighting species-specific PrrAB dependency. Notably, prrAB repression halved M. tuberculosis DAT-48 MICs, whereas overexpression increased MICs 8-fold, demonstrating a direct functional link. Molecular docking predicted DAT-48 binding to the ATP pocket of the PrrB sensor kinase, and DAT-48 synergized with bedaquiline (FICI = 0.42) and telacebec (FICI = 0.33), reinforcing PrrAB's central role in mycobacterial respiration. These findings establish PrrAB as a central regulator of energy metabolism in M. tuberculosis but dispensable in M. abscessus, defining it as a highly selective and promising target for next-generation TB therapeutics.