Drug-resistant strains of Mycobacterium tuberculosis are a major global health problem. Resistance to the front-line antibiotic isoniazid is often associated with mutations in the katG encoded bifunctional catalase-peroxidase. We hypothesised that perturbed KatG activity would generate collateral vulnerabilities in INH-resistant katG mutants, providing new pathways to combat isoniazid resistance. Here, we used whole genome CRISPRi screens, transcriptomics, and metabolomics to generate a genome-wide map of cellular vulnerabilities in a M. tuberculosis katG mutant. We discovered that metabolic and transcriptional remodelling compensates for the loss of KatG but in doing so generates vulnerabilities in ribosome biogenesis, and nucleotide and amino acid metabolism. These vulnerabilities were more sensitive to inhibition in an isoniazid-resistant katG mutant under in vitro and host-relevant conditions and translated to clinical populations. These findings provide an experimental framework for developing novel strategies to combat antimicrobial resistance in M. tuberculosis and other bacterial pathogens.

KatG inactivation generates vulnerabilities in isoniazid resistant strains of Mycobacterium tuberculosis