Dual inhibition of respiratory complexes in Mycobacterium tuberculosis results in bacterial killing

There is an urgent need for novel drug targets and combination therapies for Mycobacterium tuberculosis. The identification of inhibitors that target the mycobacterial respiratory chain, particularly when used in combination, has emerged as an exciting new avenue for addressing antibiotic resistance. However, the lack of inhibitors for respiratory complexes, possible functional redundancy between homologous enzymes, and a poor knowledge of which complexes, when simultaneously inhibited, have lethal phenotypes, have slowed progress in this space. To address these limitations, we have utilized CRISPR interference (CRISPRi) to identify bioenergetic complexes that are functionally redundant or that, when simultaneously inhibited, lead to cell death. Specifically, single, double, triple, and quadruple gene knockdowns of respiratory chain components were developed to investigate the consequences of transcriptional inhibition on cell viability. In addition to functional redundancy being identified between homologous groups of respiratory enzymes, we identify a network of synthetic lethal interactions, including the demonstration of a synthetic lethal interaction between the terminal oxidase cytochrome bcc_-aa₃ (i.e., qcrB) and malate-quinone oxidoreductase (mqo). In conclusion, these findings reinforce that interactions between bioenergetic complexes are crucial to the growth of M. tuberculosis and represent promising targets for future combination therapies.IMPORTANCENew drugs and combination therapies are urgently needed to treat infections caused by Mycobacterium tuberculosis. Drugs that target the mycobacterial respiratory chain, particularly when used in combination, have emerged as an exciting new avenue for addressing antibiotic resistance. However, functional redundancy between respiratory enzymes and a poor knowledge of which respiratory complexes are simultaneously inhibited have lethal phenotypes, which have slowed the development of drugs in this field. To address these limitations, we have utilized CRISPR interference (CRISPRi) transcriptional knockdowns to identify respiratory complexes that are functionally redundant or that, when simultaneously inhibited, lead to cell death. This work demonstrates that (i) there is functional redundancy between functionally similar respiratory enzymes and (ii) there is a network of lethal interactions. These findings reinforce that interactions between bioenergetic complexes are crucial to the growth of M. tuberculosis and represent promising targets for future combination therapies.