Multiplexed transcriptional repression identifies a network of bactericidal interactions between mycobacterial respiratory complexes

Mycobacterium tuberculosis remains a leading cause of infectious disease morbidity and mortality for which new drug combination therapies are needed. Combinations of respiratory inhibitors can have synergistic or synthetic lethal interactions with sterilizing activity, suggesting that regimens with multiple bioenergetic inhibitors could shorten treatment times. However, realizing this potential requires an understanding of which combinations of respiratory complexes, when inhibited, have the strongest consequences on bacterial growth and viability. Here we have used multiplex CRISPR interference (CRISPRi) and Mycobacterium smegmatis as a physiological and molecular model for mycobacterial respiration to identify interactions between respiratory complexes. In this study, we identified synthetic lethal and synergistic interactions between respiratory complexes and demonstrated how the engineering of CRISPRi-guide sequences can be used to further explore networks of interacting gene pairs. These results provide fundamental insights into the functions of and interactions between bioenergetic complexes and the utility of CRISPRi in designing drug combinations.