Bedaquiline, an inhibitor of the mycobacterial ATP synthase, has revolutionized the treatment of Mycobacterium tuberculosis infection. Although a potent inhibitor, it is characterized by poorly understood delayed time-dependent bactericidal activity. Here, we demonstrate that in contrast to bedaquiline, the transcriptional inhibition of the ATP synthase in M. tuberculosis and Mycobacterium smegmatis has rapid bactericidal activity. These results validate the mycobacterial ATP synthase as a drug target with the potential for rapid bactericidal activity.
Summary
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.
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 suggesting that regimens with multiple bioenergetic inhibitors will drastically shorten treatment times. However, realizing this potential is hampered by a lack of on-target inhibitors and a poor understanding of which inhibitor combinations have the strongest interactions. To overcome these limitations, we have used CRISPR interference (CRISPRi) to characterize the consequences of transcriptionally inhibiting individual respiratory complexes and identify bioenergetic complexes that when simultaneously inhibited result in cell death. In this study, we identified known and novel synthetic lethal interactions and demonstrate 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.
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