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

Abstract

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.

Impaired Succinate Oxidation Prevents Growth and Influences Drug Susceptibility in Mycobacterium tuberculosis

“… M. tuberculosis with both Sdh1 and Sdh2 transcriptionally depleted was hypersusceptible to growth inhibition and/or killing by other bioenergetic inhibitors, such as BDQ, TB47, and Q203. These findings add to previous reports of synergistic interactions between ETC complexes ( 13 , 61 , 62 , 66 , 69 , 91 – 93 ) and support the proposal that targeting multiple components of mycobacterial energy generation could result in efficacious drug regimens ( 94 ).…”

“…The molecular mechanisms underlying this are unclear, although both enzymes appear to use different reaction mechanisms to drive succinate oxidation ( 35 – 37 ). Moreover, these findings are in contrast to Mycobacterium smegmatis , where Sdh2 is proposed to have a higher affinity for succinate oxidation and is essential, while Sdh1 can be deleted or transcriptionally silenced with no identifiable phenotype ( 34 , 69 ).…”

High‐throughput functional genomics: A (myco)bacterial perspective

“…A major advantage of this system is that it allows for the selection of a subset of targets, keeping the size of a combinatorial library, which grows exponentially per added target, manageable. An early demonstration of this potential includes the use of multiplexed CRISPRi for target identification in mycobacteria (McNeil et al, 2022), confirming synergies identified by drug knockdown in previous studies (Lee et al, 2021). Subsequent work by the same group again used combinatorial CRISPRi to reveal functional redundancies in key drug targets (Adolph et al, 2022;Harold et al, 2022).…”

Recent Advances in Structural Studies of Cytochrome bd and Its Potential Application as a Drug Target

“…Under aerobic conditions, this is a bcc - aa 3 supercomplex composed of cytochrome bcc (complex III or qcrBCD) and the aa 3 -type cytochrome c oxidase (complex IV or ctaBCDE). Under low oxygen concentrations, cytochrome bd (cydAB) serves as a terminal oxidase [ 111 ]. Each of the terminal segments of the respiratory chain generates a proton motive force, albeit with different efficiency, to drive ATP synthesis via the F 1 F o -ATP synthase (atpBEFHAGDC).…”