Abstract
New drugs are urgently required to combat the tuberculosis epidemic that claims over 1.5 million lives annually. The identification of bedaquiline (BDQ) as an inhibitor of the mycobacterial F1Fo ATP synthase and its subsequent clinical success has validated mycobacterial energy generation as a high priority target space for antitubercular drug development. However, continued development of this promising target space requires an increased understanding of the mechanisms Mycobacterium tuberculosis uses to generate energy, as well as the identification of novel drugs and drug targets within the mycobacterial respiratory chain.
Succinate dehydrogenase (SDH), or complex II of the electron transport chain, couples the two-electron oxidation of succinate, a TCA cycle metabolite, to the reduction of quinone (succinate + quinone ↔ fumarate + quinol), thereby directly linking central carbon metabolism to the respiratory chain. M. tuberculosis encodes multiple enzymes predicted to be capable of catalyzing succinate oxidation including two different succinate dehydrogenase enzymes, Sdh1 and Sdh2, as well as a separate fumarate reductase (Frd) with possible bi-directional behavior. The dual role of SDH within both the TCA cycle and respiratory chain of M. tuberculosis makes it a particularly attractive drug target. However, the roles and essentiality of the succinate dehydrogenase and fumarate reductase enzymes encoded by M. tuberculosis are poorly defined, and no candidate inhibitors of the mycobacterial succinate dehydrogenase have been identified.
Previous investigations into the roles and essentiality of three SDH / FRD enzymes encoded by M. tuberculosis have been limited by the use of single deletion mutants, which are unable to overcome the functional redundancy in SDH catalysis. We address this by utilizing a recently developed mycobacterial CRISPR Interference system to create single, double, and triple transcriptional knockdowns of sdh1, sdh2, and frd in M. tuberculosis. We show that succinate oxidation can be catalyzed by either Sdh1 or Sdh2, but not Frd, and demonstrate that impaired succinate oxidation prevents growth and dysregulates respiration in M. tuberculosis. Moreover, our results suggest a requirement for succinate oxidation in resistance to isoniazid and pretomanid.
Furthermore, we identify and characterize the mechanism of action of a putative succinate dehydrogenase inhibitor; BB2-50F. We demonstrate that BB2-50F has rapid bactericidal activity against M. tuberculosis and show that it binds to the catalytic subunit of SDH. Using metabolomic and transcriptomic profiling of BB2-50F-treated M. tuberculosis and M. smegmatis, we propose that the mechanism of action of BB2-50F involves the dysregulation of succinate metabolism and the production of reactive oxygen species.
Overall, this thesis provides fundamental insights into the essentiality of succinate metabolism in mycobacteria and validates succinate dehydrogenase as a potential drug target in M. tuberculosis.