Journal
JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY
Volume 70, Issue 7, Pages 2028-2037Publisher
OXFORD UNIV PRESS
DOI: 10.1093/jac/dkv054
Keywords
mycobacteria; antimycobacterial agents; F1Fo-ATP synthase; TMC207; R207910
Funding
- Lottery Health New Zealand
- University of Otago Research Grants
- Otago Medical Research Foundation
- University of Otago Doctoral Scholarship
- Lottery Health
- Marsden Grant from the Royal Society of New Zealand
- James Cook Fellowship from the Royal Society of New Zealand
- Health Research Council of New Zealand
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Objectives: It is not fully understood why inhibiting ATP synthesis in Mycobacterium species leads to death in non-replicating cells. We investigated the bactericidal mode of action of the anti-tubercular F1Fo-ATP synthase inhibitor bedaquiline (Sirturo (TM)) in order to further understand the lethality of ATP synthase inhibition. Methods: Mycobacterium smegmatis strains were used for all the experiments. Growth and survival during a bedaquiline challenge were performed in multiple media types. A time-course microarray was performed during initial bedaquiline challenge in minimal medium. Oxygen consumption and proton-motive force measurements were performed on whole cells and inverted membrane vesicles, respectively. Results: A killing of 3 log(10) cfu/mL was achieved 4-fold more quickly in minimal medium (a glycerol carbon source) versus rich medium (LB with Tween 80) during bedaquiline challenge. Assessing the accelerated killing condition, we identified a transcriptional remodelling of metabolism that was consistent with respiratory dysfunction but inconsistent with ATP depletion. In glycerol-energized cell suspensions, bedaquiline caused an immediate 2.3-fold increase in oxygen consumption. Bedaquiline collapsed the transmembrane pH gradient, but not the membrane potential, in a dose-dependent manner. Both these effects were dependent on binding to the F1Fo-ATP synthase. Conclusions: Challenge with bedaquiline results in an electroneutral uncoupling of respiration-driven ATP synthesis. This may be a determinant of the bactericidal effects of bedaquiline, while ATP depletion may be a determinant of its delayed onset of killing. We propose that bedaquiline binds to and perturbs the a-c subunit interface of the F-o, leading to futile proton cycling, which is known to be lethal to mycobacteria.
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