Abstract
The Mycobacterium tuberculosis (Mtb) electron transport chain (ETC) has received significant attention as a drug target, however its vulnerability may be affected by its flexibility in response to disruption. Here we determine the effect of the ETC inhibitors bedaquiline, Q203 and clofazimine on the Mtb ETC, and the value of the ETC as a drug target, by measuring Mtb's respiration using extracellular flux technology. We find that Mtb's ETC rapidly reroutes around inhibition by these drugs and increases total respiration to maintain ATP levels. Rerouting is possible because Mtb rapidly switches between terminal oxidases, and, unlike eukaryotes, is not susceptible to back pressure. Increased ETC activity potentiates clofazimine's production of reactive oxygen species, causing rapid killing in vitro and in a macrophage model. Our results indicate that combination therapy targeting the ETC can be exploited to enhance killing of Mtb.
Highlights
The Mycobacterium tuberculosis (Mtb) electron transport chain (ETC) has received significant attention as a drug target, its vulnerability may be affected by its flexibility in response to disruption
We examine the effect of CFZ, Q203 and BDQ combinations on cellular toxicity, and Mtb killing in a macrophage infection model
Since Mtb is an obligate aerobe dependent on its ETC, we expected its bioenergetic profile to be affected by these ETC-targeting drugs
Summary
We tested the bioenergetics of Mtb bacilli treated with the ATP synthase inhibitor N,N-dicyclohexylcarbodiimide (DCCD) and found that DCCD caused an increase in OCR (Fig. 3c). We had previously seen that BDQ causes a similar increase in OCR in wt and in Mtb cydKO (Fig. 3a), indicating that the full electron flux can flow through cytochromes bc[1] and aa[3]. We reasoned that increased ETC flux caused by BDQ or Q203 suggests a more reduced ETC, possibly potentiating CFZ’s diversion of electrons to ROS production, which would explain the increased rapid bactericidal activity To test this hypothesis, we first treated wt Mtb bacilli with all drug combinations, measuring NADH and NAD þ levels over 6 days via LC-MS/MS (Fig. 5a). As was observed in vitro, adding CFZ to BDQ and/or Q203 leads to enhanced Mtb killing in a macrophage infection model
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