Turning the respiratory flexibility of Mycobacterium tuberculosis against itself

Introduction

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), kills more people than any other bacterium. TB control is threatened by the continued spread of drug resistance; multi-drug and extensively drug resistant Mtb require longer, more costly, treatment with multiple drugs causing worse side effects and have a lower likelihood of treatment success. The urgent need for better treatment options for drug resistant Mtb has led the World Health Organization to prioritize development of not only new individual antitubercular agents, but also new drug regimens1,2,3,4,5.

Mtb is an obligate aerobe, requiring the use of its flexible, branched electron transport chain (ETC) for energy production via oxidative phosphorylation (OXPHOS)6. Even during hypoxic non-replicating persistence, Mtb uses its ETC to dispose of reducing equivalents and maintain membrane potential7,8, reinforcing the importance of the ETCin vivo. The ETC-targeting diarylquinolone bedaquiline (BDQ) is the first novel antitubercular agent to be FDA approved in 40 years, and has sparked interest in ETC-targeting agents9, although some researchers express concern about the value of the Mtb ETC as a target10. BDQ inhibits ATP synthase (ETC Complex V) by binding to subunit c11, starving the bacteria of ATP12,13. BDQ has been thought to cause ‘back pressure’ on the ETC, inhibiting proton pumping ETC complexes due to increased proton motive force (PMF), and slowing overall metabolic flux6,14,15. However, recently it has been proposed to act by allowing Complex V to act as a proton channel16. The imidazopyridine Q203, another new antitubercular drug, is currently in development. Q203 inhibits cytochrome bc1 (ETC Complex III) by binding to its QcrB subunit17, forcing the bacillus to use the less energetically efficient terminal oxidase, cytochrome bd. Both drugs cause several days of bacteriostasis and ATP depletion before becoming bactericidal. Clofazimine (CFZ) is an older antimycobacterial agent18 with good in vitro activity. CFZ shuttles electrons from the ETC enzyme type 2 NADH dehydrogenase (NDH2) to O2, generating bactericidal reactive oxygen species (ROS)19. Interest in CFZ for TB treatment continues as recent trials have evaluated CFZ in combination with other anti-tuberculosis drugs in animal20,21 models and in human22,23 clinical trials.

Energy production pathways are tightly regulated using multiple feedback loops to maintain energy homoeostasis24,25. Mtb undergoes metabolic remodelling in response to BDQ, although this has not been well-characterized14. Even less is known about Mtb’s metabolic response to Q203 and CFZ. The combination of multiple feedback loops and a flexible ETC may cause complex and even surprising responses to perturbation of one part of the system.

To clarify the ETC’s value as a drug target, Mtb’s bioenergetics response to ETC targeting must be better understood. For this purpose, we use extracellular flux analysis technology26, inverted membrane vesicle (IMV) experiments, flow cytometry and time kill curves, with wild–type (wt) and selected mutant strains of Mtb, to investigate the direct effects of ETC-targeting drugs and the downstream repercussions of ETC perturbation. We also examine the effect of CFZ, Q203 and BDQ combinations on cellular toxicity, and Mtb killing in a macrophage infection model. Together, our data shed light into the complex effects of ETC targeting and identify potential strategies for combination-targeting of the ETC to achieve synergistic rapid killing.

Results

BDQ and Q203 increase Mtb respiration

To determine the effect of BDQ, Q203 and CFZ on Mtb’s bioenergetics, we used extracellular flux (XF) analysis technology (Fig. 1a) to measure Mtb’s oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in real time as markers of OPHOS and carbon catabolism (Fig. 1b), respectively27. By adding inhibitors and substrates during each experiment, we can measure actual and maximum rates of activity of different components of energy-generating pathways.

Figure 1: Diagram of the Seahorse XF Analyzer, its function and the initial bioenergetics analysis of Mtb in the presence of the ETC inhibitors.

(a) Compounds are delivered into microplate wells via drug ports. When the probe is lowered, a transient microchamber is formed above a monolayer of bacilli. Dissolved O2 and pH are monitored by sensing probes. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) are calculated from these measurements by the instrument software. (b) ECAR represents carbon catabolism and TCA cycle activity, which produce reducing equivalents that enter the ETC. Reducing equivalents pass through NDH2 or other dehydrogenases (DHs) to the menaquinone pool (MK), and then through Complexes III (cytochrome bc1) and IV (cytochrome aa3), or through cytochrome bd to O2. This contributes to the PMF, which powers ATP synthesis by Complex V (ATP synthase). CFZ acts on NDH2. Q203 inhibits Complex III. BDQ inhibits Complex V. (c) Bioenergetic analysis of Mtb. At the indicated times, 2 g l−1 of glucose (Glc) was added, followed by BDQ, Q203, CFZ, or other drugs, followed by the uncoupler CCCP to stimulate maximum respiration. BDQ and Q203, unlike CFZ or standard antimycobacterial drugs, induce an increase in bacterial respiration, above that of their respective vehicle controls. OCR and ECAR are indicated as a percentage of baseline values. Standard deviation of three replicate wells are indicated as calculated by the Seahorse XF Wave software. One representative experiment is shown; for ETC targeting drugs, at least three replicate experiments were performed. The following inter-experiment % CVs were calculated using Microsoft Excel (Microsoft Office 2010): basal OCR 47.2±5.2; % CV 11.1 (n=6), increased OCR after BDQ addition; 129.7±5.2, % CV 4.1 (n=4) and increased OCR after Q203 addition; 104.1±11.1, % CV 10.7 (n=4). The absolute value OCR profiles are shown in Supplementary Fig. 1. During optimization it was determined that fewer than 5% of the bacteria seeded into microplate wells were dislodged from the bottom during the experiment. (d) O2 depletion from microchamber sustained for 2 h. Each condition was repeated at least four times; representative traces are shown.