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.

SUMMARY The mechanisms by which Mycobacterium tuberculosis (Mtb) maintains metabolic equilibrium to survive during infection and upon exposure to antimycobacterial drugs are poorly characterized. Ergothioneine (EGT) and mycothiol (MSH) are the major redox buffers present in Mtb, but the contribution of EGT to Mtb redox homeostasis and virulence remains unknown. We report that Mtb WhiB3, a 4Fe-4S redox sensor protein, regulates EGT production and maintains bioenergetic homeostasis. We show that central carbon metabolism and lipid precursors regulate EGT production and that EGT modulates drug sensitivity. Notably, EGT and MSH are both essential for redox and bioenergetic homeostasis. Transcriptomic analyses of EGT and MSH mutants indicate overlapping, but distinct functions of EGT and MSH. Lastly, we show that EGT is critical for Mtb survival in both macrophages and mice. This study has uncovered a dynamic balance between Mtb redox and bioenergetic homeostasis, which critically influences Mtb drug susceptibility and pathogenicity.

How Mycobacterium tuberculosis (Mtb) rewires macrophage energy metabolism to facilitate survival is poorly characterized. Here, we used extracellular flux analysis to simultaneously measure the rates of glycolysis and respiration in real time. Mtb infection induced a quiescent energy phenotype in human monocyte-derived macrophages and decelerated flux through glycolysis and the TCA cycle. In contrast, infection with the vaccine strain, M. bovis BCG, or dead Mtb induced glycolytic phenotypes with greater flux. Furthermore, Mtb reduced the mitochondrial dependency on glucose and increased the mitochondrial dependency on fatty acids, shifting this dependency from endogenous fatty acids in uninfected cells to exogenous fatty acids in infected macrophages. We demonstrate how quantifiable bioenergetic parameters of the host can be used to accurately measure and track disease, which will enable rapid quantifiable assessment of drug and vaccine efficacy. Our findings uncover new paradigms for understanding the bioenergetic basis of host metabolic reprogramming by Mtb.

Hydrogen sulfide (H 2 S) is involved in numerous pathophysiological processes and shares overlapping functions with CO and •NO. However, the importance of host-derived H 2 S in microbial pathogenesis is unknown. Here we show that Mtb-infected mice deficient in the H 2 S-producing enzyme cystathionine β-synthase (CBS) survive longer with reduced organ burden, and that pharmacological inhibition of CBS reduces Mtb bacillary load in mice. Highresolution respirometry, transcriptomics and mass spectrometry establish that H 2 S stimulates Mtb respiration and bioenergetics predominantly via cytochrome bd oxidase, and that H 2 S reverses •NO-mediated inhibition of Mtb respiration. Further, exposure of Mtb to H 2 S regulates genes involved in sulfur and copper metabolism and the Dos regulon. Our results indicate that Mtb exploits host-derived H 2 S to promote growth and disease, and suggest that host-directed therapies targeting H 2 S production may be potentially useful for the management of tuberculosis and other microbial infections.

fThe antileprosy drug clofazimine has shown potential for shortening tuberculosis treatment; however, the current dosing of the drug is not evidence based, and the optimal dosing is unknown. Our objective was to conduct a preclinical evaluation of the pharmacokinetics and pharmacodynamics of clofazimine in the mouse model of tuberculosis, with the goal of providing useful information on dosing for future studies. Pharmacokinetic parameters were evaluated in infected and uninfected BALB/c mice. Pharmacodynamic parameters were evaluated in Mycobacterium tuberculosis-infected mice that were treated for 12 weeks with one of six different clofazimine dosing regimens, i.e., doses of 6.25, 12.5, and 25 mg/kg of body weight/day and 3 regimens with loading doses. Clofazimine progressively accumulated in the lungs, livers, and spleens of the mice, reaching levels of greater than 50 g/g in all tissues by 4 weeks of administration, while serum drug levels remained low at 1 to 2 g/ml. Elimination of clofazimine was extremely slow, and the half-life was dependent on the duration of drug administration. Clofazimine exhibited dosedependent tissue and serum concentrations. At any dose, clofazimine did not have bactericidal activity during the first 2 weeks of administration but subsequently demonstrated potent, dose-independent bactericidal activity. The antituberculosis activity of clofazimine was dependent on neither the dose administered nor the drug concentrations in the tissues, suggesting that much lower doses could be effectively used for tuberculosis treatment. C lofazimine (CFZ) is a phenazine dye developed in the 1950s for the treatment of tuberculosis (TB) (1, 2). Despite promising activity against Mycobacterium tuberculosis both in vitro and in vivo, clofazimine was not advanced as a TB drug but instead became incorporated decades later into the multidrug treatment of leprosy, where it remains a key drug (3, 4). Interest in clofazimine for TB treatment has been revitalized following the 2010 report by Van Deun and colleagues that a clofazimine-containing regimen not only was highly effective for the treatment of multidrug-resistant (MDR) TB, but was also associated with a significantly decreased duration of therapy (5); 9 months of a clofazimine-containing regimen resulted in 87.9% relapse-free cure, which is in sharp contrast to the limited efficacy (less than 50% relapse-free cure) of the World Health Organization (WHO)-recommended, at least 20-month-long MDR-TB treatment regimen (6, 7). This report was complemented by experimental chemotherapy studies in which the inclusion of clofazimine in first-and secondline regimens significantly shortened the duration of treatment needed for relapse-free cure in mouse models of drug-susceptible and MDR TB, respectively (8, 9). These clinical and preclinical data suggest that clofazimine has great potential for TB treatment.Because clofazimine was abandoned as an option for TB treatment shortly after its discovery, and also because the use of clofazimine for leprosy is not ba...

The ubiquitous gasotransmitter hydrogen sulfide (H2S) has been recognized to play a crucial role in human health. Using cystathionine γ-lyase (CSE)-deficient mice, we demonstrate an unexpected role of H2S inMycobacterium tuberculosis(Mtb) pathogenesis. We showed thatMtb-infected CSE−/−mice survive longer than WT mice, and support reduced pathology and lower bacterial burdens in the lung, spleen, and liver. Similarly, in vitroMtbinfection of macrophages resulted in reduced colony forming units in CSE−/−cells. Chemical complementation of infected WT and CSE−/−macrophages using the slow H2S releaser GYY3147 and the CSE inhibitor DL-propargylglycine demonstrated that H2S is the effector molecule regulatingMtbsurvival in macrophages. Furthermore, we demonstrate that CSE promotes an excessive innate immune response, suppresses the adaptive immune response, and reduces circulating IL-1β, IL-6, TNF-α, and IFN-γ levels in response toMtbinfection. Notably,Mtbinfected CSE−/−macrophages show increased flux through glycolysis and the pentose phosphate pathway, thereby establishing a critical link between H2S and central metabolism. Our data suggest that excessive H2S produced by the infected WT mice reduce HIF-1α levels, thereby suppressing glycolysis and production of IL-1β, IL-6, and IL-12, and increasing bacterial burden. Clinical relevance was demonstrated by the spatial distribution of H2S-producing enzymes in human necrotic, nonnecrotic, and cavitary pulmonary tuberculosis (TB) lesions. In summary, CSE exacerbates TB pathogenesis by altering immunometabolism in mice and inhibiting CSE or modulating glycolysis are potential targets for host-directed TB control.

Tuberculosis chemotherapy is dependent on the use of the antibiotic pyrazinamide, which is being threatened by emerging drug resistance. Resistance is mediated through mutations in the bacterial gene pncA. Methods for testing pyrazinamide susceptibility are difficult and rarely performed, and this means that the full spectrum of pncA alleles that confer clinical resistance to pyrazinamide is unknown. Here, we performed in vitro saturating mutagenesis of pncA to generate a comprehensive library of PncA polymorphisms resultant from a single-nucleotide polymorphism. We then screened it for pyrazinamide resistance both in vitro and in an infected animal model. We identify over 300 resistance-conferring substitutions. Strikingly, these mutations map throughout the PncA structure and result in either loss of enzymatic activity and/or decrease in protein abundance. Our comprehensive mutational and screening approach should stand as a paradigm for determining resistance mutations and their mechanisms of action.

A key drug for the treatment of leprosy, clofazimine has recently been associated with highly effective and significantly shortened regimens for the treatment of multidrug-resistant tuberculosis (TB). Consequently, we hypothesized that clofazimine may also shorten the duration of treatment for drug-susceptible TB. We conducted a controlled trial in the mouse model of TB chemotherapy comparing the activity of the 6-mo standard regimen for TB treatment, i.e., 2 mo of daily rifampin, isoniazid, pyrazinamide, and ethambutol followed by 4 mo of rifampin and isoniazid, with a 4-mo clofazimine-containing regimen: 2 mo of daily rifampin, isoniazid, pyrazinamide, and clofazimine followed by 2 mo of rifampin, isoniazid, and clofazimine. Treatment efficacy was assessed on the basis of Mycobacterium tuberculosis colony counts in the lungs and spleens during treatment and on the proportion of mice with culture-positive relapse 6 mo after treatment cessation. No additive effect of clofazimine was observed after the first week of treatment, but, by the second week of treatment, the colony counts were significantly lower in the clofazimine-treated mice than in the mice receiving the standard regimen. Lung culture conversion was obtained after 3 and 5 mo in mice treated with the clofazimine-containing and standard regimens, respectively, and relapse-free cure was obtained after 3 and 6 mo of treatment with the clofazimine-containing and standard regimens, respectively. Thus, clofazimine is a promising anti-TB drug with the potential to shorten the duration of TB chemotherapy by at least half (3 mo vs. 6 mo) in the mouse model of TB.lofazimine is a riminophenazine dye developed in the 1950s by Vincent Barry and colleagues for the treatment of tuberculosis (TB) (1, 2). Despite being highly active against Mycobacterium tuberculosis in vitro and in mice, a limited number of studies led to the belief that clofazimine would be poorly active in patients with TB (3), and this drug was thus considered not useful for TB treatment. However, clofazimine was subsequently found to be highly effective against multibacillary leprosy and, for decades, has been, and continues to be, one of the key drugs in the World Health Organization (WHO)-recommended multidrug regimen for the treatment of this disease (4). Because of the increasing frequency of multidrug-resistant (MDR) TB, the search for new active drugs for the treatment of this disease has included a reassessment of the anti-TB activity of clofazimine. As a part of this reassessment, clofazimine was included in several different second-line regimens that were evaluated as possible standardized combinations for the treatment of MDR TB (5). The most effective regimen identified in this evaluation was a 9-mo, clofazimine-containing drug combination that resulted in an 87.9% relapse-free cure rate, a significant improvement from the WHO-recommended treatment, which is a minimum of 20 mo in duration and associated with only a 48% success rate in patients (6). Thus, the result of this observation...