Transcription profiling of genes encoding components of the respiratory chain and the ATP synthesizing apparatus ofMycobacterium tuberculosiswas conductedin vivoin the infected mouse lung, andin vitroin bacterial cultures subjected to gradual oxygen depletion and to nitric oxide treatment. Transcript levels changed dramatically as infection progressed from bacterial exponential multiplication (acute infection) to cessation of bacterial growth (chronic infection) in response to host immunity. The proton-pumping type-I NADH dehydrogenase and theaa3-type cytochromecoxidase were strongly down-regulated. Concurrently, the less energy-efficient cytochromebdoxidase was transiently up-regulated. The nitrate transporter NarK2 was also up-regulated, indicative of increased nitrate respiration. The reduced efficiency of the respiratory chain was accompanied by decreased expression of ATP synthesis genes. Thus, adaptation ofM. tuberculosisto host immunity involves three successive respiratory states leading to decreased energy production. Decreased bacterial counts in mice infected with acydCmutant (defective in the cytochromebdoxidase-associated transporter) at the transition to chronic infection provided initial evidence that thebdoxidase pathway is required forM. tuberculosisadaptation to host immunity.In vitro, NO treatment and hypoxia caused a switch from transcription of type I to type II NADH dehydrogenase. Moreover, cytochromebdoxidase expression increased, but cytochromecoxidase expression decreased slightly (nitric oxide) or not at all (hypoxia). These specific differences in respiratory metabolism duringM. tuberculosisgrowth arrestin vitroandin vivowill guide manipulation ofin vitroconditions to model bacterial adaptation to host immunity.

Mycobacterium tuberculosis is predicted to subsist on alternative carbon sources during persistence within the human host. Catabolism of odd- and branched-chain fatty acids, branched-chain amino acids, and cholesterol generates propionyl-coenzyme A (CoA) as a terminal, three-carbon (C ₃ ) product. Propionate constitutes a key precursor in lipid biosynthesis but is toxic if accumulated, potentially implicating its metabolism in M. tuberculosis pathogenesis. In addition to the well-characterized methylcitrate cycle, the M. tuberculosis genome contains a complete methylmalonyl pathway, including a mutAB -encoded methylmalonyl-CoA mutase (MCM) that requires a vitamin B ₁₂ -derived cofactor for activity. Here, we demonstrate the ability of M. tuberculosis to utilize propionate as the sole carbon source in the absence of a functional methylcitrate cycle, provided that vitamin B ₁₂ is supplied exogenously. We show that this ability is dependent on mutAB and, furthermore, that an active methylmalonyl pathway allows the bypass of the glyoxylate cycle during growth on propionate in vitro. Importantly, although the glyoxylate and methylcitrate cycles supported robust growth of M. tuberculosis on the C ₁₇ fatty acid heptadecanoate, growth on valerate (C ₅ ) was significantly enhanced through vitamin B ₁₂ supplementation. Moreover, both wild-type and methylcitrate cycle mutant strains grew on B ₁₂ -supplemented valerate in the presence of 3-nitropropionate, an inhibitor of the glyoxylate cycle enzyme isocitrate lyase, indicating an anaplerotic role for the methylmalonyl pathway. The demonstrated functionality of MCM reinforces the potential relevance of vitamin B ₁₂ to mycobacterial pathogenesis and suggests that vitamin B ₁₂ availability in vivo might resolve the paradoxical dispensability of the methylcitrate cycle for the growth and persistence of M. tuberculosis in mice.

Summary: Mycobacterium tuberculosis contains five resuscitation‐promoting factor (Rpf)‐like proteins, RpfA‐E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA‐E genes were shown to be collectively dispensable for growth of M. tuberculosis in broth culture. The defect in resuscitation of multiple mutants from a ‘non‐culturable’ state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild‐type organisms confirming that the phenotype was associated with rpf‐like gene loss and that the ‘non‐culturable’ cells of the mutant strains were viable. Other phenotypes uncovered by sequential deletion mutagenesis revealed a functional differentiation within this protein family. The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent, effects not observed for mutants retaining only rpfE or rpfB. Furthermore, mutants retaining rpfD or rpfE were highly attenuated for growth in mice with the latter persisting better than the former in late‐stage infection. In conjunction, these results are indicative of a hierarchy in terms of function and/or potency with the Rpf family, with RpfB and RpfE ranking above RpfD.

Drug resistant tuberculosis contributes significantly to the global burden of antimicrobial resistance, often consuming a large proportion of the healthcare budget and associated resources in many endemic countries. The rapid emergence of resistance to newer tuberculosis therapies signals the need to ensure appropriate antibiotic stewardship, together with a concerted drive to develop new regimens that are active against currently circulating drug resistant strains. Herein, we highlight that the current burden of drug resistant tuberculosis is driven by a combination of ongoing transmission and the intra-patient evolution of resistance through several mechanisms. Global control of tuberculosis will require interventions that effectively address these and related aspects. Interrupting tuberculosis transmission is dependent on the availability of novel rapid diagnostics which provide accurate results, as near-patient as is possible, together with appropriate linkage to care. Contact tracing, longitudinal follow-up for symptoms and active mapping of social contacts are essential elements to curb further community-wide spread of drug resistant strains. Appropriate prophylaxis for contacts of drug resistant index cases is imperative to limit disease progression and subsequent transmission. Preventing the evolution of drug resistant strains will require the development of shorter regimens that rapidly eliminate all populations of mycobacteria, whilst concurrently limiting bacterial metabolic processes that drive drug tolerance, mutagenesis and the ultimate emergence of resistance. Drug discovery programs that specifically target bacterial genetic determinants associated with these processes will be paramount to tuberculosis eradication. In addition, the development of appropriate clinical endpoints that quantify drug tolerant organisms in sputum, such as differentially culturable/detectable tubercle bacteria is necessary to accurately assess the potential of new therapies to effectively shorten treatment duration. When combined, this holistic approach to addressing the critical problems associated with drug resistance will support delivery of quality care to patients suffering from tuberculosis and bolster efforts to eradicate this disease.

The inherent drug susceptibility of microorganisms is determined by multiple factors, including growth state, the rate of drug diffusion into and out of the cell, and the intrinsic vulnerability of drug targets with regard to the corresponding antimicrobial agent.Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant source of global morbidity and mortality, further exacerbated by its ability to readily evolve drug resistance. It is well accepted that drug resistance inM. tuberculosisis driven by the acquisition of chromosomal mutations in genes encoding drug targets/promoter regions; however, a comprehensive description of the molecular mechanisms that fuel drug resistance in the clinical setting is currently lacking. In this context, there is a growing body of evidence suggesting that active extrusion of drugs from the cell is critical for drug tolerance.M. tuberculosisencodes representatives of a diverse range of multidrug transporters, many of which are dependent on the proton motive force (PMF) or the availability of ATP. This suggests that energy metabolism and ATP production through the PMF, which is established by the electron transport chain (ETC), are critical in determining the drug susceptibility ofM. tuberculosis. In this review, we detail advances in the study of the mycobacterial ETC and highlight drugs that target various components of the ETC. We provide an overview of some of the efflux pumps present inM. tuberculosisand their association, if any, with drug transport and concomitant effects on drug resistance. The implications of inhibiting drug extrusion, through the use of efflux pump inhibitors, are also discussed.

A solvatochromic trehalose dye conjugate allows the fast and specific detection of Mycobacterium tuberculosis in sputum samples.