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Mycobacterium tuberculosis (Mtb) disrupts glycolytic flux in infected myeloid cells through an unclear mechanism. Flux through the glycolytic pathway in myeloid cells is inextricably linked to the availability of NAD+, which is maintained by NAD+ salvage and lactate metabolism. Using lung tissue from tuberculosis (TB) patients and myeloid deficient LDHA (LdhaLysM−/−) mice, we demonstrate that glycolysis in myeloid cells is essential for protective immunity in TB. Glycolytic myeloid cells are essential for the early recruitment of multiple classes of immune cells and the protective effects of IFNγ. We identified NAD+ depletion as central to the glycolytic inhibition caused by Mtb. Lastly, we show that the NAD+ precursor nicotinamide exerts a host-directed, antimycobacterial effect, and that nicotinamide prophylaxis and treatment reduces Mtb lung burden in vivo. These findings provide new insight into how Mtb alters host metabolism through perturbation of NAD(H) homeostasis and reprogramming of glycolysis, highlighting this pathway as a potential therapeutic target.

The mechanisms whereby Mycobacterium tuberculosis (Mtb) rewires the host metabolism in vivo are surprisingly unexplored. Here, we used three high-resolution mass spectrometry platforms to track altered lung metabolic changes associated with Mtb infection of mice. The multiplatform data sets were merged using consensus orthogonal partial least squares-discriminant analysis (cOPLS-DA), an algorithm that allows for the joint interpretation of the results from a single multivariate analysis. We show that Mtb infection triggers a temporal and progressive catabolic state to satisfy the continuously changing energy demand to control infection. This causes dysregulation of metabolic and oxido-reductive pathways culminating in Mtb-associated wasting. Notably, high abundances of trimethylamine-N-oxide (TMAO), produced by the host from the bacterial metabolite trimethylamine upon infection, suggest that Mtb could exploit TMAO as an electron acceptor under anaerobic conditions. Overall, these new pathway alterations advance our understanding of the link between Mtb pathogenesis and metabolic dysregulation and could serve as a foundation for new therapeutic intervention strategies. Mass spectrometry data has been deposited in the Metabolomics Workbench repository (data-set identifier: ST001328).

Hydrogen sulfide (H2S) is involved in numerous pathophysiological processes and shares overlapping functions with CO and •NO. However, the importance of host-derived H2S in microbial pathogenesis is unknown. Here we show that Mtb-infected mice deficient in the H2S-producing enzyme cystathionine β-synthase (CBS) survive longer with reduced organ burden, and that pharmacological inhibition of CBS reduces Mtb bacillary load in mice. High-resolution respirometry, transcriptomics and mass spectrometry establish that H2S stimulates Mtb respiration and bioenergetics predominantly via cytochrome bd oxidase, and that H2S reverses •NO-mediated inhibition of Mtb respiration. Further, exposure of Mtb to H2S regulates genes involved in sulfur and copper metabolism and the Dos regulon. Our results indicate that Mtb exploits host-derived H2S to promote growth and disease, and suggest that host-directed therapies targeting H2S production may be potentially useful for the management of tuberculosis and other microbial infections., The importance of host-produced hydrogen sulfide (H2S) in microbial pathogenesis is poorly understood. Here, Saini et al. show that H2S alters Mycobacterium tuberculosis (Mtb) central metabolism, stimulates respiration to promote growth and TB disease, and upregulates the Dos regulon.

Mycobacterium tuberculosis (Mtb) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H2S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H2S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we tested the hypothesis that Mtb senses and responds to H2S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H2S binds directly to the ferric (Fe3+) heme of DosS (KD = 5.64 µM) but not the ferrous (Fe2+) form. No interaction with DosT was detected. Thus, the mechanism by which DosS senses H2S is different from that for sensing NO and CO, which bind only the ferrous forms of DosS and DosT. Steered Molecular Dynamics simulations show that H2S, and not the charged HS- species, can enter the DosS heme pocket. We also show that H2S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H2S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H2S in macrophages can induce Dos regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H2S, via DosS-Fe3+. These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase.Significance StatementHydrogen sulfide (H2S) is an important signaling molecule in eukaryotes and bacteria, and along with CO and NO, is an important part of host defense against Mycobacterium tuberculosis (Mtb). However, the mechanism(s) by which Mtb senses and responds to H2S is unknown. Here, we report that the Mtb heme sensor kinase DosS, a known sensor of CO and NO, is also a sensor of H2S. We found that H2S binds DosS in its ferric (Fe3+) state, which is considered as its inactive state, to induce the Dos dormancy regulon during infection. These data highlight the unusual capacity of Mtb to sense multiple gasotransmitters through a single sensing protein.

Metabolism plays an important role in the activation and effector functions of macrophages. Intracellular pathogens, such as Mycobacterium tuberculosis, subvert the immune functions of macrophages to establish an infection by modulating the metabolism of the macrophage. Here, we describe how the Seahorse Extracellular Flux Analyzer (XF) from Agilent Technologies can be used to study the changes in the bioenergetic metabolism of the macrophages induced by infection with mycobacteria. The XF simultaneously measures the oxygen consumption and extracellular acidification of the macrophages noninvasively in real time, and together with the addition of metabolic modulators, substrates, and inhibitors enables measurements of the rates of oxidative phosphorylation, glycolysis, and ATP production.

Significance: L-ergothioneine is synthesized in actinomycetes, cyanobacteria, methylobacteria, and some fungi. In contrast to other low-molecular-weight redox buffers, glutathione and mycothiol, ergothioneine is primarily present as a thione rather than a thiol at physiological pH, which makes it resistant to autoxidation. Ergothioneine regulates microbial physiology and enables the survival of microbes under stressful conditions encountered in their natural environments. In particular, ergothioneine enables pathogenic microbes, such as Mycobacterium tuberculosis (Mtb), to withstand hostile environments within the host to establish infection. Recent Advances: Ergothioneine has been reported to maintain bioenergetic homeostasis in Mtb and protect Mtb against oxidative stresses, thereby enhancing the virulence of Mtb in a mouse model. Furthermore, ergothioneine augments the resistance of Mtb to current frontline anti-TB drugs. Recently, an opportunistic fungus, Aspergillus fumigatus, which infects immunocompromised individuals, has been found to produce ergothioneine, which is important in conidial health and germination, and contributes to the fungal resistance against redox stresses. Critical Issues: The molecular mechanisms of the functions of ergothioneine in microbial physiology and pathogenesis are poorly understood. It is currently not known if ergothioneine is used in detoxification or antioxidant enzymatic pathways. As ergothioneine is involved in bioenergetic and redox homeostasis and antibiotic susceptibility of Mtb, it is of utmost importance to advance our understanding of these mechanisms. Future Directions: A clear understanding of the role of ergothioneine in microbes will advance our knowledge of how this thione enhances microbial virulence and resistance to the host's defense mechanisms to avoid complete eradication. Antioxid. Redox Signal. 28, 431–444.

Metabolism plays an important role in the activation and effector functions of macrophages. Intracellular pathogens, such as Mycobacterium tuberculosis, subvert the immune functions of macrophages to establish an infection by modulating the metabolism of the macrophage. Here, we describe how the Seahorse Extracellular Flux Analyzer (XF) from Agilent Technologies can be used to study the changes in the bioenergetic metabolism of the macrophages induced by infection with mycobacteria. The XF simultaneously measures the oxygen consumption and extracellular acidification of the macrophages noninvasively in real time, and together with the addition of metabolic modulators, substrates, and inhibitors enables measurements of the rates of oxidative phosphorylation, glycolysis, and ATP production.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, encounters variable and hostile environments within the host. A major component of these hostile conditions is reductive and oxidative stresses induced by factors modified by the host immune response, such as oxygen tension, NO or CO gases, reactive oxygen and nitrogen intermediates, the availability of different carbon sources and changes in pH. It is therefore essential for Mtb to continuously monitor and appropriately respond to the microenvironment. To this end, Mtb has developed various redox-sensitive systems capable of monitoring its intracellular redox environment and coordinating a response essential for virulence. Various aspects of Mtb physiology are regulated by these systems, including drug susceptibility, secretion systems, energy metabolism and dormancy. While great progress has been made in understanding the mechanisms and pathways that govern the response of Mtb to the host's redox environment, many questions in this area remain unanswered. The answers to these questions are promising avenues for addressing the tuberculosis crisis., Mycobacterium tuberculosis senses and responds to redox stresses within the host to survive and cause disease.

Summary Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that controls inflammatory responses and redox homeostasis; however, its role during pulmonary tuberculosis (TB) remains unclear. Using freshly resected human TB lung tissue, we examined the role of HO-1 within the cellular and pathological spectrum of TB. Flow cytometry and histopathological analysis of human TB lung tissues showed that HO-1 is expressed primarily in myeloid cells and that HO-1 levels in these cells were directly proportional to cytoprotection. HO-1 mitigates TB pathophysiology by diminishing myeloid cell-mediated oxidative damage caused by reactive oxygen and/or nitrogen intermediates, which control granulocytic karyorrhexis to generate a zonal HO-1 response. Using whole-body or myeloid-specific HO-1-deficient mice, we demonstrate that HO-1 is required to control myeloid cell infiltration and inflammation to protect against TB progression. Overall, this study reveals that zonation of HO-1 in myeloid cells modulates free-radical-mediated stress, which regulates human TB immunopathology., Graphical Abstract, Highlights • HO-1 is expressed in distinct microanatomic zones within the human TB lung • Macrophages and neutrophils are the major producers of HO-1 in human TB lungs • In diseased areas of human TB lungs, myeloid cells have low HO-1 and high ROS and RNS • Mice lacking HO-1 expression in myeloid cells are more susceptible to Mtb infection, Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that controls inflammation and redox homeostasis; however, its role in tuberculosis (TB) is unclear. Using freshly resected human lung tissue and HO-1-deficient mice, Chinta et al. demonstrate that HO-1 in myeloid cells is important for controlling inflammatory and free-radical-mediated tissue damage in TB.

SummaryThe 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. Last, 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.

Drug efflux pumps have been contributing factor(s) in the development of multidrug resistance in various clinically relevant bacteria. During efflux pump gene expression studies on mycobacteria, we have found a previously uncharacterized open reading frame (ORF) Rv2459 to be overexpressed in drug stressed conditions. The objective of the present study was to investigate the role of this ORF as a drug efflux pump, which might add new information in our understanding about the alternative mechanisms of drug resistance in mycobacteria.The open reading frame Rv2459 of Mycobacterium tuberculosis encoding a probable drug efflux protein has been cloned using pSD5 E.coli-Mycobacterium shuttle vector and overexpressed in M. tuberculosis H(37)Rv. This ORF was named as jefA. Overexpression of this gene in clones has been verified by real-time reverse transcription PCR. Minimum inhibitory concentrations (MICs) of recombinant as well as non-recombinant clones were determined by resazurin microtitre assay plate method (REMA) with and without efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and verapamil.In recombinant strains of M. tuberculosis, the overexpression of this gene led to an increase in MIC of anti-tubercular drugs isoniazid and ethambutol when tested by REMA. In the presence of CCCP and verapamil, the recombinant strains showed decrease in MIC for these drugs. Bioinformatic analysis has shown a close relation of JefA protein with drug efflux pumps of other clinically relevant bacteria. In homology derived structure prepared from nearest available model, it was observed that amino acids forming TMH 1, 8 and 11 participated in ethambutol specificity and those forming TMH 2, 7 and 10 participated in isoniazid specificity in JefA.The increased transcription of jefA leads to increased resistance to ethambutol and isoniazid in M. tuberculosis via efflux pump like mechanism and contributes in the development of resistance to these drugs. JefA amino acid sequence is well conserved among clinically important bacterial genera, which further provides evidence of being a potent drug efflux pump. The involvement in drug resistance and very little homology with any of the human proteins makes JefA important to be included in the list of potential drug targets.