NAD(H) homeostasis underlies host protection mediated by glycolytic myeloid cells in tuberculosis.

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 IFNγ-mediated protection. We identify NAD+ depletion as central to the glycolytic inhibition caused by Mtb. Lastly, we show that the NAD+ precursor nicotinamide exerts a host-dependent, antimycobacterial effect, and that nicotinamide prophylaxis and treatment reduce Mtb lung burden in mice. These findings provide 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.

Water-soluble tocopherol derivatives inhibit SARS-CoV-2 RNA-dependent RNA polymerase.

The recent emergence of a novel coronavirus, SARS-CoV-2, has led to the global pandemic of the severe disease COVID-19 in humans. While efforts to quickly identify effective antiviral therapies have focused largely on repurposing existing drugs 1-4 , the current standard of care, remdesivir, remains the only authorized antiviral intervention of COVID-19 and provides only modest clinical benefits 5 . Here we show that water-soluble derivatives of α-tocopherol have potent antiviral activity and synergize with remdesivir as inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Through an artificial-intelligence-driven in silico screen and in vitro viral inhibition assay, we identified D-α-tocopherol polyethylene glycol succinate (TPGS) as an effective antiviral against SARS-CoV-2 and ß-coronaviruses more broadly that also displays strong synergy with remdesivir. We subsequently determined that TPGS and other water-soluble derivatives of α-tocopherol inhibit the transcriptional activity of purified SARS-CoV-2 RdRp and identified affinity binding sites for these compounds within a conserved, hydrophobic interface between SARS-CoV-2 nonstructural protein 7 and nonstructural protein 8 that is functionally implicated in the assembly of the SARS-CoV-2 RdRp 6 . In summary, we conclude that solubilizing modifications to α-tocopherol allow it to interact with the SARS-CoV-2 RdRp, making it an effective antiviral molecule alone and even more so in combination with remdesivir. These findings are significant given that many tocopherol derivatives, including TPGS, are considered safe for humans, orally bioavailable, and dramatically enhance the activity of the only approved antiviral for SARS-CoV-2 infection 7-9 .

Heme Oxygenase-1 as a Pharmacological Target for Host-Directed Therapy to Limit Tuberculosis Associated Immunopathology.

Excessive inflammation and tissue damage are pathological hallmarks of chronic pulmonary tuberculosis (TB). Despite decades of research, host regulation of these clinical consequences is poorly understood. A sustained effort has been made to understand the contribution of heme oxygenase-1 (HO-1) to this process. HO-1 is an essential cytoprotective enzyme in the host that controls inflammation and oxidative stress in many pathological conditions. While HO-1 levels are upregulated in animals and patients infected with Mycobacterium tuberculosis (Mtb), how it regulates host responses and disease pathology during TB remains unclear. This lack of clarity is due in part to contradictory studies arguing that HO-1 induction contributes to both host resistance as well as disease progression. In this review, we discuss these conflicting studies and the role of HO-1 in modulating myeloid cell functions during Mtb disease progression. We argue that HO-1 is a promising target for host-directed therapy to improve TB immunopathology.

Hydrogen sulfide stimulates Mycobacterium tuberculosis respiration, growth and pathogenesis.

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 ß-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.

Microanatomic Distribution of Myeloid Heme Oxygenase-1 Protects against Free Radical-Mediated Immunopathology in Human Tuberculosis.

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.

Host-pathogen redox dynamics modulate Mycobacterium tuberculosis pathogenesis.

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.

Ferritin H Deficiency in Myeloid Compartments Dysregulates Host Energy Metabolism and Increases Susceptibility to Mycobacterium tuberculosis Infection.

Iron is an essential factor for the growth and virulence of Mycobacterium tuberculosis (Mtb). However, little is known about the mechanisms by which the host controls iron availability during infection. Since ferritin heavy chain (FtH) is a major intracellular source of reserve iron in the host, we hypothesized that the lack of FtH would cause dysregulated iron homeostasis to exacerbate TB disease. Therefore, we used knockout mice lacking FtH in myeloid-derived cell populations to study Mtb disease progression. We found that FtH plays a critical role in protecting mice against Mtb, as evidenced by increased organ burden, extrapulmonary dissemination, and decreased survival in Fth-/- mice. Flow cytometry analysis showed that reduced levels of FtH contribute to an excessive inflammatory response to exacerbate disease. Extracellular flux analysis showed that FtH is essential for maintaining bioenergetic homeostasis through oxidative phosphorylation. In support of these findings, RNAseq and mass spectrometry analyses demonstrated an essential role for FtH in mitochondrial function and maintenance of central intermediary metabolism in vivo. Further, we show that FtH deficiency leads to iron dysregulation through the hepcidin-ferroportin axis during infection. To assess the clinical significance of our animal studies, we performed a clinicopathological analysis of iron distribution within human TB lung tissue and showed that Mtb severely disrupts iron homeostasis in distinct microanatomic locations of the human lung. We identified hemorrhage as a major source of metabolically inert iron deposition. Importantly, we observed increased iron levels in human TB lung tissue compared to healthy tissue. Overall, these findings advance our understanding of the link between iron-dependent energy metabolism and immunity and provide new insight into iron distribution within the spectrum of human pulmonary TB. These metabolic mechanisms could serve as the foundation for novel host-directed strategies.

Role of Ergothioneine in Microbial Physiology and Pathogenesis.

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.

The emerging role of gasotransmitters in the pathogenesis of tuberculosis.

Mycobacterium tuberculosis (Mtb) is a facultative intracellular pathogen and the second largest contributor to global mortality caused by an infectious agent after HIV. In infected host cells, Mtb is faced with a harsh intracellular environment including hypoxia and the release of nitric oxide (NO) and carbon monoxide (CO) by immune cells. Hypoxia, NO and CO induce a state of in vitro dormancy where Mtb senses these gases via the DosS and DosT heme sensor kinase proteins, which in turn induce a set of ∼47 genes, known as the Mtb Dos dormancy regulon. On the contrary, both iNOS and HO-1, which produce NO and CO, respectively, have been shown to be important against mycobacterial disease progression. In this review, we discuss the impact of O2, NO and CO on Mtb physiology and in host responses to Mtb infection as well as the potential role of another major endogenous gas, hydrogen sulfide (H2S), in Mtb pathogenesis.

Ergothioneine Maintains Redox and Bioenergetic Homeostasis Essential for Drug Susceptibility and Virulence of Mycobacterium tuberculosis.

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. 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.