Hydrogen sulfide dysregulates the immune response by suppressing central carbon metabolism to promote tuberculosis.

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 in Mycobacterium tuberculosis (Mtb) pathogenesis. We showed that Mtb-infected CSE-/- mice survive longer than WT mice, and support reduced pathology and lower bacterial burdens in the lung, spleen, and liver. Similarly, in vitro Mtb infection 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 regulating Mtb survival in macrophages. Furthermore, we demonstrate that CSE promotes an excessive innate immune response, suppresses the adaptive immune response, and reduces circulating IL-1ß, IL-6, TNF-α, and IFN-γ levels in response to Mtb infection. Notably, Mtb infected 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-1ß, 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.

Correction: Mycobacterium tuberculosis arrests host cycle at the G1/S transition to establish long term infection.

[This corrects the article DOI: 10.1371/journal.ppat.1006389.].

Mycobacterium tuberculosis arrests host cycle at the G1/S transition to establish long term infection.

Signals modulating the production of Mycobacterium tuberculosis (Mtb) virulence factors essential for establishing long-term persistent infection are unknown. The WhiB3 redox regulator is known to regulate the production of Mtb virulence factors, however the mechanisms of this modulation are unknown. To advance our understanding of the mechanisms involved in WhiB3 regulation, we performed Mtb in vitro, intraphagosomal and infected host expression analyses. Our Mtb expression analyses in conjunction with extracellular flux analyses demonstrated that WhiB3 maintains bioenergetic homeostasis in response to available carbon sources found in vivo to establish Mtb infection. Our infected host expression analysis indicated that WhiB3 is involved in regulation of the host cell cycle. Detailed cell-cycle analysis revealed that Mtb infection inhibited the macrophage G1/S transition, and polyketides under WhiB3 control arrested the macrophages in the G0-G1 phase. Notably, infection with the Mtb whiB3 mutant or polyketide mutants had little effect on the macrophage cell cycle and emulated the uninfected cells. This suggests that polyketides regulated by Mtb WhiB3 are responsible for the cell cycle arrest observed in macrophages infected with the wild type Mtb. Thus, our findings demonstrate that Mtb WhiB3 maintains bioenergetic homeostasis to produce polyketide and lipid cyclomodulins that target the host cell cycle. This is a new mechanism whereby Mtb modulates the immune system by altering the host cell cycle to promote long-term persistence. This new knowledge could serve as the foundation for new host-directed therapeutic discovery efforts that target the host cell cycle.

Genital and systemic immune effects of the injectable, contraceptive norethisterone enanthate (NET-EN), in South African women.

PROBLEM: Injectable hormonal contraceptives (IHC) have been associated with altered mucosal and systemic milieu which might increase HIV risk, but most studies have focused on DMPA and not NET-EN, despite the growing popularity and lower HIV risk associated with the latter in observational studies. METHOD OF STUDY: We used high-performance liquid chromatography in combination with tandem triple quadrupole mass spectrometry (HPLC-LC-MS/MS) to measure steroid hormones in plasma samples of CAPRISA004 study participants. Concentrations of 48 cytokines were measured in the cervicovaginal lavage (CVL) and plasma, and their expression was compared between participants with detectable NET-EN (n = 201) versus non-detectable IHC (n = 90). Each log10 cytokine concentration was tested as an outcome in linear-mixed models, with NET-EN detection as the main explanatory variable. Multivariable models were adjusted for potential confounders. RESULTS: In bivariate analysis, detectable NET-EN was associated with reduced cervicovaginal M-CSF (P = 0.008), GM-CSF (P = 0.025) and G-CSF (P = 0.039), and elevated levels MIF (P = 0.008), IL-18 (P = 0.011), RANTES (P = 0.005) and IL-1Rα (P < 0.001). Lower G-CSF (P = 0.011) and elevated IL-1Rα (P = 0.008) remained significant in adjusted models. Multivariable analyses of plasma samples obtained from NET-EN-detectable women showed a significant increase in IP-10 (P = 0.026) and reductions in TNF-ß (P = 0.037), RANTES (P = 0.009), and M-CSF (P < 0.001). While similar growth factor reduction in CVL was noted for both DMPA and NET-EN, similar trends were not observed for endogenous progesterone. CONCLUSIONS: Detectable NET-EN was associated with reduced growth factors in the plasma and genital tract; particularly G-CSF and M-CSF. Our results suggest that while NET-EN is not inflammatory, it may have important immunological effects.

Mycobacterium tuberculosis H2S Functions as a Sink to Modulate Central Metabolism, Bioenergetics, and Drug Susceptibility.

H2S is a potent gasotransmitter in eukaryotes and bacteria. Host-derived H2S has been shown to profoundly alter M. tuberculosis (Mtb) energy metabolism and growth. However, compelling evidence for endogenous production of H2S and its role in Mtb physiology is lacking. We show that multidrug-resistant and drug-susceptible clinical Mtb strains produce H2S, whereas H2S production in non-pathogenic M. smegmatis is barely detectable. We identified Rv3684 (Cds1) as an H2S-producing enzyme in Mtb and show that cds1 disruption reduces, but does not eliminate, H2S production, suggesting the involvement of multiple genes in H2S production. We identified endogenous H2S to be an effector molecule that maintains bioenergetic homeostasis by stimulating respiration primarily via cytochrome bd. Importantly, H2S plays a key role in central metabolism by modulating the balance between oxidative phosphorylation and glycolysis, and it functions as a sink to recycle sulfur atoms back to cysteine to maintain sulfur homeostasis. Lastly, Mtb-generated H2S regulates redox homeostasis and susceptibility to anti-TB drugs clofazimine and rifampicin. These findings reveal previously unknown facets of Mtb physiology and have implications for routine laboratory culturing, understanding drug susceptibility, and improved diagnostics.

Irreversible depletion of intestinal CD4+ T cells is associated with T cell activation during chronic HIV infection.

HIV infection in the human gastrointestinal (GI) tract is thought to be central to HIV progression, but knowledge of this interaction is primarily limited to cohorts within Westernized countries. Here, we present a large cohort recruited from high HIV endemic areas in South Africa and found that people living with HIV (PLWH) presented at a younger age for investigation in the GI clinic. We identified severe CD4+ T cell depletion in the GI tract, which was greater in the small intestine than in the large intestine and not correlated with years on antiretroviral treatment (ART) or plasma viremia. HIV-p24 staining showed persistent viral expression, particularly in the colon, despite full suppression of plasma viremia. Quantification of mucosal antiretroviral (ARV) drugs revealed no differences in drug penetration between the duodenum and colon. Plasma markers of gut barrier breakdown and immune activation were elevated irrespective of HIV, but peripheral T cell activation was inversely correlated with loss of gut CD4+ T cells in PLWH alone. T cell activation is a strong predictor of HIV progression and independent of plasma viral load, implying that the irreversible loss of GI CD4+ T cells is a key event in the HIV pathogenesis of PLWH in South Africa, yet the underlying mechanisms remain unknown.

Bedaquiline reprograms central metabolism to reveal glycolytic vulnerability in Mycobacterium tuberculosis.

The approval of bedaquiline (BDQ) for the treatment of tuberculosis has generated substantial interest in inhibiting energy metabolism as a therapeutic paradigm. However, it is not known precisely how BDQ triggers cell death in Mycobacterium tuberculosis (Mtb). Using 13C isotopomer analysis, we show that BDQ-treated Mtb redirects central carbon metabolism to induce a metabolically vulnerable state susceptible to genetic disruption of glycolysis and gluconeogenesis. Metabolic flux profiles indicate that BDQ-treated Mtb is dependent on glycolysis for ATP production, operates a bifurcated TCA cycle by increasing flux through the glyoxylate shunt, and requires enzymes of the anaplerotic node and methylcitrate cycle. Targeting oxidative phosphorylation (OXPHOS) with BDQ and simultaneously inhibiting substrate level phosphorylation via genetic disruption of glycolysis leads to rapid sterilization. Our findings provide insight into the metabolic mechanism of BDQ-induced cell death and establish a paradigm for the development of combination therapies that target OXPHOS and glycolysis.

Plasma concentration of injectable contraceptive correlates with reduced cervicovaginal growth factor expression in South African women.

Long-acting injectable contraceptives have been associated with mucosal immune changes and increased HIV acquisition, but studies have often been hampered by the inaccuracy of self-reported data, unknown timing of injection, and interactions with mucosal transmission co-factors. We used mass spectrometry to quantify the plasma concentrations of injectable contraceptives in women from the CAPRISA004 study (n = 664), with parallel quantification of 48 cytokines and >500 host proteins in cervicovaginal lavage. Higher DMPA levels were associated with reduced CVL concentrations of GCSF, MCSF, IL-16, CTACK, LIF, IL-1α, and SCGF-ß in adjusted linear mixed models. Dose-dependent relationships between DMPA concentration and genital cytokines were frequently observed. Unsupervised clustering of host proteins by DMPA concentration suggest that women with low DMPA had increases in proteins associated with mucosal fluid function, growth factors, and keratinization. Although DMPA was not broadly pro-inflammatory, DMPA was associated with increased IP-10 in HSV-2 seropositive and older women. DMPA-cytokine associations frequently differed by vaginal microbiome; in non-Lactobacillus-dominant women, DMPA was associated with elevated IL-8, MCP-1, and IP-10 concentrations. These data confirm a direct, concentration-dependant effect of DMPA on functionally important immune factors within the vaginal compartment. The biological effects of DMPA may vary depending on age, HSV-2 status, and vaginal microbiome composition.

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.

Mycobacterium tuberculosis induces decelerated bioenergetic metabolism in human macrophages.

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.

A comprehensive characterization of PncA polymorphisms that confer resistance to pyrazinamide.

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.The antibiotic pyrazinamide is central to tuberculosis treatment regimens, globally. Despite its efficacy, resistance to the drug is increasing. Here, Eric Rubin and colleagues characterise the genetic basis of pyrazinamide resistance.

Turning the respiratory flexibility of Mycobacterium tuberculosis against itself.

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.

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.