Hepatitis B virus e antigen induces atypical metabolism and differentially regulates programmed cell deaths of macrophages.

Macrophages can undergo M1-like proinflammatory polarization with low oxidative phosphorylation (OXPHOS) and high glycolytic activities or M2-like anti-inflammatory polarization with the opposite metabolic activities. Here we show that M1-like macrophages induced by hepatitis B virus (HBV) display high OXPHOS and low glycolytic activities. This atypical metabolism induced by HBV attenuates the antiviral response of M1-like macrophages and is mediated by HBV e antigen (HBeAg), which induces death receptor 5 (DR5) via toll-like receptor 4 (TLR4) to induce death-associated protein 3 (DAP3). DAP3 then induces the expression of mitochondrial genes to promote OXPHOS. HBeAg also enhances the expression of glutaminases and increases the level of glutamate, which is converted to α-ketoglutarate, an important metabolic intermediate of the tricarboxylic acid cycle, to promote OXPHOS. The induction of DR5 by HBeAg leads to apoptosis of M1-like and M2-like macrophages, although HBeAg also induces pyroptosis of the former. These findings reveal novel activities of HBeAg, which can reprogram mitochondrial metabolism and trigger different programmed cell death responses of macrophages depending on their phenotypes to promote HBV persistence.

Bacterial Protein Reshapes Host Defense toward Antiviral Responses.

In this issue of Molecular Cell, Penn et al. (2018) report the protein interactome between Mycobacterium tuberculosis (Mtb) secreted effectors and macrophage cytosolic proteins. This Resource reveals that the interaction of Mtb effector LpqN with host CBL counteracts antibacterial defense but causes a reciprocal enhancement of antiviral defense.

Long-term cell fate and functional maintenance of human hepatocyte through stepwise culture configuration.

Human hepatocyte culture system represents by far the most physiologically relevant model for our understanding of liver biology and diseases; however, its versatility has been limited due to the rapid and progressive loss of genuine characteristics, indicating the inadequacy of in vitro milieu for fate maintenance. This study, therefore, is designed to define environmental requirements necessary to sustain the homeostasis of terminally differentiated hepatocytes. Our study reveals that the supplementation of dimethyl sulfoxide (DMSO) is indispensable in mitigating fate deterioration and promoting adaptation to the in vitro environment, resulting in the restoration of tight cell-cell contact, cellular architecture, and polarity. The morphological recovery was overall accompanied by the restoration of hepatocyte marker gene expression, highlighting the interdependence between the cellular architecture and the maintenance of cell fate. However, beyond the recovery phase culture, DMSO supplementation is deemed detrimental due to the potent inhibitory effect on a multitude of hepatocyte functionalities while its withdrawal results in the loss of cell fate. In search of DMSO substitute, our screening of organic substances led to the identification of dimethyl sulfone (DMSO2), which supports the long-term maintenance of proper morphology, marker gene expression, and hepatocytic functions. Moreover, hepatocytes maintained DMSO2 exhibited clinically relevant toxicity in response to prolonged exposure to xenobiotics as well as alcohol. These observations suggest that the stepwise culture configuration consisting of the consecutive supplementation of DMSO and DMSO2 confers the microenvironment essential for the fate and functional maintenance of terminally differentiated human hepatocytes.

Phosphoenolpyruvate depletion mediates both growth arrest and drug tolerance of Mycobacterium tuberculosis in hypoxia.

Mycobacterium tuberculosis (Mtb) infection is difficult to treat because Mtb spends the majority of its life cycle in a nonreplicating (NR) state. Since NR Mtb is highly tolerant to antibiotic effects and can mutate to become drug resistant (DR), our conventional tuberculosis (TB) treatment is not effective. Thus, a novel strategy to kill NR Mtb is required. Accumulating evidence has shown that repetitive exposure to sublethal doses of antibiotics enhances the level of drug tolerance, implying that NR Mtb is formed by adaptive metabolic remodeling. As such, metabolic modulation strategies to block the metabolic remodeling needed to form NR Mtb have emerged as new therapeutic options. Here, we modeled in vitro NR Mtb using hypoxia, applied isotope metabolomics, and revealed that phosphoenolpyruvate (PEP) is nearly completely depleted in NR Mtb. This near loss of PEP reduces PEP-carbon flux toward multiple pathways essential for replication and drug sensitivity. Inversely, supplementing with PEP restored the carbon flux and the activities of the foregoing pathways, resulting in growth and heightened drug susceptibility of NR Mtb, which ultimately prevented the development of DR. Taken together, PEP depletion in NR Mtb is associated with the acquisition of drug tolerance and subsequent emergence of DR, demonstrating that PEP treatment is a possible metabolic modulation strategy to resensitize NR Mtb to conventional TB treatment and prevent the emergence of DR.

Oncogenic human herpesvirus hijacks proline metabolism for tumorigenesis.

Three-dimensional (3D) cell culture is well documented to regain intrinsic metabolic properties and to better mimic the in vivo situation than two-dimensional (2D) cell culture. Particularly, proline metabolism is critical for tumorigenesis since pyrroline-5-carboxylate (P5C) reductase (PYCR/P5CR) is highly expressed in various tumors and its enzymatic activity is essential for in vitro 3D tumor cell growth and in vivo tumorigenesis. PYCR converts the P5C intermediate to proline as a biosynthesis pathway, whereas proline dehydrogenase (PRODH) breaks down proline to P5C as a degradation pathway. Intriguingly, expressions of proline biosynthesis PYCR gene and proline degradation PRODH gene are up-regulated directly by c-Myc oncoprotein and p53 tumor suppressor, respectively, suggesting that the proline-P5C metabolic axis is a key checkpoint for tumor cell growth. Here, we report a metabolic reprogramming of 3D tumor cell growth by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Metabolomic analyses revealed that KSHV infection increased nonessential amino acid metabolites, specifically proline, in 3D culture, not in 2D culture. Strikingly, the KSHV K1 oncoprotein interacted with and activated PYCR enzyme, increasing intracellular proline concentration. Consequently, the K1-PYCR interaction promoted tumor cell growth in 3D spheroid culture and tumorigenesis in nude mice. In contrast, depletion of PYCR expression markedly abrogated K1-induced tumor cell growth in 3D culture, not in 2D culture. This study demonstrates that an increase of proline biosynthesis induced by K1-PYCR interaction is critical for KSHV-mediated transformation in in vitro 3D culture condition and in vivo tumorigenesis.

Δ20 IFITM2 differentially restricts X4 and R5 HIV-1.

CCR5 (R5)-tropic, but not CXCR4 (X4)-tropic, HIV-1 is associated with primary HIV-1 infection and transmission. Recent studies have shown that IFN-induced transmembrane (IFITM) proteins, including IFITM1, IFITM2, and IFITM3, restrict a broad range of viruses. Here, we demonstrate that an IFITM2 isoform (Δ20 IFITM2) lacking 20 amino acids at the N terminus differentially restricts X4 and R5 HIV-1. Δ20 IFITM2 suppresses replication of X4 HIV-1 strains by inhibiting their entry. High levels of Δ20 IFITM2 expression could be detected in CD4+ T cells and in monocytes. Infection of X4 viruses in monocyte-derived macrophages and dendritic cells is enhanced upon depletion of IFITM2 isoforms. Furthermore, we also show that coreceptor use is the determining factor for differential HIV-1 restriction of Δ20 IFITM2. When we replace the C terminus of CCR5 with the C terminus of CXCR4, R5 viruses become more susceptible to Δ20 IFITM2-mediated restriction. In contrast to previous studies, our research reveals that neither X4 nor R5 HIV-1 is suppressed by IFITM2 and IFITM3. The multifactor gatekeeping model has been proposed to explain restriction of X4 viruses in the early stage of HIV-1 diseases. Our findings indicate that Δ20 IFITM2 may serve as a major contributor to this gatekeeping mechanism.

Glyoxylate detoxification is an essential function of malate synthase required for carbon assimilation in Mycobacterium tuberculosis.

The glyoxylate shunt is a metabolic pathway of bacteria, fungi, and plants used to assimilate even-chain fatty acids (FAs) and has been implicated in persistence of Mycobacterium tuberculosis (Mtb). Recent work, however, showed that the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), may mediate survival of Mtb during the acute and chronic phases of infection in mice through physiologic functions apart from fatty acid metabolism. Here, we report that malate synthase (MS), the second enzyme of the glyoxylate shunt, is essential for in vitro growth and survival of Mtb on even-chain fatty acids, in part, for a previously unrecognized activity: mitigating the toxicity of glyoxylate excess arising from metabolism of even-chain fatty acids. Metabolomic profiling revealed that MS-deficient Mtb cultured on fatty acids accumulated high levels of the ICL aldehyde endproduct, glyoxylate, and increased levels of acetyl phosphate, acetoacetyl coenzyme A (acetoacetyl-CoA), butyryl CoA, acetoacetate, and ß-hydroxybutyrate. These changes were indicative of a glyoxylate-induced state of oxaloacetate deficiency, acetate overload, and ketoacidosis. Reduction of intrabacterial glyoxylate levels using a chemical inhibitor of ICL restored growth of MS-deficient Mtb, despite inhibiting entry of carbon into the glyoxylate shunt. In vivo depletion of MS resulted in sterilization of Mtb in both the acute and chronic phases of mouse infection. This work thus identifies glyoxylate detoxification as an essential physiologic function of Mtb malate synthase and advances its validation as a target for drug development.

Methylcitrate cycle defines the bactericidal essentiality of isocitrate lyase for survival of Mycobacterium tuberculosis on fatty acids.

Few mutations attenuate Mycobacterium tuberculosis (Mtb) more profoundly than deletion of its isocitrate lyases (ICLs). However, the basis for this attenuation remains incompletely defined. Mtb's ICLs are catalytically bifunctional isocitrate and methylisocitrate lyases required for growth on even and odd chain fatty acids. Here, we report that Mtb's ICLs are essential for survival on both acetate and propionate because of its methylisocitrate lyase (MCL) activity. Lack of MCL activity converts Mtb's methylcitrate cycle into a "dead end" pathway that sequesters tricarboxylic acid (TCA) cycle intermediates into methylcitrate cycle intermediates, depletes gluconeogenic precursors, and results in defects of membrane potential and intrabacterial pH. Activation of an alternative vitamin B12-dependent pathway of propionate metabolism led to selective corrections of TCA cycle activity, membrane potential, and intrabacterial pH that specifically restored survival, but not growth, of ICL-deficient Mtb metabolizing acetate or propionate. These results thus resolve the biochemical basis of essentiality for Mtb's ICLs and survival on fatty acids.

Effects of fluconazole on the metabolomic profile of Candida albicans.

BACKGROUND: Little is known about the effects of fluconazole on the metabolism of Candida albicans. We performed LC/MS-based metabolomic profiling of the response of C. albicans cells to increasing doses of fluconazole. METHODS: C. albicans cells were cultured to mid-logarithmic growth phase in liquid medium and then inoculated in replicate on to nitrocellulose filters under vacuum filtration. Organisms were cultured to mid-logarithmic growth phase and treated with 0-4 mg/L fluconazole. Following metabolic quenching at mid-logarithmic growth phase, intracellular metabolites were extracted and analysed by LC/MS. Changes in pool sizes of individual metabolites were verified by Student's t-test, adjusted for multiple hypothesis testing by Benjamini-Hochberg correction. Distribution of metabolites was analysed by the Kyoto Encyclopedia of Genes and Genomes metabolic pathways database. RESULTS: We reproducibly detected 64 metabolites whose identities were confirmed by comparison against a pure standard and a library of accurate mass-retention time pairs. These 64 metabolites were broadly representative of eukaryotic central metabolic pathways. Among them 12 had their mean abundance significantly altered in response to increasing fluconazole concentrations. Pool sizes of four intermediates of central carbon metabolism (α-ketoglutarate, glucose-6-phosphate, phenylpyruvate and ribose-5-phosphate) and mevalonate were increased by 0.5-1.5-fold (P ≤ 0.05). Five amino acids (glycine, proline, tryptophan, aminoisobutanoate and asparagine) and guanine were decreased by 0.5-0.75-fold (P ≤ 0.05). CONCLUSIONS: Fluconazole treatment of C. albicans resulted in increased central carbon and decreased amino acid synthesis intermediates, suggesting a rerouting of metabolic pathways. The function of these metabolomic changes remains to be elucidated; however, they may represent previously unrecognized mechanisms of metabolic injury induced by fluconazole against C. albicans.

Inactivation of fructose-1,6-bisphosphate aldolase prevents optimal co-catabolism of glycolytic and gluconeogenic carbon substrates in Mycobacterium tuberculosis.

Metabolic pathways used by Mycobacterium tuberculosis (Mtb) to establish and maintain infections are important for our understanding of pathogenesis and the development of new chemotherapies. To investigate the role of fructose-1,6-bisphosphate aldolase (FBA), we engineered an Mtb strain in which FBA levels were regulated by anhydrotetracycline. Depletion of FBA resulted in clearance of Mtb in both the acute and chronic phases of infection in vivo, and loss of viability in vitro when cultured on single carbon sources. Consistent with prior reports of Mtb's ability to co-catabolize multiple carbon sources, this in vitro essentiality could be overcome when cultured on mixtures of glycolytic and gluconeogenic carbon sources, enabling generation of an fba knockout (Δfba). In vitro studies of Δfba however revealed that lack of FBA could only be compensated for by a specific balance of glucose and butyrate in which growth and metabolism of butyrate were determined by Mtb's ability to co-catabolize glucose. These data thus not only evaluate FBA as a potential drug target in both replicating and persistent Mtb, but also expand our understanding of the multiplicity of in vitro conditions that define the essentiality of Mtb's FBA in vivo.

Multifunctional essentiality of succinate metabolism in adaptation to hypoxia in Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a chronic, facultative intracellular pathogen that spends the majority of its decades-long life cycle in a non- or slowly replicating state. However, the bacterium remains poised to resume replicating so that it can transmit itself to a new host. Knowledge of the metabolic adaptations used to facilitate entry into and exit from nonreplicative states remains incomplete. Here, we apply (13)C-based metabolomic profiling to characterize the activity of M. tuberculosis tricarboxylic acid cycle during adaptation to and recovery from hypoxia, a physiologically relevant condition associated with nonreplication. We show that, as M. tuberculosis adapts to hypoxia, it slows and remodels its tricarboxylic acid cycle to increase production of succinate, which is used to flexibly sustain membrane potential, ATP synthesis, and anaplerosis, in response to varying degrees of O2 limitation and the presence or absence of the alternate electron acceptor nitrate. This remodeling is mediated by the bifunctional enzyme isocitrate lyase acting in a noncanonical role distinct from fatty acid catabolism. Isocitrate lyase-dependent production of succinate affords M. tuberculosis with a unique and bioenergetically efficient metabolic means of entry into and exit from hypoxia-induced quiescence.

Mycobacterium dormancy and antibiotic tolerance within the retinal pigment epithelium of ocular tuberculosis.

Tuberculosis (TB) is a leading cause of death among infectious diseases worldwide due to latent TB infection, which is the critical step for the successful pathogenic cycle. In this stage, Mycobacterium tuberculosis resides inside the host in a dormant and antibiotic-tolerant state. Latent TB infection can also lead to multisystemic diseases because M. tuberculosis invades virtually all organs, including ocular tissues. Ocular tuberculosis (OTB) occurs when the dormant bacilli within the ocular tissues reactivate, originally seeded by hematogenous spread from pulmonary TB. Histological evidence suggests that retinal pigment epithelium (RPE) cells play a central role in immune privilege and in protection from antibiotic effects, making them an anatomical niche for invading M. tuberculosis. RPE cells exhibit high tolerance to environmental redox stresses, allowing phagocytosed M. tuberculosis bacilli to maintain viability in a dormant state. However, the microbiological and metabolic mechanisms determining the interaction between the RPE intracellular environment and phagocytosed M. tuberculosis are largely unknown. Here, liquid chromatography-mass spectrometry metabolomics were used to illuminate the metabolic state within RPE cells reprogrammed to harbor dormant M. tuberculosis bacilli and enhance antibiotic tolerance. Timely and accurate diagnosis as well as efficient chemotherapies are crucial in preventing the poor visual outcomes of OTB patients. Unfortunately, the efficacy of current methods is highly limited. Thus, the results will lead to propose a novel therapeutic option to synthetically kill the dormant M. tuberculosis inside the RPE cells by modulating the phenotypic state of M. tuberculosis and laying the foundation for a new, innovative regimen for treating OTB. IMPORTANCE: Understanding the metabolic environment within the retinal pigment epithelium (RPE) cells altered by infection with Mycobacterium tuberculosis and mycobacterial dormancy is crucial to identify new therapeutic methods to cure ocular tuberculosis. The present study showed that RPE cellular metabolism is altered to foster intracellular M. tuberculosis to enter into the dormant and drug-tolerant state, thereby blunting the efficacy of anti-tuberculosis chemotherapy. RPE cells serve as an anatomical niche as the cells protect invading bacilli from antibiotic treatment. LC-MS metabolomics of RPE cells after co-treatment with H2O2 and M. tuberculosis infection showed that the intracellular environment within RPE cells is enriched with a greater level of oxidative stress. The antibiotic tolerance of intracellular M. tuberculosis within RPE cells can be restored by a metabolic manipulation strategy such as co-treatment of antibiotic with the most downstream glycolysis metabolite, phosphoenolpyruvate.

Mycobacterium dormancy and antibiotic tolerance within the retinal pigment epithelium of ocular tuberculosis.

Tuberculosis (TB) is a leading cause of death among infectious diseases worldwide due to latent TB infection, which is the critical step for the successful pathogenic cycle. In this stage, Mycobacterium tuberculosis resides inside the host in a dormant and antibiotic-tolerant state. Latent TB infection can lead to a multisystemic diseases because M. tuberculosis invades virtually all organs, including ocular tissues. Ocular tuberculosis (OTB) occurs when the dormant bacilli within ocular tissues reactivate, originally seeded by hematogenous spread from pulmonary TB. Timely and accurate diagnosis as well as efficient chemotherapies are crucial in preventing poor visual outcomes of OTB patients. Histological evidence suggests that retinal pigment epithelium (RPE) cells play a central role in immune privilege and in the protection from the antibiotic effects, making them an anatomical niche for invading M. tuberculosis . RPE cells exhibit high tolerance to environmental redox stresses, allowing phagocytosed M. tuberculosis bacilli to maintain viability in a dormant state. However, the microbiological and metabolic mechanisms determining the interaction between the RPE intracellular environment and phagocytosed M. tuberculosis are largely unknown. Here, liquid chromatography mass spectrometry (LC-MS) metabolomics was used to illuminate the metabolic state within RPE cells reprogrammed to harbor dormant M. tuberculosis bacilli and enhance the antibiotic tolerance. The results have led to propose a novel therapeutic option to synthetically kill the dormant M. tuberculosis inside the RPE cells by modulating the phenotypic state of M. tuberculosis , thus laying the foundation for a new, innovative regimen for treating OTB. Importance: Understanding the metabolic environment within the retinal pigment epithelium (RPE) cells altered by infection with M. tuberculosis and mycobacterial dormancy is crucial to identify new therapeutic methods to cure OTB. The present study showed that RPE cellular metabolism is altered to foster intracellular M. tuberculosis to enter into the dormant and drug tolerant state, thereby blunting the efficacy of anti-TB chemotherapy. RPE cells serve as an anatomical niche as the cells protect invading bacilli from antibiotic treatment. LC-MS metabolomics of RPE cells after co-treatment with H2O2 and M. tuberculosis infection showed that intracellular environment within RPE cells is enriched with greater level of oxidative stress. The antibiotic tolerance of intracellular M. tuberculosis within RPE cells can be restored by a metabolic manipulation strategy such as co-treatment of antibiotic with the most downstream glycolysis metabolite, phosphoenolpyruvate.

NOTCH localizes to mitochondria through the TBC1D15-FIS1 interaction and is stabilized via blockade of E3 ligase and CDK8 recruitment to reprogram tumor-initiating cells.

The P53-destabilizing TBC1D15-NOTCH protein interaction promotes self-renewal of tumor-initiating stem-like cells (TICs); however, the mechanisms governing the regulation of this pathway have not been fully elucidated. Here, we show that TBC1D15 stabilizes NOTCH and c-JUN through blockade of E3 ligase and CDK8 recruitment to phosphodegron sequences. Chromatin immunoprecipitation (ChIP-seq) analysis was performed to determine whether TBC1D15-dependent NOTCH1 binding occurs in TICs or non-TICs. The TIC population was isolated to evaluate TBC1D15-dependent NOTCH1 stabilization mechanisms. The tumor incidence in hepatocyte-specific triple knockout (Alb::CreERT2;Tbc1d15Flox/Flox;Notch1Flox/Flox;Notch2Flox/Flox;HCV-NS5A) Transgenic (Tg) mice and wild-type mice was compared after being fed an alcohol-containing Western diet (WD) for 12 months. The NOTCH1-TBC1D15-FIS1 interaction resulted in recruitment of mitochondria to the perinuclear region. TBC1D15 bound to full-length NUMB and to NUMB isoform 5, which lacks three Ser phosphorylation sites, and relocalized NUMB5 to mitochondria. TBC1D15 binding to NOTCH1 blocked CDK8- and CDK19-mediated phosphorylation of the NOTCH1 PEST phosphodegron to block FBW7 recruitment to Thr-2512 of NOTCH1. ChIP-seq analysis revealed that TBC1D15 and NOTCH1 regulated the expression of genes involved in mitochondrial metabolism-related pathways required for the maintenance of TICs. TBC1D15 inhibited CDK8-mediated phosphorylation to stabilize NOTCH1 and protect it from degradation The NUMB-binding oncoprotein TBC1D15 rescued NOTCH1 from NUMB-mediated ubiquitin-dependent degradation and recruited NOTCH1 to the mitochondrial outer membrane for the generation and expansion of liver TICs. A NOTCH-TBC1D15 inhibitor was found to inhibit NOTCH-dependent pathways and exhibited potent therapeutic effects in PDX mouse models. This unique targeting of the NOTCH-TBC1D15 interaction not only normalized the perinuclear localization of mitochondria but also promoted potent cytotoxic effects against TICs to eradicate patient-derived xenografts through NOTCH-dependent pathways.

Targeting Mycobacterium tuberculosis Persistence through Inhibition of the Trehalose Catalytic Shift.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death worldwide by infectious disease. Treatment of Mtb infection requires a six-month course of multiple antibiotics, an extremely challenging regimen necessitated by Mtb's ability to form drug-tolerant persister cells. Mtb persister formation is dependent on the trehalose catalytic shift, a stress-responsive metabolic remodeling mechanism in which the disaccharide trehalose is liberated from cell surface glycolipids and repurposed as an internal carbon source to meet energy and redox demands. Here, using a biofilm-persister model, metabolomics, and cryo-electron microscopy (EM), we found that azidodeoxy- and aminodeoxy-d-trehalose analogues block the Mtb trehalose catalytic shift through inhibition of trehalose synthase TreS (Rv0126), which catalyzes the isomerization of trehalose to maltose. Out of a focused eight-member compound panel constructed by chemoenzymatic synthesis, the natural product 2-trehalosamine exhibited the highest potency and significantly potentiated first- and second-line TB drugs in broth culture and macrophage infection assays. We also report the first structure of TreS bound to a substrate analogue inhibitor, obtained via cryo-EM, which revealed conformational changes likely essential for catalysis and inhibitor binding that can potentially be exploited for future therapeutic development. Our results demonstrate that inhibition of the trehalose catalytic shift is a viable strategy to target Mtb persisters and advance trehalose analogues as tools and potential adjunctive therapeutics for investigating and targeting mycobacterial persistence.

TXNIP-mediated crosstalk between oxidative stress and glucose metabolism.

Thioredoxin-interacting protein (TXNIP) has emerged as a key player in cancer and diabetes since it targets thioredoxin (TRX)-mediated redox regulation and glucose transporter (GLUT)-mediated metabolism. TXNIP consists of two arrestin (ARR, N-ARR and C-ARR) domains at its amino-terminus and two PPxY (PY) motifs and a di-leucine (LL) motif for endocytosis at its carboxyl-terminus. Here, we report that TXNIP shuffles between TRX and GLUTs to regulate homeostasis of intracellular oxidative stress and glucose metabolism. While TXNIP functions as a gatekeeper of TRX by default, it robustly interacted with class I GLUTs through its C-ARR domain upon increase of intracellular reactive oxygen species. This interaction prompted the surface expression downregulation and lysosomal degradation of GLUTs by its carboxyl-terminal LL endocytic signaling motif to attenuate glucose uptake. Consequently, TXNIP expression significantly limited glucose uptake, leading to the suppression of glycolysis, hexosamine biosynthesis, and the pentose phosphate pathway. Our findings establish a fundamental link between ROS and glucose metabolism through TXNIP and provide a promising target for the drug development against GLUT-related metabolic disorders.

Evaluating the sensitivity of Mycobacterium tuberculosis to biotin deprivation using regulated gene expression.

In the search for new drug targets, we evaluated the biotin synthetic pathway of Mycobacterium tuberculosis (Mtb) and constructed an Mtb mutant lacking the biotin biosynthetic enzyme 7,8-diaminopelargonic acid synthase, BioA. In biotin-free synthetic media, ΔbioA did not produce wild-type levels of biotinylated proteins, and therefore did not grow and lost viability. ΔbioA was also unable to establish infection in mice. Conditionally-regulated knockdown strains of Mtb similarly exhibited impaired bacterial growth and viability in vitro and in mice, irrespective of the timing of transcriptional silencing. Biochemical studies further showed that BioA activity has to be reduced by approximately 99% to prevent growth. These studies thus establish that de novo biotin synthesis is essential for Mtb to establish and maintain a chronic infection in a murine model of TB. Moreover, these studies provide an experimental strategy to systematically rank the in vivo value of potential drug targets in Mtb and other pathogens.

Metabolically distinct roles of NAD synthetase and NAD kinase define the essentiality of NAD and NADP in Mycobacterium tuberculosis.

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated derivative (NADP) are essential cofactors that participate in hundreds of biochemical reactions and have emerged as therapeutic targets in cancer, metabolic disorders, neurodegenerative diseases, and infections, including tuberculosis. The biological basis for the essentiality of NAD(P) in most settings, however, remains experimentally unexplained. Here, we report that inactivation of the terminal enzyme of NAD synthesis, NAD synthetase (NadE), elicits markedly different metabolic and microbiologic effects than those of the terminal enzyme of NADP biosynthesis, NAD kinase (PpnK), in Mycobacterium tuberculosis (Mtb). Inactivation of NadE led to parallel reductions of both NAD and NADP pools and Mtb viability, while inactivation of PpnK selectively depleted NADP pools but only arrested growth. Inactivation of each enzyme was accompanied by metabolic changes that were specific for the affected enzyme and associated microbiological phenotype. Bacteriostatic levels of NAD depletion caused a compensatory remodeling of NAD-dependent metabolic pathways in the absence of an impact on NADH/NAD ratios, while bactericidal levels of NAD depletion resulted in a disruption of NADH/NAD ratios and inhibition of oxygen respiration. These findings reveal a previously unrecognized physiologic specificity associated with the essentiality of two evolutionarily ubiquitous cofactors. IMPORTANCE The current course for cure of Mycobacterium tuberculosis (Mtb)-the etiologic agent of tuberculosis (TB)-infections is lengthy and requires multiple antibiotics. The development of shorter, simpler treatment regimens is, therefore, critical to the goal of eradicating TB. NadE, an enzyme required for the synthesis of the ubiquitous cofactor NAD, is essential for survival of Mtb and regarded as a promising drug target. However, the basis of this essentiality was not clear due to its role in the synthesis of both NAD and NADP. Here, we resolve this ambiguity through a combination of gene silencing and metabolomics. We specifically show that NADP deficiency is bacteriostatic, while NAD deficiency is bactericidal due to its role in Mtb's respiratory capacity. These results argue for a prioritization of NAD biosynthesis inhibitors in anti-TB drug development.

Central carbon metabolism remodeling as a mechanism to develop drug tolerance and drug resistance in Mycobacterium tuberculosis.

Suboptimal efficacy of the current antibiotic regimens and frequent emergence of antibiotic-resistant Mycobacterium tuberculosis (Mtb), an etiological agent of tuberculosis (TB), render TB the world's deadliest infectious disease before the COVID-19 outbreak. Our outdated TB treatment method is designed to eradicate actively replicating populations of Mtb. Unfortunately, accumulating evidence suggests that a small population of Mtb can survive antimycobacterial pressure of antibiotics by entering a "persister" state (slowly replicating or non-replicating and lacking a stably heritable antibiotic resistance, termed drug tolerance). The formation of drug-tolerant Mtb persisters is associated with TB treatment failure and is thought to be an adaptive strategy for eventual development of permanent genetic mutation-mediated drug resistance. Thus, the molecular mechanisms behind persister formation and drug tolerance acquisition are a source of new antibiotic targets to eradicate both Mtb persisters and drug-resistant Mtb. As Mtb persisters are genetically identical to antibiotic susceptible populations, metabolomics has emerged as a vital biochemical tool to differentiate these populations by determining phenotypic shifts and metabolic reprogramming. Metabolomics, which provides detailed insights into the molecular basis of drug tolerance and resistance in Mtb, has unique advantages over other techniques by its ability to identify specific metabolic differences between the two genetically identical populations. This review summarizes the recent advances in our understanding of the metabolic adaptations used by Mtb persisters to achieve intrinsic drug tolerance and facilitate the emergence of drug resistance. These findings present metabolomics as a powerful tool to identify previously unexplored antibiotic targets and improved combinations of drug regimens against drug-resistant TB infection.

Fatty acid metabolism of Mycobacterium tuberculosis: A double-edged sword.

Unlike other heterotrophic bacteria, Mycobacterium tuberculosis (Mtb) can co-catabolize a range of carbon sources simultaneously. Evolution of Mtb within host nutrient environment allows Mtb to consume the host's fatty acids as a main carbon source during infection. The fatty acid-induced metabolic advantage greatly contributes to Mtb's pathogenicity and virulence. Thus, the identification of key enzymes involved in Mtb's fatty acid metabolism is urgently needed to aid new drug development. Two fatty acid metabolism enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and isocitrate lyase (ICL) have been intensively studied as promising drug targets, but recently, Quinonez et al. (mBio, doi: 10.1128/mbio.03559-21) highlighted a link between the fatty acid-induced dormancy-like state and drug tolerance. Using metabolomics profiling of a PEPCK-deficient mutant, Quinonez et al. identified that over-accumulation of methylcitrate cycle (MCC) intermediates are phenotypically associated with enhanced drug tolerance against first- and second- line TB antibiotics. This finding was further corroborated by metabolomics and phenotypic characterization of Mtb mutants lacking either ICL or 2-methylcitrate dehydratase. Fatty acid metabolism induced drug-tolerance was also recapitulated in wildtype Mtb after treatment with authentic 2-methylisocitrate, an MCC intermediate. Together, the fatty acid-induced dormancy-like state and drug tolerance are attributed to dysregulated MCC activity.