A "plus one" strategy impacts replication of felid alphaherpesvirus 1, Mycoplasma and Chlamydia, and the metabolism of coinfected feline cells.

Coinfections are known to play an important role in disease progression and severity. Coinfections are common in cats, but no coinfection studies have investigated the in vitro dynamics between feline viral and bacterial pathogens. In this study, we performed co-culture and invasion assays to investigate the ability of common feline bacterial respiratory pathogens, Chlamydia felis and Mycoplasma felis, to replicate in and invade into Crandell-Rees feline kidney cells. We subsequently investigated how coinfection of these feline cells with each bacterium (C. felis or M. felis) and the common feline viral pathogen, felid alphaherpesvirus 1 (FHV-1), affects replication of each agent in this cell culture system. We also investigated the metabolic impact of each co-pathogen using metabolomic analysis of infected and coinfected cells. C. felis was able to invade and replicate in CRFKs, while M. felis had little capacity to invade. During coinfection, FHV-1 replication was minimally affected by the presence of either bacterial pathogen, but bacterial replication kinetics were more affected, particularly in M. felis. Both C. felis and M. felis replicated to higher levels in the presence of a secondary pathogen. Coinfections resulted in reprogramming of the glycolysis pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle. The distinct metabolic profiles of coinfected cells compared to those of cells infected with just one of these three pathogens, as well as the impact of coinfections on viral or bacterial load, suggest strong interactions between these three pathogens and possible synergistic mechanisms enhancing virulence that need further investigation.IMPORTANCEIn the natural world, respiratory pathogens coexist within their hosts, but their dynamics and interactions remain largely unexplored. Herpesviruses, mycoplasmas, and chlamydias are common and significant causes of acute and chronic respiratory and system disease in animals and people, and these diseases are increasingly found to be polymicrobial. This study investigates how coinfection of feline cells between three respiratory pathogens of cats impact each other as well as the host innate metabolic response to infection. Each of these pathogens have been implicated in the induction of feline upper respiratory tract disease in cats, which is the leading cause of euthanasia in shelters. Understanding how coinfection impacts co-pathogenesis and host responses is critical for improving disease management.

Metabolic Plasticity of Glioblastoma Cells in Response to DHODH Inhibitor BAY2402234 Treatment.

Glioblastoma (IDH-wildtype) represents a formidable challenge in oncology, lacking effective chemotherapeutic or biological interventions. The metabolic reprogramming of cancer cells is a hallmark of tumor progression and drug resistance, yet the role of metabolic reprogramming in glioblastoma during drug treatment remains poorly understood. The dihydroorotate dehydrogenase (DHODH) inhibitor BAY2402234 is a blood-brain barrier penetrant drug showing efficiency in in vivo models of many brain cancers. In this study, we investigated the effect of BAY2402234 in regulating the metabolic phenotype of EGFRWT and EGFRvIII patient-derived glioblastoma cell lines. Our findings reveal the selective cytotoxicity of BAY2402234 toward EGFRWT glioblastoma subtypes with minimal effect on EGFRvIII patient cells. At sublethal doses, BAY2402234 induces triglyceride synthesis at the expense of membrane lipid synthesis and fatty acid oxidation in EGFRWT glioblastoma cells, while these effects are not observed in EGFRvIII glioblastoma cells. Furthermore, BAY2402234 reduced the abundance of signaling lipid species in EGFRWT glioblastoma. This study elucidates genetic mutation-specific metabolic plasticity and efficacy in glioblastoma cells in response to drug treatment, offering insights into therapeutic avenues for precision medicine approaches.

Aerobic glycolysis but not GLS1-dependent glutamine metabolism is critical for anti-tumor immunity and response to checkpoint inhibition.

Tumor cells undergo uncontrolled proliferation driven by enhanced anabolic metabolism including glycolysis and glutaminolysis. Targeting these pathways to inhibit cancer growth is a strategy for cancer treatment. Critically, however, tumor-responsive T cells share metabolic features with cancer cells, making them susceptible to these treatments as well. Here, we assess the impact on anti-tumor T cell immunity and T cell exhaustion by genetic ablation of lactate dehydrogenase A (LDHA) and glutaminase1 (GLS1), key enzymes in aerobic glycolysis and glutaminolysis. Loss of LDHA severely impairs expansion of T cells in response to tumors and chronic infection. In contrast, T cells lacking GLS1 can compensate for impaired glutaminolysis by engaging alternative pathways, including upregulation of asparagine synthetase, and thus efficiently respond to tumor challenge and chronic infection as well as immune checkpoint blockade. Targeting GLS1-dependent glutaminolysis, but not aerobic glycolysis, may therefore be a successful strategy in cancer treatment, particularly in combination with immunotherapy.

The AMPK activator ATX-304 alters cellular metabolism to protect against cisplatin-induced acute kidney injury.

Acute kidney injury (AKI) disrupts energy metabolism. Targeting metabolism through AMP-activated protein kinase (AMPK) may alleviate AKI. ATX-304, a pan-AMPK activator, was evaluated in C57Bl/6 mice and tubular epithelial cell (TEC) cultures. Mice received ATX-304 (1 mg/g) or control chow for 7 days before cisplatin-induced AKI (CI-AKI). Primary cultures of tubular epithelial cells (TECs) were pre-treated with ATX-304 (20 µM, 4 h) prior to exposure to cisplatin (20 µM, 23 h). ATX-304 increased acetyl-CoA carboxylase phosphorylation, indicating AMPK activation. It protected against CI-AKI measured by serum creatinine (control 0.05 + 0.03 mM vs ATX-304 0.02 + 0.01 mM, P = 0.03), western blot for neutrophil gelatinase-associated lipocalin (NGAL) (control 3.3 + 1.8-fold vs ATX-304 1.2 + 0.55-fold, P = 0.002), and histological injury (control 3.5 + 0.59 vs ATX-304 2.7 + 0.74, P = 0.03). In TECs, pre-treatment with ATX-304 protected against cisplatin-mediated injury, as measured by lactate dehydrogenase release, MTS cell viability, and cleaved caspase 3 expression. ATX-304 protection against cisplatin was lost in AMPK-null murine embryonic fibroblasts. Metabolomic analysis in TECs revealed that ATX-304 (20 µM, 4 h) altered 66/126 metabolites, including fatty acids, tricarboxylic acid cycle metabolites, and amino acids. Metabolic studies of live cells using the XFe96 Seahorse analyzer revealed that ATX-304 increased the basal TEC oxygen consumption rate by 38%, whereas maximal respiration was unchanged. Thus, ATX-304 protects against cisplatin-mediated kidney injury via AMPK-dependent metabolic reprogramming, revealing a promising therapeutic strategy for AKI.

Plasma beta-hydroxy-beta-methylbutyrate availability after enteral administration during critical illness after trauma: An exploratory study.

BACKGROUND: During critical illness skeletal muscle wasting occurs rapidly. Although beta-hydroxy-beta-methylbutyrate (HMB) is a potential treatment to attenuate this process, the plasma appearance and muscle concentration is uncertain. METHODS: This was an exploratory study nested within a blinded, parallel group, randomized clinical trial in which critically ill patients after trauma received enteral HMB (3 g daily) or placebo. Plasma samples were collected at 0, 60, and 180 min after study supplement administration on day 1. Needle biopsies of the vastus lateralis muscle were collected (baseline and day 7 of the HMB treatment intervention period). An external standard curve was used to calculate HMB concentrations in plasma and muscle. RESULTS: Data were available for 16 participants (male n = 12 (75%), median [interquartile range] age 50 [29-58] years) who received placebo and 18 participants (male n = 14 (78%), age 49 [34-55] years) who received HMB. Plasma HMB concentrations were similar at baseline but increased after HMB (T = 60 min: placebo 0.60 [0.44-1.31] µM; intervention 51.65 [22.76-64.72] µM). Paired muscle biopsies were collected from 11 participants (placebo n = 7, HMB n = 4). Muscle HMB concentrations were similar at baseline between groups (2.35 [2.17-2.95]; 2.07 [1.78-2.31] µM). For participants in the intervention group who had the repeat biopsy within 4 h of HMB administration, concentrations were greater (7.2 and 12.3 µM) than those who had the repeat biopsy >4 h after HMB (2.7 and 2.1 µM). CONCLUSION: In this exploratory study, enteral HMB administration increased plasma HMB availability. The small sample size limits interpretation of the muscle HMB findings.

Matrix Selection for the Visualization of Small Molecules and Lipids in Brain Tumors Using Untargeted MALDI-TOF Mass Spectrometry Imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging allows for the study of metabolic activity in the tumor microenvironment of brain cancers. The detectable metabolites within these tumors are contingent upon the choice of matrix, deposition technique, and polarity setting. In this study, we compared the performance of three different matrices, two deposition techniques, and the use of positive and negative polarity in two different brain cancer types and across two species. Optimal combinations were confirmed by a comparative analysis of lipid and small-molecule abundance by using liquid chromatography-mass spectrometry and RNA sequencing to assess differential metabolites and enzymes between normal and tumor regions. Our findings indicate that in the tumor-bearing brain, the recrystallized α-cyano-4-hydroxycinnamic acid matrix with positive polarity offered superior performance for both detected metabolites and consistency with other techniques. Beyond these implications for brain cancer, our work establishes a workflow to identify optimal matrices for spatial metabolomics studies.

Gas Chromatography-Mass Spectrometry-Based Targeted Metabolomics of Hard Coral Samples.

Gas chromatography-mass spectrometry (GC-MS)-based approaches have proven to be powerful for elucidating the metabolic basis of the cnidarian-dinoflagellate symbiosis and how coral responds to stress (i.e., during temperature-induced bleaching). Steady-state metabolite profiling of the coral holobiont, which comprises the cnidarian host and its associated microbes (Symbiodiniaceae and other protists, bacteria, archaea, fungi, and viruses), has been successfully applied under ambient and stress conditions to characterize the holistic metabolic status of the coral. However, to answer questions surrounding the symbiotic interactions, it is necessary to analyze the metabolite profiles of the coral host and its algal symbionts independently, which can only be achieved by physical separation and isolation of the tissues, followed by independent extraction and analysis. While the application of metabolomics is relatively new to the coral field, the sustained efforts of research groups have resulted in the development of robust methods for analyzing metabolites in corals, including the separation of the coral host tissue and algal symbionts. This paper presents a step-by-step guide for holobiont separation and the extraction of metabolites for GC-MS analysis, including key optimization steps for consideration. We demonstrate how, once analyzed independently, the combined metabolite profile of the two fractions (coral and Symbiodiniaceae) is similar to the profile of the whole (holobiont), but by separating the tissues, we can also obtain key information about the metabolism of and interactions between the two partners that cannot be obtained from the whole alone.

Reversion of mutations in a live mycoplasma vaccine alters its metabolism.

The live attenuated temperature sensitive vaccine strain MS-H (Vaxsafe® MS, Bioproperties Pty. Ltd., Australia) is widely used to control disease associated with M. synoviae infection in commercial poultry. MS-H was derived from a field strain (86079/7NS) through N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced mutagenesis. Whole genomic sequence analysis of the MS-H and comparison with that of the 86079/7NS have found that MS-H contains 32 single nucleotide polymorphisms (SNPs). Three of these SNPs, found in the obgE, oppF and gapdh genes, have been shown to be prone to reversion under field condition, albeit at a low frequency. Three MS-H reisolates containing the 86079/7NS genotype in obgE (AS2), obgE and oppF (AB1), and obgE, oppF and gapdh (TS4), appeared to be more immunogenic and transmissible compared to MS-H in chickens. To investigate the influence of these reversions in the in vitro fitness of M. synoviae, the growth kinetics and steady state metabolite profiles of the MS-H reisolates, AS2, AB1 and TS4, were compared to those of the vaccine strain. Steady state metabolite profiling of the reisolates showed that changes in ObgE did not significantly influence the metabolism, while changes in OppF was associated with significant alterations in uptake of peptides and/or amino acids into the M. synoviae cell. It was also found that GAPDH plays a role in metabolism of the glycerophospholipids as well as an arginine deiminase (ADI) pathway. This study underscores the role of ObgE, OppF and GAPDH in M. synoviae metabolism, and suggests that the impaired fitness arising from variations in ObgE, OppF and GAPDH contributes to attenuation of MS-H.

Feeding an unsalable carrot total-mixed ration altered bacterial amino acid degradation in the rumen of lambs.

The objective of this study was to determine the influence of a total-mixed ration including unsalable carrots at 45% DM on the rumen microbiome; and the plasma, rumen and liver metabolomes. Carrots discarded at processing were investigated as an energy-dense substitute for barley grain in a conventional feedlot diet, and improved feed conversion efficiency by 25%. Here, rumen fluid was collected from 34 Merino lambs at slaughter (n = 16 control; n = 18 carrot) after a feeding period of 11-weeks. The V4 region of the 16S rRNA gene was sequenced to profile archaeal and bacterial microbe communities. Further, a comprehensive, targeted profile of known metabolites was constructed for blood plasma, rumen fluid and biopsied liver metabolites using a gas chromatography mass spectrometry (GC-MS) metabolomics approach. An in vitro batch culture was used to characterise ruminal fermentation including gas and methane (CH4) production. In vivo rumen microbial community structure of carrot fed lambs was dissimilar (P < 0.01; PERMANOVA), and all measures of alpha diversity were greater (P < 0.01), compared to those fed the control diet. Unclassified genera in Bacteroidales (15.9 ± 6.74% relative abundance; RA) were more abundant (P < 0.01) in the rumen fluid of carrot-fed lambs, while unclassified taxa in the Succinivibrionaceae family (11.1 ± 3.85% RA) were greater (P < 0.01) in the control. The carrot diet improved in vitro ruminal fermentation evidenced as an 8% increase (P < 0.01) in DM digestibility and a 13.8% reduction (P = 0.01) in CH4 on a mg/ g DM basis, while the control diet increased (P = 0.04) percentage of propionate within total VFA by 20%. Fourteen rumen fluid metabolites and 27 liver metabolites were influenced (P ≤ 0.05) by diet, while no effect (P ≥ 0.05) was observed in plasma metabolites. The carrot diet enriched (impact value = 0.13; P = 0.01) the tyrosine metabolism pathway (acetoacetic acid, dopamine and pyruvate), while the control diet enriched (impact value = 0.42; P ≤ 0.02) starch and sucrose metabolism (trehalose and glucose) in rumen fluid. This study demonstrated that feeding 45% DM unsalable carrots diversified bacterial communities in the rumen. These dietary changes influenced pathways of tyrosine degradation, such that previous improvements in feed conversion efficiency in lambs could be explained.

Remodeling of Carbon Metabolism during Sulfoglycolysis in Escherichia coli.

Sulfoquinovose (SQ) is a major metabolite in the global sulfur cycle produced by nearly all photosynthetic organisms. One of the major pathways involved in the catabolism of SQ in bacteria such as Escherichia coli is a variant of the glycolytic Embden-Meyerhof-Parnas (EMP) pathway termed the sulfoglycolytic EMP (sulfo-EMP) pathway, which leads to the consumption of three of the six carbons of SQ and the excretion of 2,3-dihydroxypropanesulfonate (DHPS). Comparative metabolite profiling of aerobically glucose (Glc)-grown and SQ-grown E. coli cells was undertaken to identify the metabolic consequences of the switch from glycolysis to sulfoglycolysis. Sulfoglycolysis was associated with the diversion of triose phosphates (triose-P) to synthesize sugar phosphates (gluconeogenesis) and an unexpected accumulation of trehalose and glycogen storage carbohydrates. Sulfoglycolysis was also associated with global changes in central carbon metabolism, as indicated by the changes in the levels of intermediates in the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway (PPP), polyamine metabolism, pyrimidine metabolism, and many amino acid metabolic pathways. Upon entry into stationary phase and the depletion of SQ, E. coli cells utilize their glycogen, indicating a reversal of metabolic fluxes to allow glycolytic metabolism. IMPORTANCE The sulfosugar sulfoquinovose is estimated to be produced on a scale of 10 billion metric tons per annum, making it a major organosulfur species in the biosulfur cycle. The microbial degradation of sulfoquinovose through sulfoglycolysis allows the utilization of its carbon content and contributes to the biomineralization of its sulfur. However, the metabolic consequences of microbial growth on sulfoquinovose are unclear. We use metabolomics to identify the metabolic adaptations that Escherichia coli undergoes when grown on sulfoquinovose versus glucose. This revealed the increased flux into storage carbohydrates through gluconeogenesis and the reduced flux of carbon into the TCA cycle and downstream metabolism. These changes are relieved upon entry into stationary phase and reversion to glycolytic metabolism. This work provides new insights into the metabolic consequences of microbial growth on an abundant sulfosugar.

Multi-substrate Metabolic Tracing Reveals Marked Heterogeneity and Dependency on Fatty Acid Metabolism in Human Prostate Cancer.

Cancer cells undergo metabolic reprogramming to meet increased bioenergetic demands. Studies in cells and mice have highlighted the importance of oxidative metabolism and lipogenesis in prostate cancer; however, the metabolic landscape of human prostate cancer remains unclear. To address this knowledge gap, we performed radiometric (14C) and stable (13C) isotope tracing assays in precision-cut slices of patient-derived xenografts (PDX). Glucose, glutamine, and fatty acid oxidation was variably upregulated in malignant PDXs compared with benign PDXs. De novo lipogenesis (DNL) and storage of free fatty acids into phospholipids and triacylglycerols were increased in malignant PDXs. There was no difference in substrate utilization between localized and metastatic PDXs and hierarchical clustering revealed marked metabolic heterogeneity across all PDXs. Mechanistically, glucose utilization was mediated by acetyl-CoA production rather than carboxylation of pyruvate, while glutamine entered the tricarboxylic acid cycle through transaminase reactions before being utilized via oxidative or reductive pathways. Blocking fatty acid uptake or fatty acid oxidation with pharmacologic inhibitors was sufficient to reduce cell viability in PDX-derived organoids, whereas blockade of DNL, or glucose or glutamine oxidation induced variable and limited therapeutic efficacy. These findings demonstrate that human prostate cancer, irrespective of disease stage, can effectively utilize all metabolic substrates, albeit with marked heterogeneity across tumors. We also confirm that fatty acid uptake and oxidation are targetable metabolic dependencies in human prostate cancer. IMPLICATIONS: Prostate cancer utilizes multiple substrates to fuel energy requirements, yet pharmacologic targeting of fatty acid uptake and oxidation reveals metabolic dependencies in localized and metastatic tumors.

Estrogen receptor alpha deficiency in cardiomyocytes reprograms the heart-derived extracellular vesicle proteome and induces obesity in female mice.

Dysregulation of estrogen receptor alpha (ERα) has been linked with increased metabolic and cardiovascular disease risk. Here, we generate and characterize cardiomyocyte-specific ERα knockout (ERαHKO) mice to assess the role of ERα in the heart. The most striking phenotype was obesity in female ERαHKO but not male ERαHKO mice. Female ERαHKO mice showed cardiac dysfunction, mild glucose and insulin intolerance and reduced ERα gene expression in skeletal muscle and white adipose tissue. Transcriptomic, proteomic, lipidomic and metabolomic analyses revealed evidence of contractile and/or metabolic dysregulation in heart, skeletal muscle and white adipose tissue. We show that heart-derived extracellular vesicles from female ERαHKO mice contain a distinct proteome associated with lipid and metabolic regulation, and have the capacity to metabolically reprogram the target skeletal myocyte proteome with functional impacts on glycolytic capacity and reserve. This multi-omics study uncovers a cardiac-initiated and sex-specific cardiometabolic phenotype regulated by ERα and provides insights into extracellular vesicle-mediated interorgan communication.

Blocking AMPK signalling to acetyl-CoA carboxylase increases cisplatin-induced acute kidney injury and suppresses the benefit of metformin.

BACKGROUND: Acute kidney injury (AKI) is accompanied by dysregulation of cellular energy metabolism and accumulation of intracellular lipid. Phosphorylation of acetyl-CoA carboxylase (ACC) by AMP-activated protein kinase (AMPK) inhibits fatty acid synthesis and promotes fatty acid oxidation (FAO), vital for kidney tubular epithelial cells (TECs). The diabetes drug metformin is protective in models of AKI; however, it is not known whether ACC phosphorylation plays a role. METHODS: Cisplatin-induced AKI (CI-AKI) was established in ACC1/2 double knock-in (ACC1/2DKI) mice, harbouring mutations that disrupt fatty acid metabolism, and the role of metformin was studied in this model. Outcomes measured included serum biochemistry, expression of kidney injury markers such as neutrophil gelatinase-associated lipocalin (NGAL), and metabolomic analysis. FINDINGS: ACC1/2DKI mice demonstrated more severe CI-AKI than wild type (WT), as assessed by serum urea and creatinine, histological injury, and expression of NGAL and interleukin-6. Metformin protected against AKI in WT mice, shown by reduced NGAL, but this effect was absent in ACC1/2DKI mice. In cultured TECs exposed to cisplatin, metformin reduced expression of cleaved caspase-3, however, this effect was diminished in ACC1/2DKI TECs. Analysis of kidney polar metabolites found numerous differences between metformin-treated CI-AKI in ACC1/2DKI and WT mice, involving multiple pathways of amino acid, nucleoside, and energy metabolism. INTERPRETATION: Severity of CI-AKI is exacerbated by the inability to regulate metabolism via phosphorylation of ACC. ACC phosphorylation contributes to the protective effect of metformin against AKI, influencing multiple mechanisms involved in the pathogenesis of kidney injury.

Cystathionine-ß-synthase is essential for AKT-induced senescence and suppresses the development of gastric cancers with PI3K/AKT activation.

Hyperactivation of oncogenic pathways downstream of RAS and PI3K/AKT in normal cells induces a senescence-like phenotype that acts as a tumor-suppressive mechanism that must be overcome during transformation. We previously demonstrated that AKT-induced senescence (AIS) is associated with profound transcriptional and metabolic changes. Here, we demonstrate that human fibroblasts undergoing AIS display upregulated cystathionine-ß-synthase (CBS) expression and enhanced uptake of exogenous cysteine, which lead to increased hydrogen sulfide (H2S) and glutathione (GSH) production, consequently protecting senescent cells from oxidative stress-induced cell death. CBS depletion allows AIS cells to escape senescence and re-enter the cell cycle, indicating the importance of CBS activity in maintaining AIS. Mechanistically, we show this restoration of proliferation is mediated through suppressing mitochondrial respiration and reactive oxygen species (ROS) production by reducing mitochondrial localized CBS while retaining antioxidant capacity of transsulfuration pathway. These findings implicate a potential tumor-suppressive role for CBS in cells with aberrant PI3K/AKT pathway activation. Consistent with this concept, in human gastric cancer cells with activated PI3K/AKT signaling, we demonstrate that CBS expression is suppressed due to promoter hypermethylation. CBS loss cooperates with activated PI3K/AKT signaling in promoting anchorage-independent growth of gastric epithelial cells, while CBS restoration suppresses the growth of gastric tumors in vivo. Taken together, we find that CBS is a novel regulator of AIS and a potential tumor suppressor in PI3K/AKT-driven gastric cancers, providing a new exploitable metabolic vulnerability in these cancers.

Lipid Metabolism Is Dysregulated in the Motor Cortex White Matter in Amyotrophic Lateral Sclerosis.

Lipid metabolism is profoundly dysregulated in amyotrophic lateral sclerosis (ALS), yet the lipid composition of the white matter, where the myelinated axons of motor neurons are located, remains uncharacterised. We aimed to comprehensively characterise how myelin is altered in ALS by assessing its lipid and protein composition. We isolated white matter from the motor cortex from post-mortem tissue of ALS patients (n = 8 sporadic ALS cases and n = 6 familial ALS cases) and age- and sex-matched controls (n = 8) and conducted targeted lipidomic analyses, qPCR for gene expression of relevant lipid metabolising enzymes and Western blotting for myelin proteins. We also quantified myelin density by using spectral confocal reflectance microscopy (SCoRe). Whilst myelin protein composition was similar in ALS and control tissue, both the lipid levels and the expression of their corresponding enzymes were dysregulated, highlighting altered lipid metabolism in the white matter as well as a likely change in myelin composition. Altered myelin composition could contribute to motor neuron dysfunction, and this highlights how oligodendrocytes may play a critical role in ALS pathogenesis.

Type I interferon antagonism of the JMJD3-IRF4 pathway modulates macrophage activation and polarization.

Metabolic adaptations can directly influence the scope and scale of macrophage activation and polarization. Here we explore the impact of type I interferon (IFNß) on macrophage metabolism and its broader impact on cytokine signaling pathways. We find that IFNß simultaneously increased the expression of immune-responsive gene 1 and itaconate production while inhibiting isocitrate dehydrogenase activity and restricting α-ketoglutarate accumulation. IFNß also increased the flux of glutamine-derived carbon into the tricarboxylic acid cycle to boost succinate levels. Combined, we identify that IFNß controls the cellular α-ketoglutarate/succinate ratio. We show that by lowering the α-ketoglutarate/succinate ratio, IFNß potently blocks the JMJD3-IRF4-dependent pathway in GM-CSF and IL-4 activated macrophages. The suppressive effects of IFNß on JMJD3-IRF4-dependent responses, including M2 polarization and GM-CSF-induced inflammatory pain, were reversed by supplementation with α-ketoglutarate. These results reveal that IFNß modulates macrophage activation and polarization through control of the cellular α-ketoglutarate/succinate ratio.

The long noncoding RNA glycoLINC assembles a lower glycolytic metabolon to promote glycolysis.

Non-covalent complexes of glycolytic enzymes, called metabolons, were postulated in the 1970s, but the concept has been controversial. Here we show that a c-Myc-responsive long noncoding RNA (lncRNA) that we call glycoLINC (gLINC) acts as a backbone for metabolon formation between all four glycolytic payoff phase enzymes (PGK1, PGAM1, ENO1, and PKM2) along with lactate dehydrogenase A (LDHA). The gLINC metabolon enhances glycolytic flux, increases ATP production, and enables cell survival under serine deprivation. Furthermore, gLINC overexpression in cancer cells promotes xenograft growth in mice fed a diet deprived of serine, suggesting that cancer cells employ gLINC during metabolic reprogramming. We propose that gLINC makes a functional contribution to cancer cell adaptation and provide the first example of a lncRNA-facilitated metabolon.

Characterisation of putative lactate synthetic pathways of Coxiella burnetii.

The zoonotic pathogen Coxiella burnetii, the causative agent of the human disease Q fever, is an ever-present danger to global public health. Investigating novel metabolic pathways necessary for C. burnetii to replicate within its unusual intracellular niche may identify new therapeutic targets. Recent studies employing stable isotope labelling established the ability of C. burnetii to synthesize lactate, despite the absence of an annotated synthetic pathway on its genome. A noncanonical lactate synthesis pathway could provide a novel anti-Coxiella target if it is essential for C. burnetii pathogenesis. In this study, two C. burnetii proteins, CBU1241 and CBU0823, were chosen for analysis based on their similarities to known lactate synthesizing enzymes. Recombinant GST-CBU1241, a putative malate dehydrogenase (MDH), did not produce measurable lactate in in vitro lactate dehydrogenase (LDH) activity assays and was confirmed to function as an MDH. Recombinant 6xHis-CBU0823, a putative NAD+-dependent malic enzyme, was shown to have both malic enzyme activity and MDH activity, however, did not produce measurable lactate in either LDH or malolactic enzyme activity assays in vitro. To examine potential lactate production by CBU0823 more directly, [13C]glucose labelling experiments compared label enrichment within metabolic pathways of a cbu0823 transposon mutant and the parent strain. No difference in lactate production was observed, but the loss of CBU0823 significantly reduced 13C-incorporation into glycolytic and TCA cycle intermediates. This disruption to central carbon metabolism did not have any apparent impact on intracellular replication within THP-1 cells. This research provides new information about the mechanism of lactate biosynthesis within C. burnetii, demonstrating that CBU1241 is not multifunctional, at least in vitro, and that CBU0823 also does not synthesize lactate. Although critical for normal central carbon metabolism of C. burnetii, loss of CBU0823 did not significantly impair replication of the bacterium inside cells.

A Mycoplasma gallisepticum Glycerol ABC Transporter Involved in Pathogenicity.

MalF has been shown to be required for virulence in the important avian pathogen Mycoplasma gallisepticum To characterize the function of MalF, predicted to be part of a putative ABC transporter, we compared metabolite profiles of a mutant with a transposon inserted in malF (MalF-deficient ST mutant 04-1; ΔmalF) with those of wild-type bacteria using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Of the substrates likely to be transported by an ABC transport system, glycerol was detected at significantly lower abundance in the ΔmalF mutant, compared to the wild type. Stable isotope labeling using [U-13C]glycerol and reverse transcription-quantitative PCR analysis indicated that MalF was responsible for the import of glycerol into M. gallisepticum and that, in the absence of MalF, the transcription of gtsA, which encodes a second transporter, GtsA, was upregulated, potentially to increase the import of glycerol-3-phosphate into the cell to compensate for the loss of MalF. The loss of MalF appeared to have a global effect on glycerol metabolism, suggesting that it may also play a regulatory role, and cellular morphology was also affected, indicating that the change to glycerol metabolism may have a broader effect on cellular organization. Overall, this study suggests that the reduced virulence of the ΔmalF mutant is due to perturbed glycerol uptake and metabolism and that the operon including malF should be reannotated as golABC to reflect its function in glycerol transport.IMPORTANCE Many mycoplasmas are pathogenic and cause disease in humans and animals. M. gallisepticum causes chronic respiratory disease in chickens and infectious sinusitis in turkeys, resulting in economic losses in poultry industries throughout the world. Expanding our knowledge about the pathogenesis of mycoplasma infections requires better understanding of the specific gene functions of these bacteria. In this study, we have characterized the metabolic function of a protein involved in the pathogenicity of M. gallisepticum, as well as its effect on expression of selected genes, cell phenotype, and H2O2 production. This study is a key step forward in elucidating why this protein plays a key role in virulence in chickens. This study also emphasizes the importance of functional characterization of mycoplasma proteins, using tools such as metabolomics, since prediction of function based on homology to other bacterial proteins is not always accurate.

Reprogrammed mRNA translation drives resistance to therapeutic targeting of ribosome biogenesis.

Elevated ribosome biogenesis in oncogene-driven cancers is commonly targeted by DNA-damaging cytotoxic drugs. Our previous first-in-human trial of CX-5461, a novel, less genotoxic agent that specifically inhibits ribosome biogenesis via suppression of RNA polymerase I (Pol I) transcription, revealed single-agent efficacy in refractory blood cancers. Despite this clinical response, patients were not cured. In parallel, we demonstrated a marked improvement in the in vivo efficacy of CX-5461 in combination with PI3K/AKT/mTORC1 pathway inhibitors. Here, we reveal the molecular basis for this improved efficacy observed in vivo, which is associated with specific suppression of translation of mRNAs encoding regulators of cellular metabolism. Importantly, acquired resistance to this cotreatment is driven by translational rewiring that results in dysregulated cellular metabolism and induction of a cAMP-dependent pathway critical for the survival of blood cancers including lymphoma and acute myeloid leukemia. Our studies thus identify key molecular mechanisms underpinning the response of blood cancers to selective inhibition of ribosome biogenesis and define metabolic vulnerabilities that will facilitate the rational design of more effective regimens for Pol I-directed therapies.