Metabolic Fingerprints of Effective Fluoxetine Treatment in the Prefrontal Cortex of Chronically Socially Isolated Rats: Marker Candidates and Predictive Metabolites.

The increasing prevalence of depression requires more effective therapy and the understanding of antidepressants' mode of action. We carried out untargeted metabolomics of the prefrontal cortex of rats exposed to chronic social isolation (CSIS), a rat model of depression, and/or fluoxetine treatment using liquid chromatography-high resolution mass spectrometry. The behavioral phenotype was assessed by the forced swim test. To analyze the metabolomics data, we employed univariate and multivariate analysis and biomarker capacity assessment using the receiver operating characteristic (ROC) curve. We also identified the most predictive biomarkers using a support vector machine with linear kernel (SVM-LK). Upregulated myo-inositol following CSIS may represent a potential marker of depressive phenotype. Effective fluoxetine treatment reversed depressive-like behavior and increased sedoheptulose 7-phosphate, hypotaurine, and acetyl-L-carnitine contents, which were identified as marker candidates for fluoxetine efficacy. ROC analysis revealed 4 significant marker candidates for CSIS group discrimination, and 10 for fluoxetine efficacy. SVM-LK with accuracies of 61.50% or 93.30% identified a panel of 7 or 25 predictive metabolites for depressive-like behavior or fluoxetine effectiveness, respectively. Overall, metabolic fingerprints combined with the ROC curve and SVM-LK may represent a new approach to identifying marker candidates or predictive metabolites for ongoing disease or disease risk and treatment outcome.

Alternative routes for production of the drug candidate d-chiro-inositol with Corynebacterium glutamicum using endogenous or promiscuous plant enzymes.

d-chiro-Inositol (DCI) is a promising drug candidate for treating insulin resistance and associated diseases such as type 2 diabetes or polycystic ovary syndrome. In this study, we developed two production processes for DCI using Corynebacterium glutamicum as host. In the first process, myo-inositol (MI) is oxidized to 2-keto-myo-inositol (2KMI) by the inositol dehydrogenase (IDH) IolG and then isomerized to 1-keto-d-chiro-inositol (1KDCI) by the isomerases Cg0212 or Cg2312, both of which were identified in this work. 1KDCI is then reduced to DCI by IolG. Overproduction of IolG and Cg0212 in a chassis strain unable to degrade inositols allowed the production of 1.1 g/L DCI from 10 g/L MI. As both reactions involved are reversible, only a partial conversion of MI to DCI can be achieved. To enable higher conversion ratios, a novel route towards DCI was established by utilizing the promiscuous activity of two plant-derived enzymes, the NAD+-dependent d-ononitol dehydrogenase MtOEPa and the NADPH-dependent d-pinitol dehydrogenase MtOEPb from Medicago truncatula (barrelclover). Heterologous production of these enzymes in the chassis strain led to the production of 1.6 g/L DCI from 10 g/L MI. For replacing the substrate MI by glucose, the two plant genes were co-expressed with the endogenous myo-inositol-1-phosphate synthase gene ino1 either as a synthetic operon or using a novel, bicistronic T7-based expression vector. With the single operon construct, 0.75 g/L DCI was formed from 20 g/L glucose, whereas with the bicistronic construct 1.2 g/L DCI was obtained, disclosing C. glutamicum as an attractive host for of d-chiro-inositol production.

Engineered plant control of associative nitrogen fixation.

Engineering N2-fixing symbioses between cereals and diazotrophic bacteria represents a promising strategy to sustainably deliver biologically fixed nitrogen (N) in agriculture. We previously developed novel transkingdom signaling between plants and bacteria, through plant production of the bacterial signal rhizopine, allowing control of bacterial gene expression in association with the plant. Here, we have developed both a homozygous rhizopine producing (RhiP) barley line and a hybrid rhizopine uptake system that conveys upon our model bacterium Azorhizobium caulinodans ORS571 (Ac) 103-fold improved sensitivity for rhizopine perception. Using this improved genetic circuitry, we established tight rhizopine-dependent transcriptional control of the nitrogenase master regulator nifA and the N metabolism σ-factor rpoN, which drove nitrogenase expression and activity in vitro and in situ by bacteria colonizing RhiP barley roots. Although in situ nitrogenase activity was suboptimally effective relative to the wild-type strain, activation was specific to RhiP barley and was not observed on the roots of wild-type plants. This work represents a key milestone toward the development of a synthetic plant-controlled symbiosis in which the bacteria fix N2 only when in contact with the desired host plant and are prevented from interaction with nontarget plant species.

From Myo-inositol to D-chiro-inositol molecular pathways.

OBJECTIVE: Inositol is a carbocyclic sugar polyalcohol. By epimerization of its hydroxyl groups, nine possible stereoisomers can be generated, two of major physiological and clinical relevance: myo-inositol and D-chiro-inositol. Myo-inositol and D-chiro-inositol are normally stored in kidney, brain and liver and are necessary for functions, such as signal transduction, metabolic flux, insulin signaling, regulation of ion-channel permeability, stress response and embryo development. In this narrative review, we summarize the mechanisms by which myo-inositol and D-chiro-inositol can be synthesized and absorbed and their possible role in the etiopathogenesis of neural tube defects. MATERIALS AND METHODS: We performed an online search in the PubMed database using the following keywords: "inositol", "D-chiro-inositol", "myo-inositol", "neural tube defects and inositol". RESULTS: Inositol requirements are partly met by dietary intake, while the rest is synthesized endogenously. Inositol deficiency may be involved in the pathogenesis of diseases, such as metabolic syndrome, spina bifida (a neural tube defect), polycystic ovary syndrome and diabetes. Supplementation of the two inositol stereoisomers, D-chiro-inositol and myo-inositol is important to prevent these conditions. CONCLUSIONS: Inositol is fundamental for signal transduction in the brain, kidneys, reproductive organs and other tissues in response to neurotransmitters, hormones and growth factors. Various genes are involved in inositol metabolism and associated pathways. Altered inositol concentrations are observed in several diseases. Analysis of the genes involved in inositol metabolism may provide important information for the clinical management of these conditions.

Adventures with Mike.

Here are a few personal recollections of my 'big adventure' with Mike.

Rice EARLY SENESCENCE 2, encoding an inositol polyphosphate kinase, is involved in leaf senescence.

BACKGROUND: Early leaf senescence influences yield and yield quality by affecting plant growth and development. A series of leaf senescence-associated molecular mechanisms have been reported in rice. However, the complex genetic regulatory networks that control leaf senescence need to be elucidated. RESULTS: In this study, an early senescence 2 (es2) mutant was obtained from ethyl methanesulfonate mutagenesis (EMS)-induced mutational library for the Japonica rice cultivar Wuyugeng 7 (WYG7). Leaves of es2 showed early senescence at the seedling stage and became severe at the tillering stage. The contents of reactive oxygen species (ROS) significantly increased, while chlorophyll content, photosynthetic rate, catalase (CAT) activity significantly decreased in the es2 mutant. Moreover, genes which related to senescence, ROS and chlorophyll degradation were up-regulated, while those associated with photosynthesis and chlorophyll synthesis were down-regulated in es2 mutant compared to WYG7. The ES2 gene, which encodes an inositol polyphosphate kinase (OsIPK2), was fine mapped to a 116.73-kb region on chromosome 2. DNA sequencing of ES2 in the mutant revealed a missense mutation, ES2 was localized to nucleus and plasma membrane of cells, and expressed in various tissues of rice. Complementation test and overexpression experiment confirmed that ES2 completely restored the normal phenotype, with chlorophyll contents and photosynthetic rate increased comparable with the wild type. These results reveal the new role of OsIPK2 in regulating leaf senescence in rice and therefore will provide additional genetic evidence on the molecular mechanisms controlling early leaf senescence. CONCLUSIONS: The ES2 gene, encoding an inositol polyphosphate kinase localized in the nucleus and plasma membrane of cells, is essential for leaf senescence in rice. Further study of ES2 will facilitate the dissection of the genetic mechanisms underlying early leaf senescence and plant growth.

Glutathione: A powerful but rare cofactor among Actinobacteria.

Glutathione (γ-l-glutamyl-l-cysteinylglycine, GSH) is a powerful cellular redox agent. In nature only the l,l-form is common among the tree of life. It serves as antioxidant or redox buffer system, protein regeneration and activation by interaction with thiol groups, unspecific reagent for conjugation during detoxification, marker for amino acid or peptide transport even through membranes, activation or solubilization of compounds during degradative pathways or just as redox shuttle. However, the role of GSH production and utilization in bacteria is more complex and especially little is known for the Actinobacteria. Some recent reports on GSH use in degradative pathways came across and this is described herein. GSH is used by transferases to activate and solubilize epoxides. It allows funneling epoxides as isoprene oxide or styrene oxide into central metabolism. Thus, the distribution of GSH synthesis, recycling and application among bacteria and especially Actinobacteria are highlighted including the pathways and contributing enzymes.

Characterization and engineering control of the effects of reactive oxygen species on the conversion of sterols to steroid synthons in Mycobacterium neoaurum.

The conversion of sterols to steroid synthons by engineered mycobacteria comprises one of the basic ways for the production of steroid medications in the pharmaceutical industry. Here, we revealed that high amounts of reactive oxygen species (ROS) generate during the conversion process of sterols, which impairs the cell viability of mycobacterial cells and thus hinders the conversion of sterols to steroid synthons. Accordingly, the endogenous antioxidants for detoxifying ROS in mycobacteria, ROS scavenging enzymes and low molecular weight thiols, were examined. The results revealed that three antioxidants, catalase (CAT), mycothiol (MSH), and ergothioneine (EGT), demonstrated efficacy toward neutralizing the excessive ROS produced during sterol metabolism. CAT overexpression or MSH or EGT augmentation enhanced the conversion of phytosterols to 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) by 18.9%, 23.8%, and 32.1%, respectively, and also enhanced the cell viability, indicating the benefits of these antioxidants in reducing ROS-induced stress. Further combinatorial augmentation of CAT, MSH, and EGT demonstrated enhanced effects toward intracellular ROS scavenging, resulting in 54.2% greater cell viability and 47.5% enhancement in 4-HBC production. These findings indicated that the excessive ROS induces cell stress, in turn limiting the conversion of sterols, whereas neutralization of the excessive ROS by combined control of CAT, MSH, and EGT serves as an effective strategy to boost the conversion productivity of sterols to steroid synthons.

Engineering transkingdom signalling in plants to control gene expression in rhizosphere bacteria.

The root microbiota is critical for agricultural yield, with growth-promoting bacteria able to solubilise phosphate, produce plant growth hormones, antagonise pathogens and fix N2. Plants control the microorganisms in their immediate environment and this is at least in part through direct selection, the immune system, and interactions with other microorganisms. Considering the importance of the root microbiota for crop yields it is attractive to artificially regulate this environment to optimise agricultural productivity. Towards this aim we express a synthetic pathway for the production of the rhizopine scyllo-inosamine in plants. We demonstrate the production of this bacterial derived signal in both Medicago truncatula and barley and show its perception by rhizosphere bacteria, containing bioluminescent and fluorescent biosensors. This study lays the groundwork for synthetic signalling networks between plants and bacteria, allowing the targeted regulation of bacterial gene expression in the rhizosphere for delivery of useful functions to plants.

Water Deficit Elicits a Transcriptional Response of Genes Governing d-pinitol Biosynthesis in Soybean (Glycine max).

d-pinitol is the most commonly accumulated sugar alcohol in the Leguminosae family and has been observed to increase significantly in response to abiotic stress. While previous studies have identified genes involved in d-pinitol synthesis, no study has investigated transcript expression in planta. The present study quantified the expression of several genes involved in d-pinitol synthesis in different plant tissues and investigated the accumulation of d-pinitol, myo-inositol and other metabolites in response to a progressive soil drought in soybean (Glycine max). Expression of myo-inositol 1-phosphate synthase (INPS), the gene responsible for the conversion of glucose-6-phosphate to myo-inositol-1-phosphate, was significantly up regulated in response to a water deficit for the first two sampling weeks. Expression of myo-inositol O-methyl transferase (IMT1), the gene responsible for the conversion of myo-inositol into d-ononitol was only up regulated in stems at sampling week 3. Assessment of metabolites showed significant changes in their concentration in leaves and stems. d-Pinitol concentration was significantly higher in all organs sampled from water deficit plants for all three sampling weeks. In contrast, myo-inositol, had significantly lower concentrations in leaf samples despite up regulation of INPS suggesting the transcriptionally regulated flux of carbon through the myo-inositol pool is important during water deficit.

Impaired GCR1 transcription resulted in defective inositol levels, vacuolar structure and autophagy in Saccharomyces cerevisiae.

In yeast, the GCR1 transcription factor is involved in the regulation of glycolysis and its deletion exhibited growth defect, reduced inositol and phosphatidylinositol (PI) levels compared to WT cells. We observed a down regulation of the INO1 and PIS1 expression in gcr1∆ cells under both I- and I+ conditions and the over expression of GCR1 in gcr1∆ cells restored the growth, retrieved the expression of INO1, and PIS1 comparable to WT cells. In the gel shift assay, the Gcr1p binds to its consensus sequence CTTCC in PIS1 promoter and regulates its expression but not in INO1 transcription. The WT cells, under I- significantly reduced the expression of GCR1 and PIS1, but increased the expression of KCS1 and de-repressed INO1. The Kcs1p expression was reduced in gcr1∆ cells; this reduced INO1 expression resulting in abnormal vacuolar structure and reduced autophagy in Saccharomyces cerevisiae.

Response of high-risk MDS to azacitidine and lenalidomide is impacted by baseline and acquired mutations in a cluster of three inositide-specific genes.

Specific myeloid-related and inositide-specific gene mutations can be linked to myelodysplastic syndromes (MDS) pathogenesis and therapy. Here, 44 higher-risk MDS patients were treated with azacitidine and lenalidomide and mutations analyses were performed at baseline and during the therapy. Results were then correlated to clinical outcome, overall survival (OS), leukemia-free-survival (LFS) and response to therapy. Collectively, 34/44 patients were considered evaluable for response, with an overall response rate of 76.25% (26/34 cases): 17 patients showed a durable response, 9 patients early lost response and 8 patients never responded. The most frequently mutated genes were ASXL1, TET2, RUNX1, and SRSF2. All patients early losing response, as well as cases never responding, acquired the same 3 point mutations during therapy, affecting respectively PIK3CD (D133E), AKT3 (D280G), and PLCG2 (Q548R) genes, that regulate cell proliferation and differentiation. Moreover, Kaplan-Meier analyses revealed that this mutated cluster was significantly associated with a shorter OS, LFS, and duration of response. All in all, a common mutated cluster affecting 3 inositide-specific genes is significantly associated with loss of response to azacitidine and lenalidomide therapy in higher risk MDS. Further studies are warranted to confirm these data and to further analyze the functional role of this 3-gene cluster.

The small RNA RssR regulates myo-inositol degradation by Salmonella enterica.

Small noncoding RNAs (sRNAs) with putative regulatory functions in gene expression have been identified in the enteropathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). Two sRNAs are encoded by the genomic island GEI4417/4436 responsible for myo-inositol (MI) degradation, suggesting a role in the regulation of this metabolic pathway. We show that a lack of the sRNA STnc2160, termed RssR, results in a severe growth defect in minimal medium (MM) with MI. In contrast, the second sRNA STnc1740 was induced in the presence of glucose, and its overexpression slightly attenuated growth in the presence of MI. Constitutive expression of RssR led to an increased stability of the reiD mRNA, which encodes an activator of iol genes involved in MI utilization, via interaction with its 5'-UTR. SsrB, a response regulator contributing to the virulence properties of salmonellae, activated rssR transcription by binding the sRNA promoter. In addition, the absence of the RNA chaperone Hfq resulted in strongly decreased levels of RssR, attenuated S. Typhimurium growth with MI, and reduced expression of several iol genes required for MI degradation. Considered together, the extrinsic RssR allows fine regulation of cellular ReiD levels and thus of MI degradation by acting on the reiD mRNA stability.

Generation and characterization of thiol-deficient Mycobacterium tuberculosis mutants.

Mycothiol (MSH) and ergothioneine (ERG) are thiols able to compensate for each other to protect mycobacteria against oxidative stress. Gamma-glutamylcysteine (GGC), another thiol and an intermediate in ERG biosynthesis has detoxification abilities. Five enzymes are involved in ERG biosynthesis, namely EgtA, EgtB, EgtC, EgtD and EgtE. The role of these enzymes in the production of ERG had been unclear. On the other hand, the enzyme MshA is known to be essential for MSH biosynthesis. In this manuscript, we describe the raw data of the generation and characterization of Mycobacterium tuberculosis (M.tb) mutants harbouring a deletion of the gene coding for each of these enzymes, and the raw data of the phenotypic characterization of the obtained thiol-deficient M.tb mutants. High throughput screening (HTS) of off-patent drugs and natural compounds revealed few compounds that displayed a higher activity against the thiol-deficient mutants relative to the wild-type strain. The mode of action of these drugs was further investigated. Raw data displaying these results are described here.

The functional interplay of low molecular weight thiols in Mycobacterium tuberculosis.

BACKGROUND: Three low molecular weight thiols are synthesized by Mycobacterium tuberculosis (M.tb), namely ergothioneine (ERG), mycothiol (MSH) and gamma-glutamylcysteine (GGC). They are able to counteract reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). In addition, the production of ERG is elevated in the MSH-deficient M.tb mutant, while the production of MSH is elevated in the ERG-deficient mutants. Furthermore, the production of GGC is elevated in the MSH-deficient mutant and the ERG-deficient mutants. The propensity of one thiol to be elevated in the absence of the other prompted further investigations into their interplay in M.tb. METHODS: To achieve that, we generated two M.tb mutants that are unable to produce ERG nor MSH but are able to produce a moderate (ΔegtD-mshA) or significantly high (ΔegtB-mshA) amount of GGC relative to the wild-type strain. In addition, we generated an M.tb mutant that is unable to produce GGC nor MSH but is able to produce a significantly low level of ERG (ΔegtA-mshA) relative to the wild-type strain. The susceptibilities of these mutants to various in vitro and ex vivo stress conditions were investigated and compared. RESULTS: The ΔegtA-mshA mutant was the most susceptible to cellular stress relative to its parent single mutant strains (ΔegtA and ∆mshA) and the other double mutants. In addition, it displayed a growth-defect in vitro, in mouse and human macrophages suggesting; that the complete inhibition of ERG, MSH and GGC biosynthesis is deleterious for the growth of M.tb. CONCLUSIONS: This study indicates that ERG, MSH and GGC are able to compensate for each other to maximize the protection and ensure the fitness of M.tb. This study therefore suggests that the most effective strategy to target thiol biosynthesis for anti-tuberculosis drug development would be the simultaneous inhibition of the biosynthesis of ERG, MSH and GGC.

Gamma-glutamylcysteine protects ergothioneine-deficient Mycobacterium tuberculosis mutants against oxidative and nitrosative stress.

Mycobacterium tuberculosis (M.tb.), the causative agent of tuberculosis (TB), cannot synthesize GSH, but synthesizes two major low molecular weight thiols namely mycothiol (MSH) and ergothioneine (ERG). Gamma-glutamylcysteine (GGC), an intermediate in GSH synthesis, has been implicated in the protection of lactic acid bacteria from oxidative stress in the absence of GSH. In mycobacteria, GGC is an intermediate in ERG biosynthesis, and its formation is catalysed by EgtA (GshA). GGC is subsequently used by EgtB in the formation of hercynine-sulphoxide-GGC. In this study, M.tb. mutants harbouring unmarked, in-frame deletions in each of the fives genes involved in ERG biosynthesis (egtA, egtB, egtC, egtD and egtE) or a marked deletion of the mshA gene (required for MSH biosynthesis) were generated. Liquid chromatography tandem mass spectrometry analyses (LC-MS) revealed that the production of GGC was elevated in the MSH-deficient and the ERG-deficient mutants. The ERG-deficient ΔegtB mutant which accumulated GGC was more resistant to oxidative and nitrosative stress than the ERG-deficient, GGC-deficient ΔegtA mutant. This implicates GGC in the detoxification of reactive oxygen and nitrogen species in M.tb.

A new-generation of Bacillus subtilis cell factory for further elevated scyllo-inositol production.

BACKGROUND: A stereoisomer of inositol, scyllo-inositol (SI), has been regarded as a promising therapeutic agent for Alzheimer's disease. However, this compound is relatively rare, whereas another stereoisomer of inositol, myo-inositol (MI) is abundant in nature. Bacillus subtilis 168 has the ability to metabolize inositol stereoisomers, including MI and SI. Previously, we reported a B. subtilis cell factory with modified inositol metabolism that converts MI into SI in the culture medium. The strain was constructed by deleting all genes related to inositol metabolism and overexpressing key enzymes, IolG and IolW. By using this strain, 10 g/l of MI initially included in the medium was completely converted into SI within 48 h of cultivation in a rich medium containing 2% (w/v) Bacto soytone. RESULTS: When the initial concentration of MI was increased to 50 g/l, conversion was limited to 15.1 g/l of SI. Therefore, overexpression systems of IolT and PntAB, the main transporter of MI in B. subtilis and the membrane-integral nicotinamide nucleotide transhydrogenase in Escherichia coli respectively, were additionally introduced into the B. subtilis cell factory, but the conversion efficiency hardly improved. We systematically determined the amount of Bacto soytone necessary for ultimate conversion, which was 4% (w/v). As a result, the conversion of SI reached to 27.6 g/l within 48 h of cultivation. CONCLUSIONS: The B. subtilis cell factory was improved to yield a SI production rate of 27.6 g/l/48 h by simultaneous overexpression of IolT and PntAB, and by addition of 4% (w/v) Bacto soytone in the conversion medium. The concentration of SI was increased even in the stationary phase perhaps due to nutrients in the Bacto soytone that contribute to the conversion process. Thus, MI conversion to SI may be further optimized via identification and control of these unknown nutrients.

An in vitro synthetic biology platform for the industrial biomanufacturing of myo-inositol from starch.

Myo-Inositol (vitamin B8) is widely used in the drug, cosmetic, and food & feed industries. Here, we present an in vitro non-fermentative enzymatic pathway that converts starch to inositol in one vessel. This in vitro pathway is comprised of four enzymes that operate without ATP or NAD+ supplementation. All enzyme BioBricks are carefully selected from hyperthermophilic microorganisms, that is, alpha-glucan phosphorylase from Thermotoga maritima, phosphoglucomutase from Thermococcus kodakarensis, inositol 1-phosphate synthase from Archaeoglobus fulgidus, and inositol monophosphatase from T. maritima. They were expressed efficiently in high-density fermentation of Escherichia coli BL21(DE3) and easily purified by heat treatment. The four-enzyme pathway supplemented with two other hyperthermophilic enzymes (i.e., 4-α-glucanotransferase from Thermococcus litoralis and isoamylase from Sulfolobus tokodaii) converts branched or linear starch to inositol, accomplishing a very high product yield of 98.9 ± 1.8% wt./wt. This in vitro (aeration-free) biomanufacturing has been successfully operated on 20,000-L reactors. Less costly inositol would be widely added in heath food, low-end soft drink, and animal feed, and may be converted to other value-added biochemicals (e.g., glucarate). This biochemical is the first product manufactured by the in vitro synthetic biology platform on an industrial scale. Biotechnol. Bioeng. 2017;114: 1855-1864. © 2017 Wiley Periodicals, Inc.

Integrated transcriptomic and metabolomic analysis shows that disturbances in metabolism of tumor cells contribute to poor survival of RCC patients.

PURPOSE: Cellular metabolism of renal cell carcinoma (RCC) tumors is disturbed. The clinical significance of these alterations is weakly understood. We aimed to find if changes in metabolic pathways contribute to survival of RCC patients. MATERIAL AND METHODS: 35 RCC tumors and matched controls were used for metabolite profiling using gas chromatography-mass spectrometry and transcriptomic analysis with qPCR-arrays targeting the expression of 93 metabolic genes. The clinical significance of obtained data was validated on independent cohort of 468 RCC patients with median follow-up of 43.22months. RESULTS: The levels of 31 metabolites were statistically significantly changed in RCC tumors compared with controls. The top altered metabolites included beta-alanine (+4.2-fold), glucose (+3.4-fold), succinate (-11.0-fold), myo-inositol (-4.6-fold), adenine (-4.2-fold), uracil (-3.7-fold), and hypoxanthine (-3.0-fold). These disturbances were associated with altered expression of 53 metabolic genes. ROC curve analysis revealed that the top metabolites discriminating between tumor and control samples included succinate (AUC=0.91), adenine (AUC=0.89), myo-inositol (AUC=0.87), hypoxanthine (AUC=0.85), urea (AUC=0.85), and beta-alanine (AUC=0.85). Poor survival of RCC patients correlated (p<0.0001) with altered expression of genes involved in metabolism of succinate (HR=2.7), purines (HR=2.4), glucose (HR=2.4), beta-alanine (HR=2.5), and myo-inositol (HR=1.9). CONCLUSIONS: We found that changes in metabolism of succinate, beta-alanine, purines, glucose and myo-inositol correlate with poor survival of RCC patients.

Mycobacterium tuberculosis WhiB3 Responds to Vacuolar pH-induced Changes in Mycothiol Redox Potential to Modulate Phagosomal Maturation and Virulence.

The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (EMSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in EMSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic EMSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, MtbΔwhiB3 displayed intramacrophage survival defect, which can be rescued bypharmacological inhibition of phagosomal acidification. Last, MtbΔwhiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis.