Activity of Lymphostatin, A Lymphocyte Inhibitory Virulence Factor of Pathogenic Escherichia coli, is Dependent on a Cysteine Protease Motif.

Lymphostatin (LifA) is a 366 kDa protein expressed by attaching & effacing Escherichia coli. It plays an important role in intestinal colonisation and inhibits the mitogen- and antigen-stimulated proliferation of lymphocytes and the synthesis of proinflammatory cytokines. LifA exhibits N-terminal homology with the glycosyltransferase domain of large clostridial toxins (LCTs). A DTD motif within this region is required for lymphostatin activity and binding of the sugar donor uridine diphosphate N-acetylglucosamine. As with LCTs, LifA also contains a cysteine protease motif (C1480, H1581, D1596) that is widely conserved within the YopT-like superfamily of cysteine proteases. By analogy with LCTs, we hypothesised that the CHD motif may be required for intracellular processing of the protein to release the catalytic N-terminal domain after uptake and low pH-stimulated membrane insertion of LifA within endosomes. Here, we created and validated a C1480A substitution mutant in LifA from enteropathogenic E. coli strain E2348/69. The purified protein was structurally near-identical to the wild-type protein. In bovine T lymphocytes treated with wild-type LifA, a putative cleavage product of approximately 140 kDa was detected. Appearance of the putative cleavage product was inhibited in a concentration-dependent manner by bafilomycin A1 and chloroquine, which inhibit endosome acidification. The cleavage product was not observed in cells treated with the C1480A mutant of LifA. Lymphocyte inhibitory activity of the purified C1480A protein was significantly impaired. The data indicate that an intact cysteine protease motif is required for cleavage of lymphostatin and its activity against T cells.

How the molecular weight affects the in vivo fate of exogenous hyaluronan delivered intravenously: A stable-isotope labelling strategy.

There is inconsistent information regarding the size effects of exogenously given hyaluronan on its in vivo fate. The data are often biased by the poor quality of hyaluronan and non-ideal labelling strategies used for resolving exogenous/endogenous hyaluronan, which only monitor the label and not hyaluronan itself. To overcome these drawbacks and establish the pharmacokinetics of intravenous hyaluronan in relation to its Mw, 13C-labelled HA of five Mws from 13.6-1562 kDa was prepared and administered to mice at doses 25-50 mg kg-1. The elimination efficiency increased with decreasing Mw. Low Mw hyaluronan was rapidly eliminated as small hyaluronan fragments in urine, while high Mw hyaluronan exhibited saturable kinetics and complete metabolization within 48 h. All tested Mws exhibited a similar uptake by liver cells and metabolization into activated sugars. 13C-labelling combined with LC-MS provides an excellent approach to elucidating in vivo fate and biological activities of hyaluronan.

Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1.

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and via phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interferes with UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the interactions of the two GFAT-1 domains to control catalytic activity. Notably, Ser205 phosphorylation has two discernible effects: it lowers baseline GFAT-1 activity and abolishes UDP-GlcNAc feedback inhibition. PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.

The folate cycle enzyme MTHFD2 induces cancer immune evasion through PD-L1 up-regulation.

Metabolic enzymes and metabolites display non-metabolic functions in immune cell signalling that modulate immune attack ability. However, whether and how a tumour's metabolic remodelling contributes to its immune resistance remain to be clarified. Here we perform a functional screen of metabolic genes that rescue tumour cells from effector T cell cytotoxicity, and identify the embryo- and tumour-specific folate cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2). Mechanistically, MTHFD2 promotes basal and IFN-γ-stimulated PD-L1 expression, which is necessary for tumourigenesis in vivo. Moreover, IFN-γ stimulates MTHFD2 through the AKT-mTORC1 pathway. Meanwhile, MTHFD2 drives the folate cycle to sustain sufficient uridine-related metabolites including UDP-GlcNAc, which promotes the global O-GlcNAcylation of proteins including cMYC, resulting in increased cMYC stability and PD-L1 transcription. Consistently, the O-GlcNAcylation level positively correlates with MTHFD2 and PD-L1 in pancreatic cancer patients. These findings uncover a non-metabolic role for MTHFD2 in cell signalling and cancer biology.

The hexosamine biosynthetic pathway and cancer: Current knowledge and future therapeutic strategies.

The hexosamine biosynthetic pathway (HBP) is a glucose metabolism pathway that results in the synthesis of a nucleotide sugar UDP-GlcNAc, which is subsequently used for the post-translational modification (O-GlcNAcylation) of intracellular proteins that regulate nutrient sensing and stress response. The HBP is carried out by a series of enzymes, many of which have been extensively implicated in cancer pathophysiology. Increasing evidence suggests that elevated activation of the HBP may act as a cancer biomarker. Inhibition of HBP enzymes could suppress tumor cell growth, modulate the immune response, reduce resistance, and sensitize tumor cells to conventional cancer therapy. Therefore, targeting the HBP may serve as a novel strategy for treating cancer patients. Here, we review the current findings on the significance of HBP enzymes in various cancers and discuss future approaches for exploiting HBP inhibition for cancer treatment.

Cell Energy Metabolism and Hyaluronan Synthesis.

Hyaluronan (HA) is a linear glycosaminoglycan (GAG) of extracellular matrix (ECM) synthesized by three hyaluronan synthases (HASes) at the plasma membrane using uridine diphosphate (UDP)-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc) as substrates. The production of HA is mainly regulated by hyaluronan synthase 2 (HAS2), that can be controlled at different levels, from epigenetics to transcriptional and post-translational modifications. HA biosynthesis is an energy-consuming process and, along with HA catabolism, is strongly connected to the maintenance of metabolic homeostasis. The cytoplasmic pool of UDP-sugars is critical for HA synthesis. UDP-GlcNAc is an important nutrient sensor and serves as donor substrate for the O-GlcNAcylation of many cytosolic proteins, including HAS2. This post-translational modification stabilizes HAS2 in the membrane and increases HA production. Conversely, HAS2 can be phosphorylated by AMP activated protein kinase (AMPK), a master metabolic regulator activated by low ATP/AMP ratios, which inhibits HA secretion. Similarly, HAS2 expression and the deposition of HA within the pericellular coat are inhibited by sirtuin 1 (SIRT1), another important energetic sensor, confirming the tight connection between nutrients availability and HA metabolism.

Functional Characterization of 3-Aminobenzoic Acid Adenylation Enzyme PctU and UDP-N-Acetyl-d-Glucosamine: 3-Aminobenzoyl-ACP Glycosyltransferase PctL in Pactamycin Biosynthesis.

Pactamycin is an antibiotic produced by Streptomyces pactum with antitumor and antimalarial properties. Pactamycin has a unique aminocyclitol core that is decorated with 3-aminoacetophenone, 6-methylsaliciate, and an N,N-dimethylcarbamoyl group. Herein, we show that the adenylation enzyme PctU activates 3-aminobenzoic acid (3ABA) with adenosine triphosphate and ligates it to the holo form of the discrete acyl carrier protein PctK to yield 3ABA-PctK. Then, 3ABA-PctK is N-glycosylated with uridine diphosphate-N-acetyl-d-glucosamine (UDP-GlcNAc) by the glycosyltransferase PctL to yield GlcNAc-3ABA-PctK. Because 3ABA is known to be a precursor of the 3-aminoacetophenone moiety, PctU appears to be a gatekeeper that selects the appropriate 3-aminobenzoate starter unit. Overall, we propose that acyl carrier protein-bound glycosylated 3ABA derivatives are biosynthetic intermediates of pactamycin biosynthesis.

Conserved Glu-47 and Lys-50 residues are critical for UDP-N-acetylglucosamine/UMP antiport activity of the mouse Golgi-associated transporter Slc35a3.

Nucleotide sugar transporters (NSTs) regulate the flux of activated sugars from the cytosol into the lumen of the Golgi apparatus where glycosyltransferases use them for the modification of proteins, lipids, and proteoglycans. It has been well-established that NSTs are antiporters that exchange nucleotide sugars with the respective nucleoside monophosphate. Nevertheless, information about the molecular basis of ligand recognition and transport is scarce. Here, using topology predictors, cysteine-scanning mutagenesis, expression of GFP-tagged protein variants, and phenotypic complementation of the yeast strain Kl3, we identified residues involved in the activity of a mouse UDP-GlcNAc transporter, murine solute carrier family 35 member A3 (mSlc35a3). We specifically focused on the putative transmembrane helix 2 (TMH2) and observed that cells expressing E47C or K50C mSlc35a3 variants had lower levels of GlcNAc-containing glycoconjugates than WT cells, indicating impaired UDP-GlcNAc transport activity of these two variants. A conservative substitution analysis revealed that single or double substitutions of Glu-47 and Lys-50 do not restore GlcNAc glycoconjugates. Analysis of mSlc35a3 and its genetic variants reconstituted into proteoliposomes disclosed the following: (i) all variants act as UDP-GlcNAc/UMP antiporters; (ii) conservative substitutions (E47D, E47Q, K50R, or K50H) impair UDP-GlcNAc uptake; and (iii) substitutions of Glu-47 and Lys-50 dramatically alter kinetic parameters, consistent with a critical role of these two residues in mSlc35a3 function. A bioinformatics analysis revealed that an EXXK motif in TMH2 is highly conserved across SLC35 A subfamily members, and a 3D-homology model predicted that Glu-47 and Lys-50 are facing the central cavity of the protein.

Identification and functional characterization of a bacterial homologue of Zeta toxin in Leishmania donovani.

Zeta-toxin is a cognate toxin of epsilon antitoxin of prokaryotic Type II toxin-antitoxin system (TA) and play an important role in cell death. An orthologue of bacterial-zeta-toxin (BzT) was identified in Leishmania donovani with similar structural and functional features. Leishmania zeta-toxin (named Ld_ζ1) harboring similar UNAG and ATP-binding pockets showed UNAG kinase and ATP-binding activity. An active Ld_ζ1 was found to express in infective extracellular promastigotes stage of L. donovani and episomal overexpression of an active Ld_ζ1domain-triggered cell death. This study demonstrates the presence of prokaryotic-like-zeta-toxin in eukaryotic parasite Leishmania and its association with cell death. Conceivably, phosphorylated UNAG or analogues, the biochemical mimics of zeta-toxin function mediating cell death can act as a novel anti-leishmanial chemotherapeutics.

SLC35A5 Protein-A Golgi Complex Member with Putative Nucleotide Sugar Transport Activity.

Solute carrier family 35 member A5 (SLC35A5) is a member of the SLC35A protein subfamily comprising nucleotide sugar transporters. However, the function of SLC35A5 is yet to be experimentally determined. In this study, we inactivated the SLC35A5 gene in the HepG2 cell line to study a potential role of this protein in glycosylation. Introduced modification affected neither N- nor O-glycans. There was also no influence of the gene knock-out on glycolipid synthesis. However, inactivation of the SLC35A5 gene caused a slight increase in the level of chondroitin sulfate proteoglycans. Moreover, inactivation of the SLC35A5 gene resulted in the decrease of the uridine diphosphate (UDP)-glucuronic acid, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine Golgi uptake, with no influence on the UDP-galactose transport activity. Further studies demonstrated that SLC35A5 localized exclusively to the Golgi apparatus. Careful insight into the protein sequence revealed that the C-terminus of this protein is extremely acidic and contains distinctive motifs, namely DXEE, DXD, and DXXD. Our studies show that the C-terminus is directed toward the cytosol. We also demonstrated that SLC35A5 formed homomers, as well as heteromers with other members of the SLC35A protein subfamily. In conclusion, the SLC35A5 protein might be a Golgi-resident multiprotein complex member engaged in nucleotide sugar transport.

Toxin ζ Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool.

Toxin ζ expression triggers a reversible state of dormancy, diminishes the pool of purine nucleotides, promotes (p)ppGpp synthesis, phosphorylates a fraction of the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG), leading to unreactive UNAG-P, induces persistence in a reduced subpopulation, and sensitizes cells to different antibiotics. Here, we combined computational analyses with biochemical experiments to examine the mechanism of toxin ζ action. Free ζ toxin showed low affinity for UNAG. Toxin ζ bound to UNAG hydrolyzed ATP·Mg2+, with the accumulation of ADP, Pi, and produced low levels of phosphorylated UNAG (UNAG-P). Toxin ζ, which has a large ATP binding pocket, may temporally favor ATP binding in a position that is distant from UNAG, hindering UNAG phosphorylation upon ATP hydrolysis. The residues D67, E116, R158 and R171, involved in the interaction with metal, ATP, and UNAG, were essential for the toxic and ATPase activities of toxin ζ; whereas the E100 and T128 residues were partially dispensable. The results indicate that ζ bound to UNAG reduces the ATP concentration, which indirectly induces a reversible dormant state, and modulates the pool of UNAG.

Distinct amino acid residues confer one of three UDP-sugar substrate specificities in Acinetobacter baumannii PglC phosphoglycosyltransferases.

Acinetobacter baumannii is an opportunistic human pathogen with the highest reported rates of multidrug resistance among Gram-negative pathogens. The capsular polysaccharide of A. baumannii is considered one of its most significant virulence factors providing resistance against complemented-mediated killing. Capsule synthesis in A. baumannii is usually initiated by the phosphoglycosyltransferase PglC. PglC transfers a phosphosugar from a nucleotide diphosphate-sugar to a polyprenol phosphate generating a polyprenol diphosphate-linked monosaccharide. Traditionally, PglC was thought to have stringent specificity towards UDP-N-N'-diacetylbacillosamine (UDP-diNAcBac). In this work we demonstrate that A. baumannii PglC has the ability to utilize three different UDP-sugar substrates: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc) or UDP-diNAcBac. Using phylogenetic analyses, we first demonstrate that A. baumannii PglC orthologs separate into three distinct clades. Moreover, all members within a clade are predicted to have the same preference for one of the three possible sugar substrates. To experimentally determine the substrate specificity of each clade, we utilized in vivo complementation models and NMR analysis. We demonstrate that UDP-diNAcBac is accommodated by all PglC orthologs, but some orthologs evolved to utilize UDP-GlcNAc or UDP-GalNAc in a clade-dependent manner. Furthermore, we show that a single point mutation can modify the sugar specificity of a PglC ortholog specific for UDP-diNAcBac and that introduction of a non-native PglC ortholog into A. baumannii can generate a new capsule serotype. Collectively, these studies begin to explain why A. baumannii strains have such highly diverse glycan repertoires.

UDP-sugar accumulation drives hyaluronan synthesis in breast cancer.

Increased uptake of glucose, a general hallmark of malignant tumors, leads to an accumulation of intermediate metabolites of glycolysis. We investigated whether the high supply of these intermediates promotes their flow into UDP-sugars, and consequently into hyaluronan, a tumor-promoting matrix molecule. We quantified UDP-N-Acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcUA) in human breast cancer biopsies, the levels of enzymes contributing to their synthesis, and their association with the hyaluronan accumulation in the tumor. The content of UDP-GlcUA was 4 times, and that of UDP-GlcNAc 12 times higher in the tumors as compared to normal glandular tissue obtained from breast reductions. The surge of UDP-GlcNAc correlated with an elevated mRNA expression of glutamine-fructose-6-phosphate aminotransferase 2 (GFAT2), one of the key enzymes in the biosynthesis of UDP-GlcNAc, and the expression of GFAT1 was also elevated. The contents of both UDP-sugars strongly correlated with tumor hyaluronan levels. Interestingly, hyaluronan content did not correlate with the mRNA levels of the hyaluronan synthases (HAS1-3), thus emphasizing the role of the UDP-sugar substrates of these enzymes. The UDP-sugars showed a trend to higher levels in ductal vs. lobular cancer subtypes. The results reveal for the first time a dramatic increase of UDP-sugars in breast cancer, and suggest that their high supply drives the accumulation of hyaluronan, a known promoter of breast cancer and other malignancies. In general, the study shows how the disturbed glucose metabolism typical for malignant tumors can influence cancer microenvironment through UDP-sugars and hyaluronan.

Bacterial RNA polymerase caps RNA with various cofactors and cell wall precursors.

Bacterial RNA polymerase is able to initiate transcription with adenosine-containing cofactor NAD+, which was proposed to result in a portion of cellular RNAs being 'capped' at the 5' end with NAD+, reminiscent of eukaryotic cap. Here we show that, apart from NAD+, another adenosine-containing cofactor FAD and highly abundant uridine-containing cell wall precursors, UDP-Glucose and UDP-N-acetylglucosamine are efficiently used to initiate transcription in vitro. We show that the affinity to NAD+ and UDP-containing factors during initiation is much lower than their cellular concentrations, and that initiation with them stimulates promoter escape. Efficiency of initiation with NAD+, but not with UDP-containing factors, is affected by amino acids of the Rifampicin-binding pocket, suggesting altered RNA capping in Rifampicin-resistant strains. However, relative affinity to NAD+ does not depend on the -1 base of the template strand, as was suggested earlier. We show that incorporation of mature cell wall precursor, UDP-MurNAc-pentapeptide, is inhibited by region 3.2 of σ subunit, possibly preventing targeting of RNA to the membrane. Overall, our in vitro results propose a wide repertoire of potential bacterial RNA capping molecules, and provide mechanistic insights into their incorporation.

Glucose and glutamine fuel protein O-GlcNAcylation to control T cell self-renewal and malignancy.

Sustained glucose and glutamine transport are essential for activated T lymphocytes to support ATP and macromolecule biosynthesis. We found that glutamine and glucose also fuel an indispensable dynamic regulation of intracellular protein O-GlcNAcylation at key stages of T cell development, transformation and differentiation. Glucose and glutamine are precursors of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a substrate for cellular glycosyltransferases. Immune-activated T cells contained higher concentrations of UDP-GlcNAc and increased intracellular protein O-GlcNAcylation controlled by the enzyme O-linked-ß-N-acetylglucosamine (O-GlcNAc) glycosyltransferase as compared with naive cells. We identified Notch, the T cell antigen receptor and c-Myc as key controllers of T cell protein O-GlcNAcylation via regulation of glucose and glutamine transport. Loss of O-GlcNAc transferase blocked T cell progenitor renewal, malignant transformation and peripheral T cell clonal expansion. Nutrient-dependent signaling pathways regulated by O-GlcNAc glycosyltransferase are thus fundamental for T cell biology.

UDP-sugar substrates of HAS3 regulate its O-GlcNAcylation, intracellular traffic, extracellular shedding and correlate with melanoma progression.

Hyaluronan content is a powerful prognostic factor in many cancer types, but the molecular basis of its synthesis in cancer still remains unclear. Hyaluronan synthesis requires the transport of hyaluronan synthases (HAS1-3) from Golgi to plasma membrane (PM), where the enzymes are activated. For the very first time, the present study demonstrated a rapid recycling of HAS3 between PM and endosomes, controlled by the cytosolic levels of the HAS substrates UDP-GlcUA and UDP-GlcNAc. Depletion of UDP-GlcNAc or UDP-GlcUA shifted the balance towards HAS3 endocytosis, and inhibition of hyaluronan synthesis. In contrast, UDP-GlcNAc surplus suppressed endocytosis and lysosomal decay of HAS3, favoring its retention in PM, stimulating hyaluronan synthesis, and HAS3 shedding in extracellular vesicles. The concentration of UDP-GlcNAc also controlled the level of O-GlcNAc modification of HAS3. Increasing O-GlcNAcylation reproduced the effects of UDP-GlcNAc surplus on HAS3 trafficking, while its suppression showed the opposite effects, indicating that O-GlcNAc signaling is associated to UDP-GlcNAc supply. Importantly, a similar correlation existed between the expression of GFAT1 (the rate limiting enzyme in UDP-GlcNAc synthesis) and hyaluronan content in early and deep human melanomas, suggesting the association of UDP-sugar metabolism in initiation of melanomagenesis. In general, changes in glucose metabolism, realized through UDP-sugar contents and O-GlcNAc signaling, are important in HAS3 trafficking, hyaluronan synthesis, and correlates with melanoma progression.

Hexosamine biosynthesis in keratinocytes: roles of GFAT and GNPDA enzymes in the maintenance of UDP-GlcNAc content and hyaluronan synthesis.

UDP-N-acetylglucosamine (UDP-GlcNAc) is a glucose metabolite with pivotal functions as a key substrate for the synthesis of glycoconjugates like hyaluronan, and as a metabolic sensor that controls cell functions through O-GlcNAc modification of intracellular proteins. However, little is known about the regulation of hexosamine biosynthesis that controls UDP-GlcNAc content. Four enzymes can catalyze the crucial starting point of the pathway, conversion of fructose-6-phosphate (Fru6P) to glucosamine-6-phosphate (GlcN6P): glutamine-fructose-6-phosphate aminotransferases (GFAT1 and 2) and glucosamine-6-phosphate deaminases (GNPDA1 and 2). Using siRNA silencing, we studied the contributions of these enzymes to UDP-GlcNAc content and hyaluronan synthesis in human keratinocytes. Depletion of GFAT1 reduced the cellular pool of UDP-GlcNAc and hyaluronan synthesis, while simultaneous blocking of both GNPDA1 and GDPDA2 exerted opposite effects, indicating that in standard culture conditions keratinocyte GNPDAs mainly catalyzed the reaction from GlcN6P back to Fru6P. However, when hexosamine biosynthesis was blocked by GFAT1 siRNA, the effect by GNPDAs was reversed, now catalyzing Fru6P towards GlcN6P, likely in an attempt to maintain UDP-GlcNAc content. Silencing of these enzymes also changed the gene expression of related enzymes: GNPDA1 siRNA induced GFAT2 which was hardly measurable in these cells under standard culture conditions, GNPDA2 siRNA increased GFAT1, and GFAT1 siRNA increased the expression of hyaluronan synthase 2 (HAS2). Silencing of GFAT1 stimulated GNPDA1 and GDPDA2, and inhibited cell migration. The multiple delicate adjustments of these reactions demonstrate the importance of hexosamine biosynthesis in cellular homeostasis, known to be deranged in diseases like diabetes and cancer.

A common sugar-nucleotide-mediated mechanism of inhibition of (glycosamino)glycan biosynthesis, as evidenced by 6F-GalNAc (Ac3).

Glycosaminoglycan (GAG) polysaccharides have been implicated in a variety of cellular processes, and alterations in their amount and structure have been associated with diseases such as cancer. In this study, we probed 11 sugar analogs for their capacity to interfere with GAG biosynthesis. One analog, with a modification not directly involved in the glycosidic bond formation, 6F-N-acetyl-d-galactosamine (GalNAc) (Ac3), was selected for further study on its metabolic and biologic effect. Treatment of human ovarian carcinoma cells with 50 µM 6F-GalNAc (Ac3) inhibited biosynthesis of GAGs (chondroitin/dermatan sulfate by ∼50-60%, heparan sulfate by ∼35%), N-acetyl-d-glucosamine (GlcNAc)/GalNAc containing glycans recognized by the lectins Datura stramonium and peanut agglutinin (by ∼74 and ∼43%, respectively), and O-GlcNAc protein modification. With respect to function, 6F-GalNAc (Ac3) treatment inhibited growth factor signaling and reduced in vivo angiogenesis by ∼33%. Although the analog was readily transformed in cells into the uridine 5'-diphosphate (UDP)-activated form, it was not incorporated into GAGs. Rather, it strongly reduced cellular UDP-GalNAc and UDP-GlcNAc pools. Together with data from the literature, these findings indicate that nucleotide sugar depletion without incorporation is a common mechanism of sugar analogs for inhibiting GAG/glycan biosynthesis.

Network integration of parallel metabolic and transcriptional data reveals metabolic modules that regulate macrophage polarization.

Macrophage polarization involves a coordinated metabolic and transcriptional rewiring that is only partially understood. By using an integrated high-throughput transcriptional-metabolic profiling and analysis pipeline, we characterized systemic changes during murine macrophage M1 and M2 polarization. M2 polarization was found to activate glutamine catabolism and UDP-GlcNAc-associated modules. Correspondingly, glutamine deprivation or inhibition of N-glycosylation decreased M2 polarization and production of chemokine CCL22. In M1 macrophages, we identified a metabolic break at Idh, the enzyme that converts isocitrate to alpha-ketoglutarate, providing mechanistic explanation for TCA cycle fragmentation. (13)C-tracer studies suggested the presence of an active variant of the aspartate-arginosuccinate shunt that compensated for this break. Consistently, inhibition of aspartate-aminotransferase, a key enzyme of the shunt, inhibited nitric oxide and interleukin-6 production in M1 macrophages, while promoting mitochondrial respiration. This systems approach provides a highly integrated picture of the physiological modules supporting macrophage polarization, identifying potential pharmacologic control points for both macrophage phenotypes.

Toxin ζ reversible induces dormancy and reduces the UDP-N-acetylglucosamine pool as one of the protective responses to cope with stress.

Toxins of the ζ/PezT family, found in the genome of major human pathogens, phosphorylate the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) leading to unreactive UNAG-3P. Transient over-expression of a PezT variant impairs cell wall biosynthesis and triggers autolysis in Escherichia coli. Conversely, physiological levels of ζ reversibly induce dormancy produce a sub-fraction of membrane-compromised cells, and a minor subpopulation of Bacillus subtilis cells become tolerant of toxin action. We report here that purified ζ is a strong UNAG-dependent ATPase, being GTP a lower competitor. In vitro, ζ toxin phosphorylates a fraction of UNAG. In vivo, ζ-mediated inactivation of UNAG by phosphorylation does not deplete the active UNAG pool, because expression of the toxin enhances the efficacy of genuine cell wall inhibitors (fosfomycin, vancomycin or ampicillin). Transient ζ expression together with fosfomycin treatment halt cell proliferation, but ε2 antitoxin expression facilitates the exit of ζ-induced dormancy, suggesting that there is sufficient UNAG for growth. We propose that ζ induces diverse cellular responses to cope with stress, being the reduction of the UNAG pool one among them. If the action of ζ is not inhibited, e.g., by de novo ε2 antitoxin synthesis, the toxin markedly enhances the efficacy of antimicrobial treatment without massive autolysis in Firmicutes.