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An important prelude to bacterial infection is the ability of a pathogen to survive independently of the host and to withstand environmental stress. The compatible solute trehalose has previously been connected with diverse abiotic stress tolerances, particularly osmotic shock. In this study, we combine molecular biology and biochemistry to dissect the trehalose metabolic network in the opportunistic human pathogen Pseudomonas aeruginosa PAO1 and define its role in abiotic stress protection. We show that trehalose metabolism in PAO1 is integrated with the biosynthesis of branched α-glucan (glycogen), with mutants in either biosynthetic pathway significantly compromised for survival on abiotic surfaces. While both trehalose and α-glucan are important for abiotic stress tolerance, we show they counter distinct stresses. Trehalose is important for the PAO1 osmotic stress response, with trehalose synthesis mutants displaying severely compromised growth in elevated salt conditions. However, trehalose does not contribute directly to the PAO1 desiccation response. Rather, desiccation tolerance is mediated directly by GlgE-derived α-glucan, with deletion of the glgE synthase gene compromising PAO1 survival in low humidity but having little effect on osmotic sensitivity. Desiccation tolerance is independent of trehalose concentration, marking a clear distinction between the roles of these two molecules in mediating responses to abiotic stress., Author summary Author summary To survive outside their host, pathogenic bacteria must withstand various environmental stresses. The sugar molecule trehalose is associated with a range of abiotic stress tolerances, particularly osmotic shock. In this study, we analyse the trehalose metabolic network in the human pathogen Pseudomonas aeruginosa PAO1 and define its role in abiotic stress protection. We show that trehalose metabolism in PAO1 is intimately connected to the biosynthesis of branched α-glucan, or glycogen. Disruption of either trehalose or glycogen biosynthesis significantly reduces the ability of PAO1 to survive on steel work surfaces. While both trehalose and glycogen are important for stress tolerance, they counter very different stresses. Trehalose is important for the osmotic stress response, and survival in conditions of elevated salt. On the other hand, glycogen is responsible for desiccation tolerance and survival in low humidity environments. Trehalose does not apparently contribute to desiccation tolerance, marking a clear distinction between the roles of trehalose and glycogen in mediating abiotic stress responses in P. aeruginosa.

α-Glucans are not only abundant in biological systems, but are also of industrial importance. For example, starch, with its α-1,4-linked amylose component, is a carbon storage compound, major dietary component and industrial feedstock with uses in the biofuel, pharmaceutical, textile and paper industries on a 109 tons year–1 scale.1,2 α-Glucans related to starch also exist in bacteria and are involved in carbon storage and immune evasion.3 α-1,6-Linked dextran and α-1,4- and α-1,6-linked pullulan are associated with microbial biofilms and have uses in foods, medicine, cosmetics and research.4,5 The composition, biosynthesis, modification and degradation of such molecules are not fully understood and this is largely because the analysis of complex biological α-glucan samples with a range of degrees of polymerisation (DP; i.e. the number of glucose units) can be challenging. Chromatography with or without derivatisation followed by compound identification is a well-established strategy that can provide useful information, but at the expense of time and sensitivity. Despite the versatility of traditional mass spectrometric methods, isobaric carbohydrates such as α-glucans add additional challenges. Several new approaches have been developed to tackle isobaric carbohydrates such as ion mobility mass spectrometry (IMMS) and energy-resolved collision-induced dissociation.6 IMMS provides a two-dimensional (2D) method of separating ions on the basis of shape through mobility within a gas-filled drift tube and then m/z. Collision cross-sections (CCSs) of ions can be determined directly with some instruments and estimated with others through calibration with standards. Most reported analyses are in the positive mode with alkali metal coordinated ions,7–16 with relatively few in the negative mode.17 Recent examples of IMMS being able to differentiate isobaric oligosaccharides include N-linked glycans associated with proteins,8–12 glycosaminoglycans18 and other small oligosaccharides14 including a few glucans with a DP of up to 5.13,15–17 It has been possible to resolve isobaric carbohydrates with different primary structures, branching patterns and anomeric/positional glycosidic linkages. More recent advances included combining IMMS with tandem mass spectrometry14,17,19 and the development of tandem IMMS.13 However, there are no reports of a systematic analysis of larger linear homo- and hetero-oligomeric α-glucans using IMMS establishing whether conformational differences in aqueous solution are retained with such large molecules in the gas phase. We now show that linear homo- and hetero-oligomeric α-glucans with a DP of up to 35, which differ in only their content of α-1,4- and α-1,6-linkages, can be resolved using IMMS in the negative mode with ion mobilities consistent with known molecular conformations in solution. In addition, calibration with α,ω-dicarboxy-terminated polystyrene20 allows the CCS of each species to be estimated. Furthermore, the ability to identify with IMMS a product of the Mycobacterium tuberculosis GlgB branching enzyme within a complex maltooligosaccharide mixture is also demonstrated.

Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of M. tuberculosis allowing the regulated gene silencing of the T6P phosphatase gene otsB2. We found that otsB2 is essential for growth of M. tuberculosis in vitro as well as for the acute infection phase in mice following aerosol infection. By contrast, otsB2 is not essential for the chronic infection phase in mice, highlighting the substantial remodelling of trehalose metabolism during infection by M. tuberculosis. Blocking OtsB2 resulted in the accumulation of its substrate T6P, which appears to be toxic, leading to the self-poisoning of cells. Accordingly, blocking T6P production in a ΔotsA mutant abrogated otsB2 essentiality. T6P accumulation elicited a global upregulation of more than 800 genes, which might result from an increase in RNA stability implied by the enhanced neutralization of toxins exhibiting ribonuclease activity. Surprisingly, overlap with the stress response caused by the accumulation of another toxic sugar phosphate molecule, maltose-1-phosphate, was minimal. A genome-wide screen for synthetic lethal interactions with otsA identified numerous genes, revealing additional potential drug targets synergistic with OtsB2 suitable for combination therapies that would minimize the emergence of resistance to OtsB2 inhibitors., Author Summary Trehalose biosynthesis is considered an attractive target for the development of new drugs against various microbial pathogens including Mycobacterium tuberculosis. In this human pathogen, two partially redundant pathways mediate trehalose biosynthesis. The OtsA-OtsB2 pathway, which dominates in culture, involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB2. While OtsA is dispensable, OtsB2 is strictly essential for growth of M. tuberculosis. Using conditional gene silencing, we here show that essentiality of OtsB2 is linked to accumulation of its substrate T6P, which exhibits direct or indirect toxic effects. Regulated gene expression in vivo revealed that OtsB2 is required to establish an acute infection of M. tuberculosis in a mouse infection model, but is surprisingly fully dispensable during the chronic infection phase. This highlights that trehalose metabolism of M. tuberculosis is substantially remodelled during infection.

GlgE is a maltosyltransferase involved in α-glucan biosynthesis in bacteria that has been genetically validated as a target for tuberculosis therapies. Crystals of the Mycobacterium tuberculosis enzyme diffract at low resolution so most structural studies have been with the very similar Streptomyces coelicolor GlgE isoform 1. Although the donor binding site for α-maltose 1-phosphate had been previously structurally defined, the acceptor site had not. Using mutagenesis, kinetics, and protein crystallography of the S. coelicolor enzyme, we have now identified the +1 to +6 subsites of the acceptor/product, which overlap with the known cyclodextrin binding site. The sugar residues in the acceptor subsites +1 to +5 are oriented such that they disfavor the binding of malto-oligosaccharides that bear branches at their 6-positions, consistent with the known acceptor chain specificity of GlgE. A secondary binding site remote from the catalytic center was identified that is distinct from one reported for the M. tuberculosis enzyme. This new site is capable of binding a branched α-glucan and is most likely involved in guiding acceptors toward the donor site because its disruption kinetically compromises the ability of GlgE to extend polymeric substrates. However, disruption of this site, which is conserved in the Streptomyces venezuelae GlgE enzyme, did not affect the growth of S. venezuelae or the structure of the polymeric product. The acceptor subsites +1 to +4 in the S. coelicolor enzyme are well conserved in the M. tuberculosis enzyme so their identification could help inform the design of inhibitors with therapeutic potential.

Mycobacterium tuberculosis synthesizes intra- and extracellular α-glucans that were believed to originate from separate pathways. The extracellular glucose polymer is the main constituent of the mycobacterial capsule that is thought to be involved in immune evasion and virulence. However, the role of the α-glucan capsule in pathogenesis has remained enigmatic due to an incomplete understanding of α-glucan biosynthetic pathways preventing the generation of capsule-deficient mutants. Three separate and potentially redundant pathways had been implicated in α-glucan biosynthesis in mycobacteria: the GlgC-GlgA, the Rv3032 and the TreS-Pep2-GlgE pathways. We now show that α-glucan in mycobacteria is exclusively assembled intracellularly utilizing the building block α-maltose-1-phosphate as the substrate for the maltosyltransferase GlgE, with subsequent branching of the polymer by the branching enzyme GlgB. Some α-glucan is exported to form the α-glucan capsule. There is an unexpected convergence of the TreS-Pep2 and GlgC-GlgA pathways that both generate α-maltose-1-phosphate. While the TreS-Pep2 route from trehalose was already known, we have now established that GlgA forms this phosphosugar from ADP-glucose and glucose 1-phosphate 1000-fold more efficiently than its hitherto described glycogen synthase activity. The two routes are connected by the common precursor ADP-glucose, allowing compensatory flux from one route to the other. Having elucidated this unexpected configuration of the metabolic pathways underlying α-glucan biosynthesis in mycobacteria, an M. tuberculosis double mutant devoid of α-glucan could be constructed, showing a direct link between the GlgE pathway, α-glucan biosynthesis and virulence in a mouse infection model., Author Summary Capsule formation is critical for the virulence of many bacterial and fungal pathogens. Mycobacterium tuberculosis cells are known to be surrounded by a capsule layer that is mainly composed of an α-glucan glucose polymer that resembles glycogen. Progress in understanding its role in the virulence of this important human pathogen has been held back by a lack of knowledge of its biosynthesis, preventing the generation of α-glucan-deficient mutants that could be tested in animal infection models. In this work, we unraveled an unexpected metabolic network configuration revealing the exclusive production of both intracellular and capsular α-glucans by the maltosyltransferase GlgE in mycobacteria. GlgE polymerizes an α-maltose 1-phosphate building block, which is generated by two alternative pathways that are connected by a common intermediate allowing rechanneling of flux from one route to the other. Elucidation of this unexpected configuration of the metabolic pathways underlying α-glucan biosynthesis allowed the rational construction of an M. tuberculosis mutant strain devoid of α-glucan, showing a direct link between the GlgE pathway, α-glucan biosynthesis and virulence in a mouse infection model.

Background Mycobacterium tuberculosis is a pathogenic prokaryote adapted to survive in hostile environments. In this organism and other Gram-positive actinobacteria, the metabolic pathways of glycogen and trehalose are interconnected. Results In this work we show the production, purification and characterization of recombinant enzymes involved in the partitioning of glucose-1-phosphate between glycogen and trehalose in M. tuberculosis H37Rv, namely: ADP-glucose pyrophosphorylase, glycogen synthase, UDP-glucose pyrophosphorylase and trehalose-6-phosphate synthase. The substrate specificity, kinetic parameters and allosteric regulation of each enzyme were determined. ADP-glucose pyrophosphorylase was highly specific for ADP-glucose while trehalose-6-phosphate synthase used not only ADP-glucose but also UDP-glucose, albeit to a lesser extent. ADP-glucose pyrophosphorylase was allosterically activated primarily by phosphoenolpyruvate and glucose-6-phosphate, while the activity of trehalose-6-phosphate synthase was increased up to 2-fold by fructose-6-phosphate. None of the other two enzymes tested exhibited allosteric regulation. Conclusions Results give information about how the glucose-1-phosphate/ADP-glucose node is controlled after kinetic and regulatory properties of key enzymes for mycobacteria metabolism. General significance This work increases our understanding of oligo and polysaccharides metabolism in M. tuberculosis and reinforces the importance of the interconnection between glycogen and trehalose biosynthesis in this human pathogen., Highlights • Nucleotide-glucose synthesis in Mycobacterium tuberculosis was analyzed. • The characterization of four enzymes involved in glucose-1P partitioning is reported. • Mycobacterial ADP-glucose pyrophosphorylase is allosterically regulated. • Trehalose-6P synthase exhibits higher catalytic efficiency for ADP-glucose. • Trehalose-6P synthase is activated by fructose-6P.

Tomato (Solanum lycopersicum), like other Solanaceous species, accumulates high levels of antioxidant caffeoylquinic acids, which are strong bioactive molecules and protect plants against biotic and abiotic stresses. Among these compounds, the monocaffeoylquinic acids (e.g. chlorogenic acid [CGA]) and the dicaffeoylquinic acids (diCQAs) have been found to possess marked antioxidative properties. Thus, they are of therapeutic interest both as phytonutrients in foods and as pharmaceuticals. Strategies to increase diCQA content in plants have been hampered by the modest understanding of their biosynthesis and whether the same pathway exists in different plant species. Incubation of CGA with crude extracts of tomato fruits led to the formation of two new products, which were identified by liquid chromatography-mass spectrometry as diCQAs. This chlorogenate:chlorogenate transferase activity was partially purified from ripe fruit. The final protein fraction resulted in 388-fold enrichment of activity and was subjected to trypsin digestion and mass spectrometric sequencing: a hydroxycinnamoyl-Coenzyme A:quinate hydroxycinnamoyl transferase (HQT) was selected as a candidate protein. Assay of recombinant HQT protein expressed in Escherichia coli confirmed its ability to synthesize diCQAs in vitro. This second activity (chlorogenate:chlorogenate transferase) of HQT had a low pH optimum and a high Km for its substrate, CGA. High concentrations of CGA and relatively low pH occur in the vacuoles of plant cells. Transient assays demonstrated that tomato HQT localizes to the vacuole as well as to the cytoplasm of plant cells, supporting the idea that in this species, the enzyme catalyzes different reactions in two subcellular compartments.

GlgE is identified as a maltosyltransferase that catalyzes the polymerization step in a previously undescribed pathway in Mycobacterium tuberculosis for converting trehalose to α-glucan. A combination of traditional and chemical genetic strategies suggest GlgE to be a viable therapeutic target. New chemotherapeutics are urgently required to control the tuberculosis pandemic. We describe a new pathway from trehalose to α-glucan in Mycobacterium tuberculosis comprising four enzymatic steps mediated by TreS, Pep2, GlgE (which has been identified as a maltosyltransferase that uses maltose 1-phosphate) and GlgB. Using traditional and chemical reverse genetics, we show that GlgE inactivation causes rapid death of M. tuberculosis in vitro and in mice through a self-poisoning accumulation of maltose 1-phosphate. Poisoning elicits pleiotropic phosphosugar-induced stress responses promoted by a self-amplifying feedback loop where trehalose-forming enzymes are upregulated. Moreover, the pathway from trehalose to α-glucan exhibited a synthetic lethal interaction with the glucosyltransferase Rv3032, which is involved in biosynthesis of polymethylated α-glucans, because key enzymes in each pathway could not be simultaneously inactivated. The unique combination of maltose 1-phosphate toxicity and gene essentiality within a synthetic lethal pathway validates GlgE as a distinct potential drug target that exploits new synergistic mechanisms to induce death in M. tuberculosis.

Summary Trehalose synthase (TreS) was thought to catalyze flux from maltose to trehalose, a precursor of essential trehalose mycolates in mycobacterial cell walls. However, we now show, using a genetic approach, that TreS is not required for trehalose biosynthesis in Mycobacterium smegmatis, whereas two alternative trehalose-biosynthetic pathways (OtsAB and TreYZ) are crucial. Consistent with this direction of flux, trehalose levels in Mycobacterium tuberculosis decreased when TreS was overexpressed. In addition, TreS was shown to interconvert the α anomer of maltose and trehalose using 1H and 19F-nuclear magnetic resonance spectroscopies using its normal substrates and deoxyfluoromaltose analogs, with the nonenzymatic mutarotation of α/β-maltose being slow. Therefore, flux through TreS in mycobacteria flows from trehalose to α-maltose, which is the appropriate anomer for maltose kinase of the GlgE α-glucan pathway, which in turn contributes to intracellular and/or capsular polysaccharide biosynthesis., Graphical Abstract Highlights ► Flux through trehalose synthase (TreS) in mycobacteria is from trehalose to maltose ► The appropriate α anomer is formed by TreS for maltose kinase of the GlgE pathway ► The specificity of TreS for α-maltose is retained with deoxyfluoro analogs ► TreS supports cytosolic/capsular α-glucan but not trehalose mycolate biosynthesis, It is currently thought that trehalose synthase contributes to the biosynthesis of trehalose mycolates destined for mycobacterial cell walls. Miah et al. show that flux through this enzyme is in the reverse direction from trehalose to maltose and that the appropriate α anomer is generated for the GlgE α-glucan pathway.