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The fall armyworm (FAW), Spodoptera frugiperda is a cold-sensitive species that overwinters in temperate climates without diapause. Overwintering in insects involves rapid cold hardening (RCH), supported by trehalose (TRE), which serves as an intermediate between glycogen (GLY) and glucose (GLU). However, both GLU and TRE help maintain homeostasis under stress. TRE is hydrolyzed by the enzyme trehalase (Treh) into GLU. This study retrieved Sf-Treh1a, Sf-Treh1b, and Sf-Treh2 from the FAW transcriptome analysis. RNA interference (RNAi) targeting these three Treh genes resulted in significant downregulation of mRNA levels and altered survival rates in RNAi-treated FAW larvae following RCH treatment. High-pressure liquid chromatography (HPLC) quantification of TRE and GLU in treated groups suggests that GLU is an essential component of the hemolymph for survival adaptation to cold conditions in S. frugiperda. This study reveals limited cold adaptability of FAW, as evidenced by lower glucose concentration levels. We found that FAW requires alternative molecules, in conjunction with glucose and trehalose for freeze tolerance and survivability. Our study aims to discover the molecular mechanisms that contribute to freeze tolerance in FAW by exploring the roles of trehalose, glucose, and glycogen., (© 2024. The Author(s).)

In the yeast Saccharomyces cerevisiae, 2 types of trehalase activities have been described. Neutral trehalases (Nth1 and Nth2) are considered to be the main proteins that catalyze intracellular trehalose mobilization. In addition to Nth1 and Nth2, studies have shown that acid trehalase Ath1 is required for extracellular trehalose degradation. Although both neutral and acid-type trehalases have been predominantly investigated in laboratory strains of S. cerevisiae, we sought to examine the phenotypic consequences of disrupting these genes in wild strains. In this study, we constructed mutants of the trehalose degradation pathway (NTH1, NTH2, and ATH1) in 5 diverse S. cerevisiae strains to examine whether published lab strain phenotypes are also exhibited by wild strains. For each mutant, we assessed a number of phenotypes for comparison to trehalose biosynthesis mutants, including trehalose production, glycogen production, cell size, acute thermotolerance, high-temperature growth, sporulation efficiency, and growth on a variety of carbon sources in rich and minimal medium. We found that all trehalase mutants including single deletion nth1Δ, nth2Δ, and ath1Δ, as well as double deletion nth1nth2Δ, accumulated higher intracellular trehalose levels compared to their isogenic wild-type cells. Also, nth1Δ and nth1Δnth2Δ mutants exhibited mild thermal sensitivity, suggesting a potential minor role for trehalose mobilization when cells recover from stress. In addition, we evaluated phenotypes more directly relevant to trehalose degradation, including both extracellular and intracellular trehalose utilization. We discovered that intracellular trehalose hydrolysis is critical for typical spore germination progression, highlighting a role for trehalose in cell cycle regulation, likely as a storage carbohydrate providing glycolytic fuel. Additionally, our work provides further evidence suggesting Ath1 is indispensable for extracellular trehalose utilization as a carbon source, even in the presence of AGT1., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

In Escherichia coli , the disaccharide trehalose can be metabolized as a carbon source or be accumulated as an osmoprotectant under osmotic stress. In hypertonic environments, E. coli accumulates trehalose in the cell by synthesis from glucose mediated by the cytosolic enzymes OtsA and OtsB. Trehalose in the periplasm can be hydrolyzed into glucose by the periplasmic trehalase TreA. We have previously shown that a treA mutant of extraintestinal E. coli strain BEN2908 displayed increased resistance to osmotic stress by 0.6 M urea, and reduced production of type 1 fimbriae, reduced invasion of avian fibroblasts, and decreased bladder colonization in a murine model of urinary tract infection. Since loss of TreA likely results in higher periplasmic trehalose concentrations, we wondered if deletion of otsA and otsB genes, which would lead to decreased internal trehalose concentrations, would reduce resistance to stress by 0.6 M urea and promote type 1 fimbriae production. The BEN2908Δ otsBA mutant was sensitive to osmotic stress by urea, but displayed an even more pronounced reduction in production of type 1 fimbriae, with the consequent reduction in adhesion/invasion of avian fibroblasts and reduced bladder colonization in the murine urinary tract. The BEN2908Δ treAotsBA mutant also showed a reduction in production of type 1 fimbriae, but in contrast to the Δ otsBA mutant, resisted better than the wild type in the presence of urea. We hypothesize that, in BEN2908, resistance to stress by urea would depend on the levels of periplasmic trehalose, but type 1 fimbriae production would be influenced by the levels of cytosolic trehalose., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Klemberg, Pavanelo, Houle, Dhakal, Pokharel, Iahnig-Jacques, Dozois and Horn.)

Bacteria possess diverse metabolic and genetic processes, resulting in the inability of certain bacteria to degrade trehalose. However, some bacteria do have the capability to degrade trehalose, utilizing it as a carbon source, and for defense against environmental stress. Trehalose, a disaccharide, serves as a carbon source for many bacteria, including some that are vital for pathogens. The degradation of trehalose is carried out by enzymes like trehalase (EC 3.2.1.28) and trehalose phosphorylase (EC 2.4.1.64/2.4.1.231), which are classified under the glycoside hydrolase families GH37, GH15, and GH65. Numerous studies and reports have explored the physiological functions, recombinant expression, enzymatic characteristics, and potential applications of these enzymes. However, further research is still being conducted to understand their roles in bacteria. This review aims to provide a comprehensive summary of the current understanding of trehalose degradation pathways in various bacteria, focusing on three key areas: (i) identifying different trehalose-degrading enzymes in Gram-positive and Gram-negative bacteria, (ii) elucidating the mechanisms employed by trehalose-degrading enzymes belonging to the glycoside hydrolases GH37, GH15, and GH65, and (iii) discussing the potential applications of these enzymes in different sectors. Notably, this review emphasizes the bacterial trehalose-degrading enzymes, specifically trehalases (GH37, GH15, and GH65) and trehalose phosphorylases (GH65), in both Gram-positive and Gram-negative bacteria, an aspect that has not been highlighted before., (© The Author(s) 2023. Published by Oxford University Press.)

Trehalase is an important enzyme in the metabolic cascades of many organisms, catalysing the hydrolysis of the disaccharide trehalose. Herein we describe the first examples of fluorometric nanoprobes for detection of trehalase, based on trehalose-functionalised quantum dots (QDs). QDs cross-linked with trehalose form aggregates, which are released upon enzymatic cleavage of the trehalose glycosidic bond proportionally to the enzyme concentration, offering a unique and efficient approach for specific sensing of this biologically important enzyme.

Trehalase is widely used in industrial fermentation, food, medicine and other fields. There is a lack of industrial varieties of trehalase with excellent performance in China. Moreover, the applied research on trehalase was not well conducted. In this study, a strain of Pectobacterium cypripedii was screened from nature, and the gene PCTre encoding an acidic trehalase was cloned and expressed in E . coli BL21(DE3). The highest enzyme activity reached 4130 U/mL after fermenting in a 5 L fermenter for 28 h. The enzymatic properties study showed that PCTre hydrolyzed trehalose specifically. The optimum pH and temperature were 5.5 and 35 ℃, respectively. 80% of the enzyme activity was retained after being treated at pH 4.0, 4.5, and 5.0 for 8 h, showing good acid tolerance. Moreover, it has good tolerance to organic solvents, 60% enzyme activity was retained after being treated with 20% ( V / V ) ethanol solution for 24 h. Furthermore, trehalose could be completely hydrolyzed within 16 h in a simulated fermentation system containing 20% ( V / V ) ethanol and 7.5% trehalose, with 500 U/L PCTre added. This indicated a good application potential for industrial ethanol fermentation.

Nonreducing disaccharide trehalose is used as a stabilizer and humectant in various products and is a potential medicinal drug, showing curative effects on the animal models of various diseases. However, its use is limited as it is hydrolyzed by trehalase, a widely expressed enzyme in multiple organisms. Several trehalose analogs are prepared, including a microbial metabolite 4-trehalosamine, and their high biological stability is confirmed. For further analysis, 4-trehalosamine is selected as it shows high producibility. Compared with trehalose, 4-trehalosamine exhibits better or comparable protective activities and a high buffer capacity around the neutral pH. Another advantage of 4-trehalosamine is its chemical modifiability: simple reactions produce its various derivatives. Labeled probes and detergents are synthesized in one-pot reactions to exemplify the feasibility of their production, and their utility is confirmed for their respective applications. The labeled probes are used for mycobacterial staining. Although the derivative detergents can be effectively used in membrane protein research, long-chain detergents show 1000-3000-fold stronger autophagy-inducing activity in cultured cells than trehalose and are expected to become a drug lead and research reagent. These results indicate that 4-trehalosamine is a useful trehalose substitute for various purposes and a material to produce new useful derivative substances., (© 2022 The Authors. Advanced Biology published by Wiley-VCH GmbH.)

The α-diglucoside trehalose has historically been known as a component of the bacterial stress response, though it more recently has been studied for its relevance in human gut health and biotechnology development. The utilization of trehalose as a nutrient source by bacteria relies on carbohydrate-active enzymes, specifically those of the glycoside hydrolase family 37 (GH37), to degrade the disaccharide into substituent glucose moieties for entry into metabolism. Environmental bacteria using oligosaccharides for nutrients often possess multiple carbohydrate-active enzymes predicted to have the same biochemical activity and therefore are thought to be functionally redundant. In this study, we characterized trehalose degradation by the biotechnologically important saprophytic bacterium Cellvibrio japonicus This bacterium possesses two predicted α-α-trehalase genes, tre37A and tre37B , and our investigation using mutational analysis found that only the former is essential for trehalose utilization by C. japonicus Heterologous expression experiments found that only the expression of the C. japonicus tre37A gene in an Escherichia coli mutant strain allowed for full utilization of trehalose. Biochemical characterization of treA mutant strain allowed for full utilization of trehalose. Biochemical characterization of C. japonicus gene product is solely responsible for cleaving trehalose and is an acidic α-α-trehalase. Bioinformatic and mutational analyses indicate that Tre37A directly cleaves trehalose to glucose in the periplasm, as tre37A gene product is solely responsible for cleaving trehalose and is an acidic α-α-trehalase. Bioinformatic and mutational analyses indicate that Tre37A directly cleaves trehalose to glucose in the periplasm, as C. japonicus does not possess a phosphotransferase system. This study facilitates the development of a comprehensive metabolic model for α-linked disaccharides in C. japonicus The metabolism of trehalose is becoming increasingly important due to the inclusion of this α-diglucoside in a number of foods and its prevalence in the environment. Bacteria able to utilize trehalose in the human gut possess a competitive advantage, as do saprophytic microbes in terrestrial environments. While the biochemical mechanism of trehalose degradation is well understood, what is less clear is how bacteria acquire this metabolite from the environment. The significance of this report is that by using the model saprophyte IMPORTANCE The metabolism of trehalose is becoming increasingly important due to the inclusion of this α-diglucoside in a number of foods and its prevalence in the environment. Bacteria able to utilize trehalose in the human gut possess a competitive advantage, as do saprophytic microbes in terrestrial environments. While the biochemical mechanism of trehalose degradation is well understood, what is less clear is how bacteria acquire this metabolite from the environment. The significance of this report is that by using the model saprophyte Cellvibrio japonicus , we were able to functionally characterize the two predicted trehalase enzymes that the bacterium possesses and determined that the two enzymes are not equivalent and are not functionally redundant. The results and approaches used to understand the complex physiology of α-diglucoside metabolism from this study can be applied broadly to other polysaccharide-degrading bacteria., (Copyright © 2020 American Society for Microbiology.)

Harmonia axyridis is a major bio-control agent of pests in agriculture and forest ecosystems. It is also a globally important invasive insect species. To test whether dark elytra colour is associated with greater cold hardiness, we compared the survival rate of prolonged cold exposure in both yellow and black colour morphs of female and male H. axyridis. We determined the trehalose and glycogen content, trehalase activity, and the dynamics of genes associated with the trehalose metabolic pathway. Yellow forms predominated before winter began, however black forms increased from 11.15 to 30.46% after overwintering. There was no significant difference in trehalose content between the females and males during overwintering. Glycogen content in over-wintering yellow females and black males increased significantly, while it decreased in black females. Soluble trehalase activity increased significantly in all the insects except black females. Membrane-bound trehalase activity increased in black males, and decreased in black females. Trehalose and glycogen content and trehalase activity were regulated by differential expression of TRE and TPS genes. Female beetles weighed more than males and survived in low temperatures for longer periods of time, regardless of elytra colour, suggesting that mass is a stronger predictor of overwintering survival rather than colour morph. Our results provide a guide for comparing cold resistance in insects and a theoretical basis for cold storage of H. axyridis for use as natural enemies of pests in biological control programs., Competing Interests: The authors declare no competing interests.

Trehalose is a stable disaccharide that consists of two glucose units linked primarily by an α,α-(1 → 1)-linkage, and it has been found in a wide variety of organisms. In these organisms, trehalose functions not only as a source of carbon energy but also as a protector against various stress conditions. In addition, this disaccharide is attractive for use in a wide range of applications due to its bioactivities. In trehalose metabolism, direct trehalose-hydrolyzing enzymes are known as trehalases, which have been reported for bacteria, archaea, and eukaryotes, and are classified into glycoside hydrolase 37 (GH37), GH65, and GH15 families according to the Carbohydrate-Active enZyme (CAZy) database. The catalytic domains (CDs) of these enzymes commonly share (α/α) 6 -barrel structures and have two amino acid residues, Asp and/or Glu, that function as catalytic residues in an inverting mechanism. In this review, I focus on diverse and common features of trehalases within different GH families and their contributions to microbial trehalose metabolism.

Objective: To examine the effect of low temperature on trehalose and trehalase levels in Culex pipiens pallens ., Methods: The fourth instar larvae and female adult mosquitoes of Cx. pipiens pallens were exposed at 4 ℃ for 0, 1, 3, 6, 12, 18, 24 h and 0, 1, 3, 6, 12, 18, 24, 36, 48, 72 h, respectively. Then, the trehalose and trehalase contents were detected by enzyme-linked immunosorbent assay (ELISA) in mosquitoes., Results: The contents of trehalose and trehalase significantly increased in the larval and female adult mosquitoes post-exposure to low temperature. The changing trend of trehalose levels was consistent in the larval and female adult mosquitoes, and the highest levels were (2.458 8 ± 0.379 2) mg/g and (2.825 7 ± 0.211 1) mg/g 3 h post-exposure to low temperature, respectively. The trehalose and trehalase levels fluctuated greatly within the first 6 h post-exposure to low temperature. Following adaptation for a period of time, the trehalose and trehalase levels remained at a relatively high level., Conclusions: Low temperature may induce the production of trehalose and trehalase in Cx. pipiens pallens , and the trehalose and trehalase may play an important role in the improvement of the cold resistance.

Trehalase catalyses the breakdown of trehalose into two glucose moieties and is ubiquitous in all organisms. Here, we provide insights into the enigmatic origin and evolution of trehalase in major species. Study of taxonomic distribution, orthology, phylogeny and functional domains indicated that trehalase possibly originates from bacteria and was transmitted to other taxa through horizontal gene transfer. Domain analysis showed that glycosyl hydrolase family 37 is present in most of the sequences and represents dominant activity during evolution, and also, illustrating that cytosolic trehalase is primitive than its transmembrane form. Furthermore, it was observed that trehalase went through domain rearrangement to facilitate its activity in adverse environmental conditions like acidic pH. Gene context analysis depicts that trehalase neighbourhood consists of sugar transport and lipid metabolism genes. This highlights their relatedness in metabolic activity and similarity in gene regulation, respectively. Evolutionary and selection pressure analysis demonstrated that trehalase genes were duplicated and evolved under purifying selection, following horizontal gene transfer. Moreover, site-specific rate of evolution emphasized conservation of functionally important residues. In comparison with acid trehalase, neutral trehalase has an extra N-terminal extension. This study serves as an instigation to understand evolution and functionality of trehalase across diverse species. Communicated by Ramaswamy H. Sarma.

Trehalose is used as an additive in thousands of foods, cosmetics, and pharmaceutical products, and it is being investigated as a therapeutic for multiple human diseases. However, its ability to be used as a carbon source by microbes is a concern, as highlighted by the recent finding that trehalose can be metabolized by and potentially enhance the virulence of epidemic Clostridioides difficile. Here, we show that trehalose analogues designed to resist enzymatic degradation are incapable of being used as carbon sources by C. difficile. Furthermore, we demonstrate that trehalose analogues, but not the known trehalase inhibitor validamycin A, inhibit native trehalose utilization by hypervirulent C. difficile. Thus, degradation-resistant trehalose analogues are valuable as trehalase inhibitors and as surrogates for or co-additives with trehalose in applications where enzymatic breakdown is a concern.

Trehalose on both sides of the bilayer is a requirement for full protection of membranes against stress. It was not known yet how trehalose, synthesized in the cytosol when dividing Saccharomyces cerevisiae cells are shifted from 28°C to 40°C, is transported to the outside and degraded when cells return to 28°C. According to our results, the lack of Agt1, a trehalose transporter, although had not affected trehalose synthesis, reduced cell tolerance to 51°C and increased lipid peroxidation. The damage was reversed when external trehalose was added during 40°C adaptation, confirming that the reason for the agt1Δ sensitivity is the absence of trehalose at the outside of the lipid bilayer. The 40-28°C condition caused cytosolic trehalase (Nth1) activation, reducing intracellular trehalose and, consequently, the survival rates after 51°C. Although lower than nth1Δ strain, cells deficient in acid trehalase (ath1Δ)  maintained increased trehalose levels after 40°C-28°C shift, which conferred protection against 51°C. Both Ath1 and Agt1 were found into vesicles near to plasma membrane in response to stress. This suggests that Agt1 containing vesicles would fuse with the membrane under 40°C to transport part of the cytosolic trehalose to the outside. By a similar mechanism, Ath1 would reach the cell surface to hydrolyze the external trehalose but only when the stress would be over. Corroborating this conclusion, Ath1 activity in soluble cell-free extracts increased after 40°C adaptation but decreased when cells returned to 28°C. During 40°C, Ath1 is confined into vesicles, avoiding the cleavage of the outside trehalose.

A disaccharide, trehalose, is a main hemolymph sugar of the legume pod borer, Maruca vitrata larvae, but its titers fluctuated with feeding activity. During diurnal feeding in the photophase, hemolymph trehalose remained at a relatively low level (69 mM) and increased (98 mM) during scotophase. Starvation significantly increased the hemolymph trehalose level, in which the elevation of trehalose titers was dependent on the non-feeding period. The down-regulation of the trehalose level during the active feeding period seemed to result from mediation of the insulin/IGF signal (IIS). Injection of a porcine insulin suppressed the trehalose level in a dose-dependent manner. Genes associated with IIS of M. vitrata were predicted from its larval transcriptome, and their expression was confirmed in different developmental stages and tissues. All seven IIS genes selected were expressed in all developmental stages and different tissues. Silencing of four IIS genes (insulin receptor, Forkhead box O, a serine-threonine protein kinase, target of rapamycin) by RNA interference significantly modulated the hemolymph trehalose level. Starvation treatment changed expression of two trehalose metabolism-associated genes (trehalose phosphate synthase (TPS) and trehalase (TRE)) as well as the IIS genes. Silencing of TPS or TRE expression significantly down- or up-regulated the hemolymph trehalose level, respectively. In addition, silencing of IIS genes altered both TPS and TRE expression, indicating a functional link between IIS and trehalose metabolism. These results suggest that nutrients obtained from feeding activate IIS of M. vitrata, which then down-regulates the hemolymph trehalose level by altering trehalose metabolism., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Previously, a cytosolic trehalase (TreH) from the hyperthermophilic archaeon Sulfolobus acidocaldarius was reported; however, the gene responsible for the trehalase activity was not identified. Two genes, saci_1816 and saci_1250 , that encode the glycoside hydrolase family 15 type glucoamylase-like proteins in S. acidocaldarius were targeted and expressed in Escherichia coli , and their abilities to hydrolyze trehalose were examined. Recombinant Saci_1816 hydrolyzed trehalose exclusively without any help from a cofactor. The mass spectrometric analysis of partially purified native TreH also confirmed that Saci_1816 was involved in proteins exhibiting trehalase activity. Optimal trehalose hydrolysis activity of the recombinant Saci_1816 was observed at pH 4.0 and 60°C. The pH dependence of the recombinant enzyme was similar to that of the native enzyme, but its optimal temperature was 20-25°C lower, and its thermostability was also slightly reduced. From the biochemical and structural results, Saci_1816 was identified as a trehalase responsible for trehalose degradation in S. acidocaldarius . Identification of the treH gene confirms that the degradation of trehalose in Sulfolobus species occurs via the TreH pathway.

Two archaeal trehalase-like genes, Saci1250 and Saci1816, belonging to glycoside hydrolase family 15 (GH15) from the acidophilic Crenarchaeon Sulfolobus acidocaldarius were expressed in Escherichia coli. The gene products showed trehalose-hydrolyzing activities, and the names SaTreH1 and SaTreH2 were assigned to Saci1816 and Saci1250 gene products, respectively. These newly identified enzymes functioned within a narrow range of acidic pH values at elevated temperatures, which is similar to the behavior of Euryarchaeota Thermoplasma trehalases. SaTreH1 displayed high K M and k cat values, whereas SaTreH2 had lower K M and k cat values despite a high degree of identity in their primary structures. A mutation analysis indicated that two glutamic acid residues in SaTreH1, E374 and E574, may be involved in trehalase catalysis because SaTreH1 E374Q and E574Q showed greatly reduced trehalose-hydrolyzing activities. Additional mutations substituting G573 and H575 residues with serine and glutamic acid residues, respectively, to mimic the TVN1315 sequence resulted in a decrease in trehalase activity and thermal stability. Taken together, the results indicated that Crenarchaea trehalases adopt active site structures that are similar to Euryarchaeota enzymes but have distinct molecular features. The identification of these trehalases could extend our understanding of the relationships between the structure and function of GH15 trehalases as well as other family enzymes and will provide insights into archaeal trehalose metabolism.

Trehalose is a major blood sugar in insects with a range of physiological functions, including an energy source and a cryoprotectant. Hemolymph trehalose concentrations are tightly regulated according to physiological conditions. An insulin-like peptide, SeILP1, downregulates hemolymph trehalose concentrations in Spodoptera exigua. Here, we identified a factor that upregulates hemolymph trehalose concentration in S. exigua. Hemolymph trehalose concentrations were significantly increased after immune challenge or under starvation in a time-dependent manner. To determine endocrine factors responsible for the upregulation, stress-associated mediators, such as octopamine, serotonin, or eicosanoids were injected, but they did not upregulate hemolymph trehalose. On the other hand, injection with Schistocerca gregaria adipokinetic hormone (AKH) significantly increased hemolymph trehalose concentration in S. exigua. During upregulation of hemolymph trehalose by AKH injection, trehalose degradation appeared to be inhibited because expression of trehalase and SeILP1 were significantly suppressed while that of trehalose phosphate synthase was not significantly changed. Interrogation of a Spodoptera genome database identified an S. exigua AKH-like gene and its expression was confirmed. During starvation, its expression concentrations were increased, although RNA interference specific to the AKH-like hypertrehalosemic factor (SeHTF) gene significantly prevented the upregulation of hemolymph trehalose concentrations during starvation. A synthetic peptide of SeHTF was prepared and injected into S. exigua larvae. At nanomolar concentration, the synthetic SeHTF peptide effectively upregulated hemolymph trehalose concentrations. Here we report a novel hypertrehalosemic factor in S. exigua (SeHTF)., (© 2017 Wiley Periodicals, Inc.)

Trehalase (TRE) is a key enzyme in trehalose degradation and has important functions in insect growth and chitin synthesis. Though validamycin has the potential for pest control by suppressing TRE activities, it is not known whether validamycin acts on both trehalose and chitin metabolism. TRE1 and TRE2 activities and glucose and glycogen contents decreased significantly after the injection of different doses of validamycin solution compared with the control group, while the trehalose content increased significantly. Overall, it showed that about 13 to 38% insects was appeared abnormal phenotypes, and 10 to 57% of insects died 48h after injection of solutions with different concentrations of validamycin; the chitin content also decreased significantly. Validamycin altered the relative expression levels of trehalose, glycogen and chitin metabolism-related genes by suppressing the activities of two TREs. We showed that the expression levels of three TRE and two trehalose-6-phosphate synthase (TPS) genes increased, while the expression levels of GP; CHS1 and its two transcripts, CHS1a, CHS1b; six chitinases, including Cht3, Cht4, Cht5, Cht6, Cht7, Cht9; and the HK, G6PI2, GFAT, GNPNA, PAGM1, UAP, VVL, CI and AP genes decreased significantly 48h after the injection of any validamycin concentration compared with the control group. These results demonstrate that by inhibiting the activities of two TREs, validamycin alters N. lugens chitin synthesis and degradation and affects trehalose and chitin metabolism-related gene expression. The development of TRE inhibitors may provide effective pest control in the future., (Copyright © 2016 Elsevier Inc. All rights reserved.)

RNA interference has been used to study insects' gene function and regulation. Glycogen synthase (GS) and glycogen phosphorylase (GP) are two key enzymes in carbohydrates' conversion in insects. Glycogen content and GP and GS gene expression in several tissues and developmental stages of the Brown planthopper Nilaparvata lugens Stål (Hemiptera: Delphacidae) were analyzed in the present study, using quantitative reverse-transcription polymerase chain reaction to determine their response to double-stranded trehalases (dsTREs), trehalose-6-phosphate synthases (dsTPSs), and validamycin injection. The highest expression of both genes was detected in the wing bud, followed by leg and head tissues, and different expression patterns were shown across the developmental stages analyzed. Glycogen content significantly decreased 48 and 72 h after dsTPSs injection and 48 h after dsTREs injection. GP expression increased 48 h after dsTREs and dsTPSs injection and significantly decreased 72 h after dsTPSs, dsTRE1-1, and dsTRE1-2 injection. GS expression significantly decreased 48 h after dsTPS2 and dsTRE2 injection and 72 h after dsTRE1-1 and dsTRE1-2 injection. GP and GS expression and glycogen content significantly decreased 48 h after validamycin injection. The GP activity significantly decreased 48 h after validamycin injection, while GS activities of dsTPS1 and dsTRE2 injection groups were significantly higher than that of double-stranded GFP (dsGFP) 48 h after injection, respectively. Thus, glycogen is synthesized, released, and degraded across several insect tissues according to the need to maintain stable trehalose levels., (© The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.)

A genetic recombinant Saccharomyces cerevisiae starter with high ethanol tolerance capacities was constructed. In this study, the gene of trehalose-6-phosphate synthase (encoded by tps1), which catalyzes the first step in trehalose synthesis, was cloned and overexpressed in S. cerevisiae. Moreover, the gene of neutral trehalase (encoded by nth1, trehalose degrading enzyme) was deleted by using a disruption cassette, which contained long flanking homology regions of nth1 gene (the upstream 0.26 kb and downstream 0.4 kb). The engineered strain increased its tolerance against ethanol and glucose stress. The growth of the wild strain was inhibited when the medium contained 6 % or more ethanol, whereas growth of the engineered strain was affected when the medium contained 10 % or more ethanol. There was no significant difference in the ethanol yield between the wild strain and the engineered strain when the fermentation broth contained 10 % glucose (p > 0.05). The engineered strain showed greater ethanol yield than the wild type strain when the medium contained more than 15 % glucose (p < 0.05). Higher intracellular trehalose accumulation by overexpression of tps1 and deletion of nth1 might provide the ability for yeast to protect against environmental stress.

Trehalose is proposed to serve multiple physiological roles in insects. However, its importance remains largely unconfirmed. In the present paper, we knocked down either a trehalose biosynthesis gene (trehalose-6-phosphate synthase, LdTPS) or each of three degradation genes (soluble trehalases LdTRE1a, LdTRE1b or membrane-bound LdTRE2) in Leptinotarsa decemlineata by RNA interference (RNAi). Knockdown of LdTPS decreased trehalose content and caused larval and pupal lethality. The LdTPS RNAi survivors consumed a greater amount of foliage, obtained a heavier body mass, accumulated more glycogen, lipid and proline, and had a smaller amount of chitin compared with the controls. Ingestion of trehalose but not glucose rescued the food consumption increase and larval mass rise, increased survivorship, and recovered glycogen, lipid and chitin to the normal levels. In contrast, silencing of LdTRE1a increased trehalose content and resulted in larval and pupal lethality. The surviving LdTRE1a RNAi hypomorphs fed a smaller quantity of food, had a lighter body weight, depleted lipid and several glucogenic amino acids, and contained a smaller amount of chitin. Neither trehalose nor glucose ingestion rescued these LdTRE1a RNAi defects. Silencing of LdTRE1b caused little effects. Knockdown of LdTRE2 caused larval death, increased trehalose contents in several tissues and diminished glycogen in the brain-corpora cardiaca-corpora allata complex (BCC). Feeding glucose but not trehalose partially rescued the high mortality rate and recovered glycogen content in the BCC. It seems that trehalose is involved in feeding regulation, sugar absorption, brain energy supply and chitin biosynthesis in L. decemlineata larvae., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Unlabelled: Dekkera bruxellensis hit the spotlight in the past decade mostly due to its rather high ability to adapt to several different fermentation processes. This yeast relies on different genetic and physiological aspects to achieve and preserve its high industrial fitness and some of these traits are shared with Saccharomyces cerevisiae. We have previously described that D. bruxellensis is unable to make use of accumulating trehalose as a strategy for cell adaptation and survival in the industrial scenario, as opposed to S. cerevisiae. Since trehalose is often involved in mechanisms related to cell protection, we aimed to investigate both cause and effect of the absence of this metabolite in the cell adaptive capacity in the industrial environment. Our results indicate that the major cause for the nonaccumulation of trehalose is the high constitutive activity of neutral trehalase. Therefore, the rate of trehalose degradation could be higher than its rate of synthesis, preventing accumulation. Altogether, our data elucidate the mechanisms involved in the lack of trehalose accumulation in D. bruxellensis as well as evaluates the implications of this feature., Significance and Impact of the Study: Dekkera bruxellensis can successfully take advantage of its peculiar physiological and genetic traits in order to adapt and survive in fermentation processes. So far, tolerance to stress has been credited to trehalose synthesis. The data presented in this work provided information on the underlying mechanism that prevents trehalose accumulation and corroborated the recent information that trehalose itself is not implicated in yeast stress tolerance. Second, it showed that D. bruxellensis responds differently to Saccharomyces cerevisiae to excess of sugar, which may explain its preference for respiration (oxidative metabolism) over fermentation (reductive metabolism) even at limited oxygen supply. These findings help to understand the drop on ethanol production in processes overtaken by this yeast., (© 2016 The Society for Applied Microbiology.)

Trehalose plays important roles in the protection of organisms against adverse environmental conditions. The growth and development of Flammulina velutipes is regulated and controlled under complex external conditions. This study investigated the effect of heat stress on trehalose metabolism in mycelia and fruiting bodies. The activities of enzymes involved in trehalose metabolism, the transcriptional levels of the corresponding genes and the trehalose content in the mycelia of Flammulina velutipes strain Dan3 under relatively high temperatures were investigated. The mycelia and fruiting bodies of a strain cultivated in a factory were collected at different stages to examine the trehalose content and expression levels of various genes. The results showed that intracellular trehalose significantly accumulated in the mycelia in response to 37 °C heat shock. Heat shock significantly stimulated the activities of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, thereby promoting the accumulation of trehalose for the first 2-6 h. The activity of neutral trehalase also decreased during this period. In addition, changes in the activities of trehalose-6-phosphate synthase, trehalose-6-phosphate phosphatase and neutral trehalase paralleled changes in the expression levels of the regulatory genes. As for the trehalose phosphorylase, the degradation of trehalose was stronger than its synthesis under heat stress. Heat shock can induce a stress response in the mycelia through the regulation of genes related to trehalose metabolism and the subsequent promotion and control of the transcription and translation of enzymes. The analysis of the trehalose and gene expression levels in the cultivated strain suggests that a substantial amount of trehalose had accumulated in the mycelia prior to induction of the primordia, and the fruiting bodies could possibly utilize degraded trehalose that translocated from the mycelia to maintain their growth.

We purified a cytosolic trehalase (TreH) from a thermoacidophilic archaeon Sulfolobus acidocaldarius. Enzyme activity in cell-free extracts indicated that trehalose degradation in the cell occurred via the hydrolytic activity of TreH, and not via TreP (phosphorolytic activity) or TreT (transfer activity). TreH was purified to near-homogeneity by DEAE anion-exchange chromatography, followed by size exclusion and HiTrap Q anion-exchange chromatography, and its molecular mass was estimated as 40 kDa. Maximum activity was observed at 85°C and pH 4.5. The half-life of TreH was 53 and 41 min at 90°C and 95°C, respectively. TreH was highly specific for trehalose and was inhibited by glucose with a Ki of 0.05 mM. Compared with TreH from other trehalases, TreH from S. acidocaldarius is the most thermostable trehalase reported so far. Furthermore, this is the first trehalase characterized in the Archaea domain., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Entomopathogenic fungi proliferate in insect hemolymph by using host nutrients after penetrating the cuticle. To improve the virulence of the locust specific fungus, Metarhizium acridum, we genetically modified the fungus to overexpress ATM1, an endogenous hydrolase of trehalose, which is the main carbon source in insect hemolymph. Compared with the wild-type strain, Metarhizium acridum overexpressing ATM1 gene secreted more acid trehalase into locust hemolymph. The trehalose concentrations in locusts infected with the ATM1-overexpressing strain were 5.5 and 6.1 mmol/l, lower than that in locusts infected with the wild-type strain at 3 and 5 days post-inoculation, representing 44.5 and 60.7 % reduction, respectively. Correspondingly, overexpressing ATM1 accelerated the growth of Metarhizium acridum in host hemolymph, and the dose causing 50 % mortality (LD50) of the ATM1-overexpressing strain was reduced by 8.3-fold compared with the wild-type strain, suggesting that increasing the utilization of host nutrients by pathogens could be a promising way to improve the virulence of biopesticides based on parasites of pests.

The emergent pathogen Candida glabrata differs from other yeasts because it assimilates only two sugars, glucose and the disaccharide trehalose. Since rapid identification tests are based on the ability of this yeast to rapidly hydrolyze trehalose, in this work a biochemical and molecular characterization of trehalose catabolism by this yeast was performed. Our results show that C. glabrata consumes and ferments trehalose, with parameters similar to those observed during glucose fermentation. The presence of glucose in the medium during exponential growth on trehalose revealed extracellular hydrolysis of the sugar by a cell surface acid trehalase with a pH optimum of 4.4. Approximately ∼30% of the total enzymatic activity is secreted into the medium during growth on trehalose or glycerol. The secreted enzyme shows an apparent molecular mass of 275 kDa in its native form, but denaturant gel electrophoresis revealed a protein with ∼130 kDa, which due to its migration pattern and strong binding to concanavalin A, indicates that it is probably a dimeric glycoprotein. The secreted acid trehalase shows high affinity and activity for trehalose, with Km and Vmax values of 3.4 mM and 80 U (mg protein)(-1), respectively. Cloning of the CgATH1 gene (CAGLOK05137g) from de C. glabrata genome, a gene showing high homology to fungal acid trehalases, allowed trehalose fermentation after heterologous expression in Saccharomyces cerevisiae., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Moniliella megachiliensis, the osmo-tolerant basidiomycetous yeast was found to accumulate intracellularly energy-storing carbohydrates (trehalose and glycogen) along with polyols (glycerol and erythritol) up to stationary growth phase. In trehalose-loaded cell, osmotic-stress resulted in the rapid generation of glycerol, and oxidative stress with menadione resulted in the rapid generation of erythritol. Under either of these conditions, the levels of the energy-storing carbohydrates were depleted, while little glucose uptake was observed. These results suggested that the intracellular pools of trehalose and glycogen were rapidly converted to glycerol in response to osmotic stress, and to erythritol in response to oxidative stress and altered redox balance. Expression of tps1 encoding trehalose synthetic enzymes paralleled trehalose accumulation in the cell during the culture in 2% glucose, in contrast, expression of tpp1 or tpp2 encoding trehalose-6-phosphate phosphatase was little increased under the same condition. Expression of tre (tre1/tre2) encoding trehalose hydrolase (trehalase) increased with time associated with depletion of trehalose during oxidative stress. From these results, we concluded that glycerol and erythritol, the compatible solutes in M. megachiliensis were metabolically interrelated to energy-storing carbohydrates such as trehalose or glycogen during conditions of osmotic or oxidative stress., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Plants respond to low carbon supply by massive reprogramming of the transcriptome and metabolome. We show here that the carbon starvation-induced NAC (for NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON) transcription factor Arabidopsis (Arabidopsis thaliana) Transcription Activation Factor1 (ATAF1) plays an important role in this physiological process. We identified TREHALASE1, the only trehalase-encoding gene in Arabidopsis, as a direct downstream target of ATAF1. Overexpression of ATAF1 activates TREHALASE1 expression and leads to reduced trehalose-6-phosphate levels and a sugar starvation metabolome. In accordance with changes in expression of starch biosynthesis- and breakdown-related genes, starch levels are generally reduced in ATAF1 overexpressors but elevated in ataf1 knockout plants. At the global transcriptome level, genes affected by ATAF1 are broadly associated with energy and carbon starvation responses. Furthermore, transcriptional responses triggered by ATAF1 largely overlap with expression patterns observed in plants starved for carbon or energy supply. Collectively, our data highlight the existence of a positively acting feedforward loop between ATAF1 expression, which is induced by carbon starvation, and the depletion of cellular carbon/energy pools that is triggered by the transcriptional regulation of downstream gene regulatory networks by ATAF1., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Trehalose has widespread use as a sweetener, humectant and stabilizer, and is now attracting attention as a promising candidate for the treatment of neurodegenerative diseases as it is an autophagy inducer and chemical chaperone. However, the bioavailability of trehalose is low because it is digested by the hydrolyzing enzyme trehalase, expressed in the intestine and kidney. Enzyme-stable analogs of trehalose would potentially solve this problem. We have previously reported an enzyme-stable analog of trehalose, lentztrehalose, and herein report two new analogs. The original lentztrehalose has been renamed lentztrehalose A and the analogs named lentztrehaloses B and C. Lentztrehalose B is a di-dehydroxylated analog and lentztrehalose C is a cyclized analog of lentztrehalose A. All the lentztrehaloses are only minimally hydrolyzed by mammalian trehalase. The production of the lentztrehaloses is high in rather dry conditions and low in wet conditions. Lentztrehalose B shows a moderate antioxidative activity. These facts suggest that the lentztrehaloses are produced as humectants or protectants for the producer microorganism under severe environmental conditions. All the lentztrehaloses induce autophagy in human cancer cells at a comparable level to trehalose. Considering the enzyme-stability, these lentztrehaloses can be regarded as promising new drug candidates for the treatment of neurodegenerative diseases and other autophagy-related diseases, such as diabetes, arteriosclerosis, cancer and heart disease.

Trehalose represents the main hemolymph sugar in many insects, and it functions in energy metabolism and protection in extreme environmental conditions. To gain an insight into trehalose functions in Delia antiqua diapausing pupae, genes encoding trehalose-6-phosphate synthase (TPS), trehalose-6-phosphatase (TPP) and trehalase (TRE) were identified and characterized. Analysis of the deduced amino acid sequences indicated that these genes were highly similar to each homolog from Diptera insects. Gene expressions and their enzyme activities were also investigated. The differential expressions of TPS and TPP shared very similar trends for summer and winter diapausing pupae. Their enzyme activities were consistent with the gene expressions. Trehalose concentrations in summer- and winter-diapausing pupae were lower at the initial phase (4.37-5.09μg/mg) but increased gradually and peaked in the maintenance phase (10.59-14.36μg/mg); the concentrations then declined in the quiescence phase. We speculated that a higher trehalose content during the maintenance stage may contribute to protein and/or biological membrane stabilization in winter or to desiccation resistance in the summertime. Diapause termination requires a decrease in the trehalose concentration to promote pupal-adult development. The glucose content also varied during the diapausing processes. Our results provide an overview of the differential expression levels of trehalose metabolic enzymes, confirming the important roles of trehalose in diapausing pupae of the onion maggot. Further work remains to explore its actual functions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Each year brings new facts concerning multiple roles of sugar pathways in plant metabolism. One of them--the trehalose pathway--has been shown to play a role in stress signalling. The last enzyme of this pathway--trehalase--has been proven to be strongly expressed in guard cells. Modifications of its abundance cause changes in stomatal closure and response to abscisic acid. Our phenotypical studies of different mutants of Arabidopsis thaliana and Musa have enabled us to propose a new function of trehalase. It might play a role in the feedback of sucrose as a closing signal for stomata in reaction to an efficient photosynthesis. To characterize the phenotype we measured: the dynamic cumulative water loss, the dynamic leaf surface temperature, and the stomatal conductance. Based on the obtained results we have determined the time points for a proteomics study. The exact role of trehalase and related proteins in the proposed mechanism will be defined with multiple analysis including mass spectrophotometry and enzymatic activities. The samples will be collected from a wide type of plants including model organism (Arabidopsis--wild type, trehalase mutant plants) and crops (banana). The final results will shed light on the complete role of trehalase and the feedback pathway.

Gluconobacter strains are specialized in the incomplete oxidation of monosaccharides. In contrast, growth and product formation from disaccharides is either very low or impossible. A pathway that allows growth on trehalose was rationally designed to broaden the substrate range of Gluconobacter oxydans. Expression vectors containing different signal sequences and the gene encoding alkaline phosphatase, phoA, from Escherichia coli were constructed. The signal peptide that exhibited the strongest periplasmic PhoA activity was used to generate a G. oxydans strain able to utilize the model disaccharide trehalose as a carbon and energy source by expressing the periplasmic trehalase TreA from E. coli. The strain had a doubling time of 3.7h and reached a final optical density of 1.7 when trehalose was used as a growth substrate. In comparison, the wild-type harboring the empty vector and the strain expressing treA without a signal sequence grew slowly to a final OD of only 0.15. The trehalose concentration in treA expressing cultures decreased continuously during the exponential growth phase indicating that the substrate was hydrolyzed to glucose by TreA. In contrast to the wild-type growing on glucose, the treA expression strain mainly formed acetate and 5-ketogluconate as end products rather than gluconate., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A new trehalose analog, lentztrehalose [4-O-(2,3-dihydroxy-3-methylbutyl)trehalose], was isolated from an actinomycete Lentzea sp. Lentztrehalose is only weakly hydrolyzed by the trehalose-hydrolyzing enzyme, trehalase, so can be regarded as an enzyme-stable analog of trehalose. Although lentztrehalose does not show apparent toxicity to mammalian cells and microbes, it has antitumor activity in mice bearing S-180 sarcoma and Ehrlich carcinoma cells. In ovariectomized mice, lentztrehalose displayed a bone reinforcement effect in the femur that was superior to trehalose and induced non-morbid suppression of weight gain comparable with trehalose. These results indicate that enzyme-stable analogs of trehalose, such as lentztrehalose, may be more beneficial for human health and thus have potential as substitutes for trehalose as a sweetener.

Trehalases (Tres) have been demonstrated to be the key enzymes that are involved in various trehalose-associated physiological processes in insects. However, little attention has been devoted to the Tres in the whitefly, Bemisia tabaci. In this study, a soluble Tre (BtTre-1) and a membrane-bound Tre (BtTre-2) were cloned in the invasive cryptic species Middle East-Asia Minor 1 (MEAM1) of the whitefly B. tabaci complex. Alignment of deduced amino acids sequences of both BtTres revealed that they share common consensus regions and residues with Tres of other insect species. Levels of BtTres expression in various stages and tissues of the whitefly suggested that BtTre-2 may play a key role in trehalose catabolism during development of the whitefly, especially for oocyte development, while BtTre-1 may prevent trehalose in salivary gland from leaking and entering into plants along with saliva. Potential roles of trehalose catabolism in response to direct and/or plant-mediated indirect effects of Tomato Yellow Leaf Curl China Virus (TYLCCNV) were also detected. Whiteflies feeding on virus-infected tobacco plants showed higher BtTres expressions and accordingly higher BtTres activity but lower trehalose content than those feeding on uninfected plants. The enhanced trehalose catabolism may be beneficial to oocyte development in ovary and attenuate plant defensive responses induced by trehalose in saliva. Viruliferous and nonviruliferous whiteflies feeding on cotton, a nonhost plant for TYLCCNV, differed significantly only in trehalose content. The higher trehalose content in viruliferous whiteflies may be conducive to resisting the stress inflicted by TYLCCNV., (© 2014 Wiley Periodicals, Inc.)

Trehalose is a non-reducing disaccharide involved in stress tolerance in plants. To understand better the role of trehalose in the osmotic stress response in linseed (Linum usitatissimum), trehalose content in leaves was studied. First, the method commonly used for sugar determination, high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), gave unsatisfactory results and the separation efficiency could not be improved by varying the elution conditions. The same problem was also found in the model plant: Arabidopsis thaliana. After clearly highlighting a co-elution of trehalose in these two species by a trehalase assay and liquid chromatography-high resolution mass spectrometry analysis, gas chromatography-mass spectrometry (GC-MS) was used as the analytical method instead. These results confirmed that trehalose content is currently overestimated by HPAEC-PAD analysis, approximately 7 and 13 times for A. thaliana and linseed respectively. Thus GC-MS gave more satisfactory results for trehalose quantification in plants. With this method, trehalose accumulation was observed in linseed during an osmotic stress (-0.30 MPa), the quantity (31.49 nmol g(-1) dry weight after 48 h) appears too low to assign an osmoprotector or osmoregulator role to trehalose in stressed linseed., (Copyright © Physiologia Plantarum 2012.)

Legume-rhizobium interactions have been widely studied and characterized, and the disaccharide trehalose has been commonly detected during this symbiotic interaction. It has been proposed that trehalose content in nodules during this symbiotic interaction might be regulated by trehalase. In the present study, we assessed the role of trehalose accumulation by down-regulating trehalase in the nodules of common bean plants. We performed gene expression analysis for trehalase (PvTRE1) during nodule development. PvTRE1 was knocked down by RNA interference (RNAi) in transgenic nodules of the common bean. PvTRE1 expression in nodulated roots is mainly restricted to nodules. Down-regulation of PvTRE1 led to increased trehalose content (78%) and bacteroid number (almost one order of magnitude). In addition, nodule biomass, nitrogenase activity, and GOGAT transcript accumulation were significantly enhanced too. The trehalose accumulation, triggered by PvTRE1 down-regulation, led to a positive impact on the legume-rhizobium symbiotic interaction. This could contribute to the agronomical enhancement of symbiotic nitrogen fixation., (© 2012 The Authors New Phytologist © 2012 New Phytologist Trust.)

In this paper, an insect biofuel cell (BFC) using trehalose included in insect hemolymph was developed. The insect BFC is based on trehalase and glucose oxidase (GOD) reaction systems which oxidize β-glucose obtained by hydrolyzing trehalose. First, we confirmed by LC-MS that a sufficient amount of trehalose was present in the cockroach hemolymph (CHL). The maximum power density obtained using the insect BFC was 6.07 μW/cm(2). The power output was kept more than 10 % for 2.5 h by protecting the electrodes with a dialysis membrane. Furthermore, the maximum power density was increased to 10.5 μW/cm(2) by using an air diffusion cathode. Finally, we succeeded in driving a melody integrated circuit (IC) and a piezo speaker by connecting five insect BFCs in series. The results indicate that the insect BFC is a promising insect-mountable battery to power environmental monitoring micro-tools.

The ability to form hyphae in the human pathogenic fungus Candida albicans is a prerequisite for virulence. It contributes to tissue infection, biofilm formation, as well as escape from phagocytes. Cell elongation triggered by human body temperature involves the essential heat shock protein Hsp90, which negatively governs a filamentation program dependent upon the Ras-protein kinase A (PKA) pathway. Tight regulation of Hsp90 function is required to ensure fast appropriate response and maintenance of a wide range of regulatory and signaling proteins. Client protein activation by Hsp90 relies on a conformational change of the chaperone, whose ATPase activity is competitively inhibited by geldanamycin. We demonstrate a novel regulatory mechanism of heat- and Hsp90-dependent induced morphogenesis, whereby the nonreducing disaccharide trehalose acts as a negative regulator of Hsp90 release. By means of a mutant strain deleted for Gpr1, the G protein-coupled receptor upstream of PKA, we demonstrate that elevated trehalose content in that strain, resulting from misregulation of enzymatic activities involved in trehalose metabolism, disrupts the filamentation program in response to heat. Addition of geldanamycin does not result in hyphal extensions at 30 °C in the gpr1Δ/gpr1Δ mutant as it does in wild type cells. In addition, validamycin, a specific inhibitor of trehalase, the trehalose-degrading enzyme, inhibits cell elongation in response to heat and geldanamycin. These results place Gpr1 as a regulator of trehalose metabolism in C. albicans and illustrate that trehalose modulates Hsp90-dependent activation of client proteins and signaling pathways leading to filamentation in the human fungal pathogen.

The thermophilic fungus Malbranchea pulchella var. sulfurea produced good amounts of extracellular trehalase activity when grown for long periods on starch, maltose or glucose as the main carbon source. Studies with young cultures suggested that the main role of the extracellular acid trehalase is utilizing trehalose as a carbon source. The specific activity of the purified enzyme in the presence of manganese (680 U/mg protein) was comparable to that of other thermophilic fungi enzymes, but many times higher than the values reported for trehalases from other microbial sources. The apparent molecular mass of the native enzyme was estimated to be 104 kDa by gel filtration and 52 kDa by SDS-PAGE, suggesting that the enzyme was composed by two subunits. The carbohydrate content of the purified enzyme was estimated to be 19 % and the pi was 3.5. The optimum pH and temperature were 5.0-5.5 and 55° C, respectively. The purified enzyme was stimulated by manganese and inhibited by calcium ions, and insensitive to ATP and ADP, and 1 mM silver ions. The apparent K(M) values for trehalose hydrolysis by the purified enzyme in the absence and presence of manganese chloride were 2.70 ± 0.29 and 2.58 ± 0.13 mM, respectively. Manganese ions affected only the apparent V(max), increasing the catalytic efficiency value by 9.2-fold. The results reported herein indicate that Malbranchea pulchella produces a trehalase with mixed biochemical properties, different from the conventional acid and neutral enzymes and also from trehalases from other thermophilic fungi.

A bienzymatic electrode incorporating trehalase (Tre) and glucose oxidase (GOx) covalently bound to the surface of Pt through a functionalized thiol linker (Tre|GOx|Pt) has been designed and assembled and its catalytic properties examined by chemical and electrochemical methods in aqueous phosphate buffer solutions (PBS, pH 7.4). Exposure of Tre|GOx|Pt to PBS containing trehalose (Tr) and subsequent polarization at 0.6 V versus Ag/AgCl yielded after a few minutes well-defined steady-state currents ascribed to the oxidation of hydrogen peroxide generated by the GOx-mediated oxidation of glucose (Gl) produced by the Tre-mediated dissociation of Tr. Plots of the steady-state currents versus [Tr] over the range examined, i.e., 5-25 mM, were found to be linear. Implications of these results toward the development of an implantable biofuel cell as an autonomous energy conversion device for insects are discussed.

Ustilago maydis is a fungal pathogen which is exposed during its life cycle to both abiotic and biotic stresses before and after the infection of maize. To cope with extreme environmental changes, microorganisms usually accumulate the disaccharide trehalose. We have investigated both the accumulation of trehalose and the activity of trehalase during the adaptation of U. maydis haploid cells to thermal, sorbitol, and NaCl stresses. Sorbitol and sodium chloride induced sustained accumulation of trehalose, while a transient increase was observed under heat stress. Sorbitol stressed cells showed higher trehalase activity compared with control cells and to those stressed by NaCl and high temperature. Addition of cycloheximide, a protein synthesis inhibitor, did not affect the trehalose accumulation during the first 15 min, but basal levels of trehalose were reached after the second period of 15 min. The proteomic analysis of the response of U. maydis to temperature, sorbitol, and salt stresses indicated a complex pattern which highlights the change of 18 proteins involved in carbohydrate and amino acid metabolism, protein folding, redox regulation, ion homeostasis, and stress response. We hypothesize that trehalose accumulation during sorbitol stress in U. maydis might be related to the adaptation of this organism during plant infection.

In Arabidopsis thaliana the induction of plant trehalase during clubroot disease was proposed to act as a defense mechanism in the susceptible accession Col-0, which could thereby cope with the accumulation of pathogen-synthesized trehalose. In the present study, we assessed trehalose activity and tolerance to trehalose in the clubroot partially resistant accession Bur-0. We compared both accessions for several trehalose-related physiological traits during clubroot infection. A quantitative trait loci (QTLs) analysis of tolerance to exogenous trehalose was also conducted on a Bur-0xCol-0 RIL progeny. Trehalase activity was not induced by clubroot in Bur-0 and the inhibition of trehalase by validamycin treatments resulted in the enhancement of clubroot symptoms only in Col-0. In pathogen-free cultures, Bur-0 showed less trehalose-induced toxicity symptoms than Col-0. A QTL analysis identified one locus involved in tolerance to trehalose overlapping the confidence interval of a QTL for resistance to Plasmodiophora brassicae. This colocalization was confirmed using heterogeneous inbred family (HIF) lines. Although not based on trehalose catabolism capacity, partial resistance to clubroot is to some extent related to the tolerance to trehalose accumulation in Bur-0. These findings support an original model where contrasting primary metabolism-related regulations could contribute to the partial resistance to a plant pathogen., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

Validamycin A was used to inhibit in vivo trehalase activity in tobacco enabling the study of subsequent changes in new C partitioning into cellulosic biomass and lignin precursors. After 12-h exposure to treatment, plants were pulse labeled using radioactive (11)CO(2), and the partitioning of isotope was traced into [(11)C]cellulose and [(11)C]hemicellulose, as well as into [(11)C]phenylalanine, the precursor for lignin. Over this time course of treatment, new carbon partitioning into hemicellulose and cellulose was increased, while new carbon partitioning into phenylalanine was decreased. This trend was accompanied by a decrease in phenylalanine ammonia-lyase activity. After 4d of exposure to validamycin A, we also measured leaf protein content and key C and N metabolite pools. Extended treatment increased foliar cellulose and starch content, decreased sucrose, and total amino acid and nitrate content, and had no effect on total protein., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

β-D-Glucose-1-phosphate (βGlc1P) is an efficient glucosyl donor for both enzymatic and chemical glycosylation reactions but is currently very costly and not available in large amounts. This article provides an efficient production method of βGlc1P from trehalose and phosphate using the thermostable trehalose phosphorylase from Thermoanaerobacter brockii. At the process temperature of 60 °C, Escherichia coli expression host cells are lysed and cell treatment prior to the reaction is, therefore, not required. In this way, the theoretical maximum yield of 26% could be easily achieved. Two different purification strategies have been compared, anion exchange chromatography or carbohydrate removal by treatment with trehalase and yeast, followed by chemical phosphate precipitation. In a next step, βGlc1P was precipitated with ethanol but this did not induce crystallization, in contrast to what is observed with other glycosylphosphates. After conversion of the product to its cyclohexylammonium salt, however, crystals could be readily obtained. Although both purification methods were quantitative (>99% recovery), a large amount of product (50%) was lost during crystallization. Nevertheless, a production process for crystalline βGlc1P is now available from the cheap substrates trehalose and inorganic phosphate.

Trehalase, an anomer-inverting glycosidase, hydrolyzes only alpha,alpha-trehalose in natural substrates to release equimolecular beta-glucose and alpha-glucose. Since the hydrolytic reaction is reversible, alpha,alpha-[1,1'-(2)H]trehalose is capable of synthesis from [1-(2)H]glucose through the reverse reaction of trehalase. alpha-Secondary deuterium kinetic isotope effects (alpha-SDKIEs) for the hydrolysis of synthesized alpha,alpha-[1,1'-(2)H]trehalose by honeybee trehalase were measured to examine the catalytic reaction mechanism. Relatively high k(H)/k(D) value of 1.53 for alpha-SDKIEs was observed. The data imply that the catalytic reaction of the trehalase occurs by the oxocarbenium ion intermediate mechanism. In addition, the hydrolytic reaction of glycosidase is discussed from the viewpoint of chemical reactivity for the hydrolysis of acetal in organic chemistry. As to the hydrolytic reaction mechanism of glycosidases, oxocarbenium ion intermediate and nucleophilic displacement mechanisms have been widely recognized, but it is pointed out for the first time that the former mechanism is rational and valid and generally the latter mechanism is unlikely to occur in the hydrolytic reaction of glycosidases.

Trehalose accumulation is a common response to several stresses in the yeast Saccharomyces cerevisiae. This metabolite protects proteins and membrane lipids from structural damage and helps cells to maintain integrity. Based on genetic studies, degradation of trehalose has been proposed as a required mechanism for growth recovery after stress, and the neutral trehalase Nth1p as the unique degradative activity involved. Here we constructed a collection of mutants for several trehalose metabolism and transport genes and analysed their growth and trehalose mobilization profiles during experiments of saline stress recovery. The behaviour of the triple Deltanth1Deltanth2Deltaath1 and quadruple Deltanth1Deltanth2Deltaath1Deltaagt1 mutant strains in these experiments demonstrates the participation of the three known yeast trehalases Nth1p, Nth2p and Ath1p in the mobilization of intracellular trehalose during growth recovery after saline stress, rules out the participation of the Agt1p H(+)-disaccharide symporter, and allows us to propose the existence of additional new mechanisms for trehalose mobilization after saline stress.

In this work, the role of trehalose as an osmoprotectant against salt stress conditions was examined in root nodules of Medicago truncatula. For this purpose, we used validamycin A, a potent trehalase inhibitor, in order to induce trehalose accumulation. Validamycin A induced an increase of trehalose concentration in root nodules of M. truncatula by inhibiting trehalase activity; no effect on trehalose concentration was observed in roots and leaves. Trehalose accumulation was accompanied by a decrease in sucrose and starch content, indicating interference with carbohydrate partitioning in the plants. Under salinity conditions, sucrose accumulation appears to be induced in M. truncatula to protect nodule functioning by the inhibition of sucrose catabolism by sucrose synthase and alkaline invertase activities. However, trehalose accumulation induced by val A in nodules improved the response to salinity by increasing plant dry weight (PDW), and no effects of validamycin A on nitrogenase activity and PDW were observed in nonsalinized plants.

We investigated the biochemical properties of the enzymes involved in trehalose metabolism in the cyanobacterium Nostoc punctiforme strain IAM M-15 to elucidate the mechanism of trehalose accumulation in response to desiccation and salt stress. There was no detectable trehalose in fully hydrated N. punctiforme cells; however, these cells accumulated trehalose upon desiccation. Moreover, NaCl treatment also induced trehalose accumulation. The three genes for trehalose metabolism, treZ (maltooligosyltrehalose trehalohydrolase, Mth), treY (maltooligosyltrehalose synthase, Mts), and treH (trehalase), were found as a gene cluster, and the mRNAs for these genes were detectable at similar levels during desiccation. Trehalase of N. punctiforme was heterologously expressed in E. coli cells in an active form with a molecular mass of 52 kDa. Trehalase activity was strongly inhibited in the presence of 10 mM NaCl while trehalose synthesis activity remained active in the presence of salt. These data suggest that the rate of trehalose production exceeds that of trehalose hydrolysis under water-stress conditions characterized by increased cellular solute concentrations. In the proposed mechanism, control of trehalase plays an important role in trehalose accumulation in terrestrial cyanobacteria under conditions of extreme desiccation.

The role of the acid trehalase encoded by the ATH1 gene in the yeast Saccharomyces cerevisiae is still unclear. In this work, we investigated the regulation of ATH1 transcription and found a clear involvement of the protein kinase Hog1p in the induction of this gene under severe stress conditions, such as high salt. We also detected changes in the acid trehalase activity and trehalose levels, indicating a role of the acid trehalase in intracellular trehalose mobilization. Finally, the growth analysis for different mutants in neutral and acid trehalases after high salt stress implicates acid trehalase activity in saline stress resistance.