To engineer trehalose metabolism in sugarcane (Saccharum spp. hybrids) two transgenes were introduced to the genome: trehalose-6-phosphate synthase- phosphatase (TPSP), to increase trehalose biosynthesis and an RNAi transgene specific for trehalase, to abrogate trehalose catabolism. In RNAi-expressing lines trehalase expression was abrogated in many plants however no decrease in trehalase activity was observed. In TPSP lines trehalase activity was significantly higher. No events of co-integration of TPSP and RNAi transgenes were observed. We suggest trehalase activity is essential to mitigate embryonic lethal effects of trehalose metabolism and discuss the implications for engineering trehalose metabolism.

Diet is a key factor in determining and structuring animal diversity and adaptive radiations. The mammalian fossil record preserves phenotypic evidence of many dietary shifts, whereas genetic changes followed by dietary diversification in mammals remain largely unknown. To test whether living mammals preserve molecular evidence of dietary shifts, we examined the trehalase gene (Treh), which encodes an enzyme capable of digesting trehalose from insect blood, in bats and other mammals with diverse diets. Bats represent the largest dietary radiation among all mammalian orders, with independent origins of frugivory, nectarivory, carnivory, omnivory, and even sanguivory in an otherwise insectivorous clade. We found that Treh has been inactivated in unrelated bat lineages that independently radiated into noninsectivorous niches. Consistently, purifying selection has been markedly relaxed in noninsectivorous bats compared with their insectivorous relatives. Enzymatic assays of intestinal trehalase in bats suggest that trehalase activity tends to be lost or markedly reduced in noninsectivorous bats compared with their insectivorous relatives. Furthermore, our survey of Treh in 119 mammal species, which represent a deeper evolutionary timeframe, additionally identified a number of other independent losses of Treh in noninsectivorous species, recapitulating the evolutionary pattern that we found in bats. These results document a molecular record of dietary diversification in mammals, and suggest that such molecular signatures of dietary shifts would help us understand both historical and modern changes of animal diets.

Like other insects, in blood-feeding mosquitoes, trehalase (TRE; EC 3.2.1.28), an enzyme that metabolizes trehalose, may influence a wide array of functions including flight, survival, reproduction, and vectorial capacity, but its role has not been investigated in detail. Here, we characterized a 1,839-bp-long transcript, encoding a 555-aa-long trehalase-2 homolog protein from the mosquito Anopheles stephensi. With a conserved insect homology, and in silico predicted membrane-bound protein, we tested whether trehalase (As-TreH) also plays a role in mosquito physiologies. Constitutive expression during aquatic development or adult mosquito tissues, and a consistent upregulation until 42 h of starvation, which was restored to basal levels after sugar supply, together indicated that As-TreH may have a key role in stress tolerance. A multifold enrichment in the midgut (p < 0.001819) and salivary glands (p < 4.37E-05) of the Plasmodium vivax-infected mosquitoes indicated that As-TreH may favor parasite development and survival in the mosquito host. However, surprisingly, after the blood meal, a consistent upregulation until 24 h in the fat body, and 48 h in the ovary, prompted to test its possible functional correlation in the reproductive physiology of the adult female mosquitoes. A functional knockdown by dsRNA-mediated silencing confers As-TreH ability to alter reproductive potential, causing a significant loss in the egg numbers (p < 0.001), possibly by impairing energy metabolism in the developing oocytes. Conclusively, our data provide initial evidence that As-TreH regulates multiple physiologies and may serve as a suitable target for designing novel strategies for vector control.

Trehalase, a physiologically important glycosidase is known for its crucial role in insect glycometabolism and stress recovery. The present study describes the molecular cloning of a gene fragment, encoding the catalytically active trehalase from Drosophila melanogaster (DmTre) and its heterologous expression in Escherichia coli. The 1275bp gene was overexpressed in two different vectors viz., pET28a and pCOLD TF and investigated for variable soluble expression, purification and activity of the recombinant enzyme with optimum pH and temperature of enzyme as 6 and 55°C, respectively. The sequence was characterized in silico by subjecting it to homology search, multiple sequence alignment and phylogenetic tree construction revealing its identity to other trehalases which belong to glycoside hydrolase family 37. The deduced amino acid sequence and modeled 3D structure of DmTre possessed all features of trehalase superfamily, including signature motifs and catalytic domain. The active site pocket of recombinant DmTre was compared with the crystal structure of E. coli trehalase identifying Glu424 and Asp226 as the putative catalytic residues. Additionally, enzyme-substrate docking suggests possible involvement of other residues in the catalysis along with Asp226. The present study holds significance in understanding the structural aspects of Drosophila trehalase in spite of unavailabilty of eukaryotic trehalase crystal structure.

Trehalase catalyzes the hydrolysis of trehalose into two glucose molecules and is present in nearly all tissues in various forms. In this study, a putative bacterial trehalase gene, encoding a glycoside hydrolase family 15 (GH15) protein was identified in Microvirga sp. strain MC18 and heterologously expressed in E. coli. The specific activity of the purified recombinant trehalase MtreH was 24 U/mg, with Km and Vmax values of 23.45 mg/mL and 184.23 μmol/mg/min, respectively. The enzyme exhibited optimal activity at 40 °C and pH 7.0, whereby Ca2+ had a considerable positive effects on the catalytic activity and thermostability. The optimized enzymatic reaction conditions for the bioconversion of trehalose using rMtreH were determined as 40 °C, pH 7.0, 10 h and 1% trehalose concentration. The characterization of this bacterial trehalase improves our understanding of the metabolism and biological role of trehalose in prokaryotic organism.

Acid trehalase (AT) has always been reported to be copurified with invertase (I) and a 40 kDa additional protein. Glucose grown stationary phase cells of Saccharomyces cerevisiae contained least I activity. So, it was attempted to purify AT from these cells (I:AT = 10.83). Studies on specific activity, percent recovery and I:AT ratio of different pools, collected during purification of AT, indicated that samples containing ratio I:AT < 2.2 were unstable. Purification methodology favouring association (DEAE-Sephadex chromatography) resulted in gaining total activity while methodology favouring dissociation (HPGPLC) resulted in tremendous loss in recovery. Active pool (Pool 1X) appeared to be electrophoretically homogeneous but dissociated into 175, 90, 68, 61, 57 (minor bands) and 37-41 (major band) molar mass (kDa) bands on SDS-PAGE. Inactive pools (Pools 1Y, 3X, 3Y) did not contain the 37-41 kDa major band. So, association of both I and a 37-41 kDa protein with AT appeared to be essential. Two bands of isoelectric pH (pI) 4.6 and 4.7 were present in pool 1X enzyme preparation. All SDS-PAGE-resolved bands of pool 1X, in an average, contained high aspartate/asparagine and low cysteine residues. AT activity appeared to be highly sensitive to the change in pH and also to agents affecting ionisation of protein, e.g., betaine, NaCl, acetate, etc. Association of AT components in presence of NaCl was demonstrated spectrophotometrically. Specific activity of AT decreased with dilution. Substrate mediated allosterism for this enzyme preparation suggested that AT existed as an equilibrium mixture of protomer-oligomer. It was suggested that reversible association-dissociation was a mechanism for the regulation of AT activity.

The non-reducing disaccharide trehalose is biosynthesized in several species but not in vertebrates. However, trehalase, the enzyme required for its cleavage, has been observed in different mammalian organs. Even in humans, trehalase was detected in the gastrointestinal tract and the kidney. Trehalase is an intrinsic glycoprotein of the small intestine and kidney that transports trehalose and hydrolyses it to two glucose molecules. To our knowledge, no information is available about the in vivo distribution and localization of trehalase in the mammalian brain. Here, we report the occurrence and distribution of trehalase in vivo in the mouse brain using Western blotting and immunohistochemical techniques. Using an antibody against trehalase, we demonstrated that the enzyme showed a band with a molecular mass of approx. 70 kDa in the hippocampus, cerebral cortex, cerebellum and olfactory bulbs. Strong trehalase immunoreactivity was found in the perikarya and dendrites of neurons located in the hippocampus, cerebral cortex, Purkinje cells and mitral cells. Interestingly, Purkinje cells of the cerebellum showed higher immunoreactivity than neurons in the hippocampus and cerebral cortex. The distribution of trehalase appeared to be mainly related to neurons and was not detected in astrocytes. Independent of the presence of trehalose in neurons, the trehalase levels in neurons should have physiological significance. Investigating whether the interactions between trehalose and trehalase act on brain energy metabolism or have other not-yet-identified effects would also be interesting.

Sharp eyespot is a crucial disease affecting cereal plants, such as bread wheat (Triticum aestivum) and barley (Hordeum vulgare), and is primarily caused by the pathogenic fungus Rhizoctonia cerealis. As disease severity has increased, it has become imperative to find an effective and reasonable control strategy. One such strategy is the use of the trehalose analog, validamycin, which has been shown to have a potent inhibitory effect on several trehalases found in both insects and fungi, and is widely used as a fungicide in agriculture. In this study, we demonstrated that 0.5 μg/mL validamycin on PDA plates had an inhibitory effect on R. cerealis strain R0301, but had no significant impact on Fusarium graminearum strain PH-1. Except for its inhibiting the trehalase activity of pathogenic fungi, little is known about its mechanism of action. Six trehalase genes were identified in the genome of R. cerealis, including one neutral trehalase and five acidic trehalase genes. Enzyme activity assays indicated that treatment with 5 μg/mL validamycin significantly reduces trehalase activity, providing evidence that validamycin treatment does indeed affect trehalase, even though the expression levels of most trehalase genes, except Rc17406, were not obviously affected. Transcriptome analysis revealed that treatment with validamycin downregulated genes involved in metabolic processes, ribosome biogenesis, and pathogenicity in the R. cerealis. KEGG pathway analysis further showed that validamycin affected genes related to the MAPK signaling pathway, with a significant decrease in ribosome synthesis and assembly. In conclusion, our results indicated that validamycin not only inhibits trehalose activity, but also affects the ribosome synthesis and MAPK pathways of R. cerealis, leading to the suppression of fungal growth and pesticidal effects. This study provides novel insights into the mechanism of action of validamycin.

The chitin biosynthesis pathway starts with trehalose in insects and the main functions of trehalases are hydrolysis of trehalose to glucose. Although insects possess two types, soluble trehalase (Tre-1) and membrane-bound trehalase (Tre-2), very little is known about Tre-2 and the difference in function between Tre-1 and Tre-2.

In order to be digested, the disaccharide trehalose needs to be cleaved by the trehalase enzyme. There were reports suggesting that trehalase deficiency was more common in high-latitude than in the temperate climate populations. New horizons were opened for the epidemiologic research of trehalase enzymopathy when it became clear that reduced trehalase activity is determined by the A allele of tTREH gene (rs2276064). The aim of this study was to analyze the frequencies of the trehalase gene alleles and genotypes among the indigenous peoples of Siberia and the Russian Far East. We genotyped 567 samples representing the indigenous peoples of Siberia and the Russian Far East and 146 samples representing Eastern Slavs as the reference dataset. We found that the frequencies of the A*TREH alleles increased to the east. The A*TREH allele frequency was 0.03 in the reference group, 0.13-0.26 in the North-West Siberian indigenous populations, 0.29-0.30 in the South Siberia, 0.43 in West Siberia, and 0.46 in the low Amur populations. The highest frequency of the A allele (0.63) was observed in the Chukchi and Koryak populations. From 1 to 5% of European origin individuals are at risk of trehalase enzymopathy. In the indigenous populations, the frequency of the A*TREH allele varies 13% to 63%, whereas the frequency of the AA*TREH genotype from 3% to 39%. Thus, the total risk of trehalase enzymopathy among the homo- and heterozygous carriers of the A*TREH allele in the studied indigenous populations may be as high as 24% to 86%.

Trehalase is an essential hydrolase of trehalose in insects. However, whether and how trehalase performs in the association of parasitoid wasps and their hosts still remains unknown. Here, the exact function of trehalase of the general cutworm Spodoptera litura after it was parasitized by its predominant endoparasitoid Meterous pulchricornis was elucidated. Two trehalase genes (SlTre1, SlTre2) were identified, and they were highly expressed five days after parasitization by M. pulchricornis. Then, we successfully silenced SlTre1 and SlTre2 in parasitized third instar S. litura larvae. The content of glucose, which is the hydrolysate of trehalose, was significantly decreased after silencing SlTres in parasitized S. litura larvae, and the activities of trehalase were also notably reduced. In addition, the cocoon weight, the emergence rate, proportion of normal adults, and the body size of parasitoid offsprings were significantly decreased in SlTre1- or SlTre2-silenced groups compared to the controls. These results implied that parasitization by parasitoids regulated the trehalase of host larvae to create a suitable nutritional environment for the parasitoid offspring. The present study broadens the knowledge of trehalase in the interaction between parasitoids and their hosts and is of benefit to biological control of S. litura acting by parasitoid wasps.

Citrus canker, caused by the bacterium Xanthomonas citri subsp. citri (Xcc), is a severe disease of citrus. Xcc presents broad spectrum of citrus hosts including economically important species whereas X. fuscans subsp. aurantifolii-type C (XauC) causes a milder disease and only infects Citrus aurantifolia. Trehalase catalyzes hydrolysis of the disaccharide trehalose, a sugar that has been reported to be related to Xcc pathogenicity. We expressed the recombinant gene product and assessed Xcc trehalase structural and kinetics data. The recombinant protein presented 42.7% of secondary structures in α-helix and 13% in β-sheets, no quaternary structure in solution, and Michaelis-Menten constant (KM) of 0.077 mM and Vmax 55.308 μMol glucose.min-1.mg protein-1 for trehalose. A Xcc mutant strain (XccΔtreA) was produced by gene deletion from Xcc genome. Enzymatic activity of trehalase was determined in Xcc, XauC and XccΔtreA cellular lysates, showing the highest values for XauC in in vitro infective condition and no activity for XccΔtreA. Finally, leaves of Citrus aurantifolia infected with XccΔtreA showed much more drenching and necrosis than those infected by wild type Xcc. We concluded that trehalase contributes to alleviate bacterial virulence and that inability for trehalose hydrolysis may promote higher Xcc infectivity.

Trehalase regulates energy metabolism in insects by converting trehalose into two glucose molecules. High amounts of trehalase are critical for insect flight and larval stress resistance. However, whether trehalase participates in the development of pesticide resistance remains unclear. In this study, we explored this phenomenon and the mechanism that underlies the regulation of Trehalase transcription. We found that overexpression of PxTreh1 and PxTreh2 induced Bacillus thuringiensis (Bt) resistance in Plutella xylostella. The promoter sequences of PxTreh1 and PxTreh2 were also cloned and identified. The dual-luciferase reporter system and RNA interference technology revealed that the expression of PxTreh1 and PxTreh2 genes is possibly regulated by the CCAAT enhancer-binding protein (C/EBPα). A yeast one-hybrid experiment confirmed the interaction between C/EBPα and the PxTreh2 promoter. The findings of this study suggest that C/EBPα mediates the adaptability of P. xylostella to adverse environmental stressors by regulating the expression of trehalase.

Protein phosphorylation enables a rapid adjustment of cellular activities to diverse intracellular and environmental stimuli. Many phosphoproteins are targeted on more than one site, which allows the integration of multiple signals and the implementation of complex responses. However, the hierarchy and interplay between multiple phospho-sites are often unknown. Here, we study multi-site phosphorylation using the yeast trehalase Nth1 and its activator, the 14-3-3 protein Bmh1, as a model. Nth1 is known to be phosphorylated by the metabolic kinase PKA on four serine residues and by the cell cycle kinase CDK on one residue. However, how these five phospho-sites adjust Nth1 activity remains unclear. Using a novel reporter construct, we investigated the contribution of the individual sites for the regulation of the trehalase and its 14-3-3 interactor. In contrast to the constitutively phosphorylated S20 and S83, the weaker sites S21 and S60 are only phosphorylated by increased PKA activity. For binding Bmh1, S83 functions as the high-affinity "gatekeeper" site, but successful binding of the Bmh1 dimer and thus Nth1 activation requires S60 as a secondary site. Under nutrient-poor conditions with low PKA activity, S60 is not efficiently phosphorylated and the cell cycle dependent phosphorylation of S66 by Cdk1 contributes to Nth1 activity, likely by providing an alternative Bmh1 binding site. Additionally, the PKA sites S20 and S21 modulate the dephosphorylation of Nth1 on downstream Bmh1 sites. In summary, our results expand our molecular understanding of Nth1 regulation and provide a new aspect of the interaction of 14-3-3 proteins with their targets.

Trehalases have potential applications in several fields, including food additives, insecticide development, and transgenic plant. In the present study, we focused on a trehalase from the marine bacterium Zunongwangia sp., which hydrolyzes trehalose to glucose.

Trehalase inhibitors are considered safe alternatives for insecticides and fungicides. However, there are no studies testing these compounds on Anopheles gambiae, a major vector of human malaria. This study predicted the three-dimensional structure of Anopheles gambiae trehalase (AgTre) and identified potential inhibitors using molecular docking and molecular dynamics methods. Robetta server, C-I-TASSER, and I-TASSER were used to predict the protein structure, while the structural assessment was carried out using SWISS-MODEL, ERRAT, and VERIFY3D. Molecular docking and screening of 3022 compounds was carried out using AutoDock Vina in PyRx, and MD simulation was carried out using NAMD. The Robetta model outperformed all other models and was used for docking and simulation studies. After a post-screening analysis and ADMET studies, uniflorine, 67837201, 10406567, and Compound 2 were considered the best hits with binding energies of -6.9, -8.9, -9, and -8.4 kcal/mol, respectively, better than validamycin A standard (-5.4 kcal/mol). These four compounds were predicted to have no eco-toxicity, Brenk, or PAINS alerts. Similarly, they were predicted to be non-mutagenic, carcinogenic, or hepatoxic. 67837201, 10406567, and Compound 2 showed excellent stability during simulation. The study highlights uniflorine, 67837201, 10406567, and Compound 2 as good inhibitors of AgTre and possible compounds for malaria vector control.

Trehalase specifically hydrolyses trehalose into two glucose units and is most important in insects and fungi. Previous evidence suggested that Spodoptera frugiperda midgut trehalase (wild type, WT) has substantial conformational changes on binding different substances. Our goal is to understand this mobility. For this, two deletion mutants were produced, lacking regions supposed to be the cause of mobility [(102 residues from the N-terminus (NT) and this portion plus 31 residues from the C-terminus (NCT)]. Circular dichroism spectra before and after denaturation of the enzymes support the assertion that they are appropriately folded. The overall results show that the removal of 102 or 133 amino acids does not greatly change the interaction with the substrate and competitive inhibitors, but leads to a considerable decrease in kcat/Km values from WT 74,500 M-1 s-1 to NT 647 M-1 s-1 and NCT 1,044 M-1 s-1. Diethyl pyrocarbonate His modification only occurs in wild and truncated trehalases in the presence of some ligands. Looking for changes in folding WT, NT, and NCT were incubated with different compounds in the presence of Sypro Orange, that binds to hydrophobic regions increasing its fluorescence. The dye fluorescence is affected by 2 compounds when WT is present, and at least by 5 compounds when NT or NCT are present, suggesting that conformational changes caused by ligand binding occur only in the vicinity of the active site. These data provide physical evidence in favor of a change in folding around the active site caused by ligand binding, in agreement to prior chemical modification and other kinetic data and challenging the hypothesis that N- and C-terminal are the mobile regions.

Candida glabrata yeast is the second cause of candidiasis worldwide. Differs from other yeasts since assimilates only glucose and trehalose (a characteristic used in rapid identification tests for this pathogen) by secreting into the medium a highly active acid trehalase encoded by the CgATH1 gene.

As one of the major hydrolases in Drosophila, trehalase (Treh) catalyzes the hydrolysis of trehalose into glucose providing energy for flight muscle activity. Treh is highly conserved from bacteria to humans, but little is known about its function during animal development. Here, we analyze the function of Treh in Drosophila optic lobe development. In the optic lobe, neuroepithelial cells (NEs) first divide symmetrically to expand the stem cell pool and then differentiate into neuroblasts, which divide asymmetrically to generate medulla neurons. We find that the knockdown of Treh leads to a loss of the lamina and a smaller medulla. Analyses of Treh RNAi-expressing clones and loss-of-function mutants indicate that the lamina and medulla phenotypes result from neuroepithelial disintegration and premature differentiation into medulla neuroblasts. Although the principal role of Treh is to generate glucose, the Treh loss-of-function phenotype cannot be rescued by exogenous glucose. Thus, our results indicate that in addition to being a hydrolase, Treh plays a role in neuroepithelial stem cell maintenance and differentiation during Drosophila optic lobe development.

Candida glabrata is an opportunistic human fungal pathogen and is frequently present in the human microbiome. It has a high relative resistance to environmental stresses and several antifungal drugs. An important component involved in microbial stress tolerance is trehalose. In this work, we characterized the three C. glabrata trehalase enzymes Ath1, Nth1 and Nth2. Single, double and triple deletion strains were constructed and characterized both in vitro and in vivo to determine the role of these enzymes in virulence. Ath1 was found to be located in the periplasm and was essential for growth on trehalose as sole carbon source, while Nth1 on the other hand was important for oxidative stress resistance, an observation which was consistent by the lower survival rate of the NTH1 deletion strain in human macrophages. No significant phenotype was observed for Nth2. The triple deletion strain was unable to establish a stable colonization of the gastrointestinal (GI) tract in mice indicating the importance of having trehalase activity for colonization in the gut.