Search

Trehalase plays an important role in insect metabolism and development by hydrolyzing blood sugar trehalose, but it seems to perform primarily an immunomodulatory function in crustaceans whose blood sugar is glucose. Metal ions as pollutants seriously affecting crustacean health, but studies on trehalase in metal immunity are still limited. In this study, a soluble trehalase (NdTre1) that could bind to multiple metals was identified from Neocaridina denticulata sinensis for investigating metal resistance. Expression profiling revealed that NdTre1 was mainly expressed in the gill and was significantly decreased following stimulation with copper (Cu²⁺) and cadmium (Cd²⁺). Transcriptomic analysis of gills revealed an increase in ecdysone synthesis after interference with NdTre1. Increased ecdysone activated the endogenous mitochondrial pathway and the mitogen activated protein kinase (MAPK) pathway to further induced apoptosis. In vitro, Escherichia coli overexpressing recombinant NdTre1 had higher survival and faster growth rates to better adapted the metal-containing medium. Overall, NdTre1 exercises an important immune function in shrimp resistance to metal stimulation by regulating apoptosis and molting. Further investigation can further explore specific response mechanisms of NdTre1 to multiple metals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Trehalase gene is mainly expressed in the digestive circulatory system for regulating energy metabolism and chitin synthesis in insects, but it is significantly expressed in gill for immunomodulation in shrimp. However, its function in regulating immunity, particularly metal resistance in crustaceans has yet to be elucidated. In this study, one Tre2 gene (NdTre2) was isolated from Neocaridina denticulata sinensis. It could bind to Cd 2+ and inhibit its toxicity. Spatiotemporal expression analysis showed that the expression of NdTre2 was highest in the gill and significantly reduced at 12 h after Cd 2+ stimulation. The transcriptomic analysis of the gill after NdTre2 knockdown showed that the expression of genes synthetizing 20E was up-regulated and the increased 20E could further induce apoptosis by activating the intrinsic mitochondrial pathway, exogenous death receptor-ligand pathway, and MAPK pathway. In vitro, overexpressing NdTre2 enhanced the tolerance of E. coli in Cd 2+ environment. In summary, these results indicate that NdTre2 plays an essential role in regulating immunity and chitin metabolism in N. denticulata sinensis., Competing Interests: Declaration of competing interest The author(s) declared no potential conflicts of interest with respect to the research, author- ship, and/or publication of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The prothoracic gland (PG) is the source of ecdysteoids in larval insects. Although numerous studies have been conducted on signaling networks involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in PGs, less is known about regulation of metabolism in PGs. In the present study, we investigated correlations between expressions of sugar transporter (St)/trehalase (Treh) genes and PTTH-stimulated ecdysteroidogenesis in Bombyx mori PGs. Our results showed that in vitro PTTH treatment stimulated expression of the St1 gene, but not other transporter genes. Expression of the Treh1 gene was also stimulated by PTTH treatment. An immunoblotting analysis showed that St1 protein levels in Bombyx PGs increased during the later stage of the last larval instar and were not affect by PTTH treatment. PTTH treatment enhanced Treh enzyme activity in a time-dependent manner. Blocking either extracellular signal-regulated kinase (ERK) signaling with U0126 or phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 decreased PTTH-stimulated Treh enzyme activity, indicating a link from the ERK and PI3K signaling pathways to Treh activity. Treatment with the Treh inhibitor, validamycin A, blocked PTTH-stimulated Treh enzyme activity and partially inhibited PTTH-stimulated ecdysteroidogenesis. Treatment with either a sugar transport inhibitor (cytochalasin B) or a specific glycolysis inhibitor (2-deoxy-D-glucose, 2-DG) partially inhibited PTTH-stimulated ecdysteroidogenesis. Taken together, these results indicate that increased expressions of St1/Treh1 and Treh activity, which lie downstream of PTTH signaling, are involved in PTTH stimulation in B. mori PGs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Considering the structure of the bacterial GH15 family glucoamylase (GA), Thermoplasma trehalase Tvn1315 may be composed of a β-sandwich domain (BD) and a catalytic domain (CD). Tvn1315 BD weakly binds to insoluble β-glucans, such as cellulose, and helps fold CD. To determine how aromatic residues contribute to proper folding and enzyme activity, we performed alanine scanning for 32 aromatic residues in the BD. The study did not identify a single residue involved in glucan binding. However, several aromatic residues were found to be involved in BD or CD folding and in modulating the activity of the full-length enzyme. Among those aromatic residue mutations, the W43A mutation led to reduced solubility of the BD and full-length protein and resulted in a full-length enzyme with significantly lower activity. The activity of W43F and W43Y was significantly higher than that of W43A. In addition, Ala substitutions of Tyr83, Tyr113, and Tyr17 led to a reduction in trehalase activity, but Phe substitutions of these residues could be tolerated, as these mutants maintained activities similar to WT activity. Thus, these aromatic residues in BD may interact with CD and modulate enzyme activity. KEY POINTS: • Aromatic residues in the BD are involved in BD and CD folding. • Aromatic residues in the BD near the CD active site modulate enzyme activity. • BD interacts with CD and closely modulates enzyme activity., (© 2024. The Author(s).)

Validamycin A (VMA) is an antifungal antibiotic derived from Streptomyces hygroscopicus commonly used in plant disease management. Surprisingly, VMA was discovered to impede the production of fumonisin B1 (FB 1 ) in agricultural settings. However, the specific target of VMA in Fusarium verticillioides remained unclear. To unravel the molecular mechanism of VMA, ultrastructural observations unveiled damage to mitochondrial membranes. Trehalase (FvNth) was pinpointed as the target of VMA by utilizing a 3D-printed surface plasmon resonance sensor. Molecular docking identified Trp 285 , Arg 447 , Asp 452 , and Phe 665 as the binding sites between VMA and FvNth. A ΔFvnth mutant lacking amino acids 250-670 was engineered through homologous recombination. Transcriptome analysis indicated that samples treated with VMA and ΔFvnth displayed similar expression patterns, particularly in the suppression of the FUM gene cluster. VMA treatment resulted in reduced trehalase and ATPase activity as well as diminished production of glucose, pyruvic acid, and acetyl-CoA. Conversely, these effects were absent in samples treated with ΔFvnth . This research proposes that VMA hinders acetyl-CoA synthesis by trehalase, thereby suppressing the FB 1 biosynthesis. These findings present a novel target for the development of mycotoxin control agents.

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.)

The cold-adapted bacterium Variovorax sp. PAMC28711 possesses two distinct glycoside hydrolase (GH) families of trehalase, GH15 and GH37. While numerous studies have explored bacterial trehalase, the presence of two different trehalase genes within a single strain has not been reported until now. Interestingly, despite both GH37 and GH15 trehalases serving the same purpose of degrading trehalose, but do not share the sequence similarity. The substrate specificity assay confirmed that Vtre37 and Vtre15 displayed hydrolytic activity on α, α-trehalose. The key catalytic sites were identified as D280 and E469 in Vtre37 and E389 and E554 in Vtre15 through site-directed mutation and confirmed these two enzymes belong to trehalase. In addition, Vtre37 exhibited a relatively high level of enzyme activity of 1306.33 (±53.091) μmolmg -1 , whereas Vtre15 showed enzyme activity of 408.39 (±12.503) μmolmg -1 . Moreover, Vtre37 performed admirably showing resistance to ethanol (10 %), with high stable at acidic pH range. Furthermore, both prediction and experimental results indicate that validoxylamine A showed a potent inhibitory activity against Vtre37 trehalase with a K i value of 16.85 nM. Therefore, we postulate that Vtre37 could be utilized as an ethanol enhancer and designed for screening inhibitors related to the trehalose degradation pathway. Additionally, we believe that characterizing these bacterial trehalase contributes to a better understanding of trehalose metabolism and its biological importance in bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Background: Insects utilize trehalases (TREs) to regulate energy metabolism and chitin biosynthesis, which are essential for their growth, development, and reproduction. TREs can therefore be used as potential targets for future insecticide development. However, the roles of TREs in Frankliniella occidentalis (Pergande), a serious widespread agricultural pest, remain unclear., Results: Three TRE genes were identified in F. occidentalis and cloned, and their functions were then investigated via feeding RNA interference (RNAi) and virus-induced gene silencing (VIGS) assays. The results showed that silencing FoTRE1-1 or FoTRE1-2 significantly decreased expression levels of FoGFAT, FoPGM, FoUAP, and FoCHS, which are members of the chitin biosynthesis pathway. Silencing FoTRE1-1 or FoTRE2 significantly down-regulated FoPFK and FoPK, which are members of the energy metabolism pathway. These changes resulted in 2-fold decreases in glucose and glycogen content, 2-fold increases in trehalose content, and 1.5- to 2.0-fold decreases in chitinase activity. Furthermore, knocking down FoTRE1-1 or FoTRE1-2 resulted in deformed nymphs and pupae as a result of hindered molting. The VIGS assay for the three FoTREs revealed that FoTRE1-1 or FoTRE2 caused shortened ovarioles, and reduced egg-laying and hatching rates., Conclusion: The results suggest that FoTRE1-1 and FoTRE1-2 play important roles in the growth and development of F. occidentalis, while FoTRE1-1 and FoTRE2 are essential for its reproduction. These three genes could be candidate targets for RNAi-based management and control of this destructive agricultural pest. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Trehalases (TREs) are pivotal enzymes involved in insect development and reproduction, making them prime targets for pest control. We investigated the inhibitory effect of three thiazolidinones with piperine skeletons (6a, 7b, and 7e) on TRE activity and assessed their impact on the growth and development of the fall armyworm (FAW), Spodoptera frugiperda . The compounds were injected into FAW larvae, while the control group was treated with 2% DMSO solvent. All three compounds effectively inhibited TRE activity, resulting in a significant extension of the pupal development stage. Moreover, the treated larvae exhibited significantly decreased survival rates and a higher incidence of abnormal phenotypes related to growth and development compared to the control group. These results suggest that these TRE inhibitors affect the molting of larvae by regulating the chitin metabolism pathway, ultimately reducing their survival rates. Consequently, these compounds hold potential as environmentally friendly insecticides.

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.)

Trehalose serves as a primary circulatory sugar in insects which is crucial in energy metabolism and stress recovery. It is hydrolyzed into two glucose molecules by trehalase. Silencing or inhibiting trehalase results in reduced fitness, developmental defects, and insect mortality. Despite its importance, the molecular response of insects to trehalase inhibition is not known. Here, we performed transcriptomic analyses of Helicoverpa armigera treated with validamycin A (VA), a trehalase inhibitor. VA ingestion resulted in increased mortality, developmental delay, and reduced ex vivo trehalase activity. Pathway enrichment and gene ontology analyses suggest that key genes involved in carbohydrate, protein, fatty acid, and mitochondria-related metabolisms are deregulated. The activation of protein and fat degradation may be necessary to fulfil energy requirements, evidenced by the dysregulated expression of critical genes in these metabolisms. Co-expression analysis supports the notion that trehalase inhibition leads to putative interaction with key regulators of other pathways. Metabolomics correlates with transcriptomics to show reduced levels of key energy metabolites. VA generates an energy-deficient condition, and insects activate alternate pathways to facilitate the energy demand. Overall, this study provides insights into the molecular mechanisms underlying the response of insects to trehalase inhibition and highlights potential targets for insect control.

Verticillum dahliae is a soil-borne phytopathogenic fungus causing destructive Verticillium wilt disease. We previously found a trehalase-encoding gene ( VdPT1 ) in V. dahliae being significantly up-regulated after sensing root exudates from a susceptible cotton variety. In this study, we characterized the function of VdPT1 in the growth and virulence of V. dahliae using its deletion-mutant strains. The VdPT1 deletion mutants ( ΔVdPT1 ) displayed slow colony expansion and mycelial growth, reduced conidial production and germination rate, and decreased mycelial penetration ability and virulence on cotton, but exhibited enhanced stress resistance, suggesting that VdPT1 is involved in the growth, pathogenesis, and stress resistance of V. dahliae . Host-induced silencing of VdPT1 in cotton reduced fungal biomass and enhanced cotton resistance against V. dahliae . Comparative transcriptome analysis between wild-type and mutant identified 1480 up-regulated and 1650 down-regulated genes in the ΔVdPT1 strain. Several down-regulated genes encode plant cell wall-degrading enzymes required for full virulence of V. dahliae to cotton, and down-regulated genes related to carbon metabolism, DNA replication, and amino acid biosynthesis seemed to be responsible for the decreased growth of the ΔVdPT1 strain. In contrast, up-regulation of several genes related to glycerophospholipid metabolism in the ΔVdPT1 strain enhanced the stress resistance of the mutated strain.

Nosema bombycis, an obligate intracellular parasite, is a single-celled eukaryote known to infect various tissues of silkworms, leading to the manifestation of pebrine. Trehalase, a glycosidase responsible for catalyzing the hydrolysis of trehalose into two glucose molecules, assumes a crucial role in thermal stress tolerance, dehydration, desiccation stress, and asexual development. Despite its recognized importance in these processes, the specific role of trehalase in N. bombycis remains uncertain. This investigation focused on exploring the functions of trehalase 3 in N. bombycis (NbTre3). Immunofluorescence analysis of mature (dormant) spores indicated that NbTre3 primarily localizes to the spore membrane or spore wall, suggesting a potential involvement in spore germination. Reverse transcription-quantitative polymerase chain reaction results indicated that the transcriptional level of NbTre3 peaked at 6 h post N. bombycis infection, potentially contributing to energy storage for proliferation. Throughout the life cycle of N. bombycis within the host cell, NbTre3 was detected in sporoplasm during the proliferative stage rather than the sporulation stage. RNA interference experiments revealed a substantial decrease in the relative transcriptional level of NbTre3, accompanied by a certain reduction in the relative transcriptional level of Nb16S rRNA. These outcomes suggest that NbTre3 may play a role in the proliferation of N. bombycis. The application of the His pull-down technique identified 28 proteins interacting with NbTre3, predominantly originating from the host silkworm. This finding implies that NbTre3 may participate in the metabolism of the host cell, potentially utilizing the host cell's energy resources., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 t TREH 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%.

The general cutworm, Spodoptera litura (Lepidoptera: Noctuidae) is a worldwide destructive omnivorous pest and the endoparasitoid wasp Meteorus pulchricornis (Hymenoptera: Braconidae) is the dominant endoparasitoid of S. litura larvae. Trehalase is a key enzyme in insect trehalose metabolism and plays an important role in the growth and development of insects. However, the specific function of trehalase in parasitoid and host associations has been less reported. In this study, we obtained two trehalase genes (SlTre1 and SlTre2) from our previously constructed S. litura transcriptome database; they were highly expressed in 3rd instar larvae. SlTre1 was mainly expressed in the midgut, and SlTre2 was expressed highest in the head. SlTre1 and SlTre2 were highly expressed 5 days after parasitization by M. pulchricornis. Treatment with the trehalase inhibitor validamycin A significantly inhibited the expression levels of SlTre1 and SlTre2, and the trehalase activity. Besides, the content of trehalose was increased but the content of glucose was decreased 24 h after validamycin A treatment in parasitized S. litura larvae. In addition, the immune-related genes in phenoloxidase (PO) pathway and fatty acid synthesis-related genes in lipid metabolism were upregulated in parasitized host larvae after validamycin A treatment. Importantly, the emergence rate, proportion of normal adults, and body size of parasitoid offspring was decreased in parasitized S. litura larvae after validamycin A treatment, indicating that validamycin A disrupts the trehalose metabolism of parasitized host and thus reduces the fitness of parasitoid offspring. The present study provides a novel perspective for coordinating the application of biocontrol and antibiotics in agroecosystem., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In Candida parapsilosis, homozygous disruption of the two genes encoding trehalase activity increased the susceptibility to Itraconazole compared with the isogenic parental strain. The fungicidal effect of this azole can largely be counteracted by preincubating growing cells with rotenone and the protonophore 2,4-Dinitrophenol. In turn, measurement of endogenous reactive oxygen species formation by flow cytometry confirmed that Itraconazole clearly induced an internal oxidative stress, which can be significantly abolished in rotenone-exposed cells. Analysis of the antioxidant enzymatic activities of catalase and superoxide dismutase pointed to a moderate decrease of catalase in trehalase-deficient mutant cells compared to the wild type, with an additional increase upon addition of rotenone. These enzymatic changes were imperceptible in the case of superoxide dismutase. Alternative assays with Voriconazole led to a similar profile in the results regarding cell growth and antioxidant activities. Collectively, our data suggest that the antifungal action of Itraconazole on C. parapsilosis is dependent on a functional mitochondrial activity. They also suggest that the central metabolic pathways in pathogenic fungi should be considered as preferential antifungal targets in new research., (© 2023. The Author(s).)

To date, it has been established that the patient's genotype plays a significant role in the formation of trehalase enzymopathy: the level of enzyme activity decreases when the G→A allele replacement occurs in the rs2276064 locus of the TREH gene. To assess the prevalence of trehalase deficiency, extensive population-based studies are needed. Clinical observations show that the reduced activity of bowel trehalase is more common in the Arctic than in European populations. The aim of this research was to analyze the frequency of the alleles and variants of trehalase gene (rs2276064 TREH) in the indigenous small-numbered populations of Siberia and the Russian Far East. Material and methods . Using the Infinium iSelect HD Custom BeadChip biochip on the iScan platform and real-time polymerase chain reaction on a Bio-Rad CFX96 Touch amplifier, genotyping of 1068 DNA samples was carried out, of which 711 represent 10 ethnic groups of the indigenous people of the North of Siberia and the Far East of the Russian Federation. Two reference groups of Russians (n=311) and Yakuts (n=46) represented the "Caucasoid" and "Mongoloid" poles of the Russian population. Results . The reduced trehalase activity that the heterozygous GA*TREH genotype determines can manifest itself in 19.8-53.7% of indigenous northerners. An additional 1.0 to 19.7% of the population are carriers of the AA*TREH genotype, which is associated with apparent trehalose malabsorption. The carriers may experience nausea, abdominal pain, and other dyspeptic symptoms after eating trehalose containing foods. The total risk of trehalase enzymopathy among the indigenous northerners in the Asian part of the Russian Federation is very high and can reach 60-70%. There is a gradient in the A*TREH allele frequencies in the small-numbered indigenous northern groups of Russia from the west (Khanty, Mansi, Nenets) to the east (peoples of the Far East). Conclusion . The results are consistent with previously reported data on the higher carriage of the A*TREH mutant allele in Mongoloid populations compared to Caucasoid groups. It was hypothesized that, while the initial A*TREH allele prevalence in Mongoloid groups was moderately high, an adaptation to a low-sugar protein-lipid "high-latitude" diet led to a weaker control over the maintenance of the carriage of the ancestral G allele. Trehalose malabsorption requires special attention of specialists in the field of nutrition, gastroenterology, public health, and medical genetics working in high-latitude regions., Competing Interests: The authors declare no conflicts of interest., (Copyright© GEOTAR-Media Publishing Group.)

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.

Although four Shigella species ( S. flexneri, S . sonnei , S. dysenteriae , and S. boydii ) have been reported, S . sp. PAMC 28760, an Antarctica isolate, is the only one with a complete genome deposited in NCBI database as an uncharacterized isolate. Because it is the world's driest, windiest, and coldest continent, Antarctica provides an unfavourable environment for microorganisms. Computational analysis of genomic sequences of four Shigella species and our uncategorized Antarctica isolates Shigella sp. PAMC28760 was performed using MP3 (offline version) program to predict trehalase encoding genes as a pathogenic or non-pathogenic form. Additionally, we employed RAST and Prokka (offline version) annotation programs to determine locations of periplasmic ( treA ) and cytoplasmic ( treF ) trehalase genes in studied genomes. Our results showed that only 56 out of 134 Shigella strains had two different trehalase genes ( treF and treA ). It was revealed that the treF gene tends to be prevalent in Shigella species. In addition, both treA and treF genes were present in our strain S . sp. PAMC28760. The main objective of this study was to predict the prevalence of two different trehalase genes ( treF and treA ) in the complete genome of Shigella sp. PAMC28760 and other complete genomes of Shigella species. Till date, it is the first study to show that two types of trehalase genes are involved in Shigella species, which could offer insight on how the bacteria use accessible carbohydrate like glucose produced from the trehalose degradation pathway, and importance of periplasmic trehalase involvement in bacterial virulence.

Trehalase (Tre) is a crucial enzyme involved in trehalose metabolism, and it plays pivotal roles in insect development and metamorphosis. However, the biological function of Tre genes in Sogatella furcifera remains unclear. In the present study, two Tre genes- SfTre1 and SfTre2 -were cloned and identified based on the S. furcifera transcriptome data. Bioinformatic analysis revealed that the full-length complementary DNA of SfTre1 and SfTre2 genes were 3700 and 2757 bp long, with 1728- and 1902-bp open reading frame encoding 575 and 633 amino acid residues, respectively. Expression analysis indicated that SfTre1 and SfTre2 were expressed at all developmental stages, with the highest expression in day two adults. Furthermore, the highest expression levels of SfTre1 and SfTre2 were observed in the ovary; enriched expression was also noted in head tissues. The knockdown of SfTre1 and SfTre2 via injecting double-stranded RNAs decreased the transcription levels of the corresponding mRNAs and led to various malformed phenotypes and high lethality rates. The results of our present study indicate that SfTre1 and SfTre2 play crucial roles in S. furcifera growth and development, which can provide referable information for Tre genes as a potential target for planthopper control.

AbstractTrehalose is a nonreducing disaccharide that is a primary storage and energy source in prokaryotes, yeasts, fungi, and invertebrates. Vertebrates digest trehalose with the intestinal brush border membrane (BBM) enzyme trehalase. Intestinal trehalase activity is reported to be either very low or absent in several bird species. We assayed trehalase activity in 19 avian species, used proteomic analysis to quantify its abundance in the intestinal BBM, and used analyses of available genomes to detect the presence of the gene that codes for trehalase ( Treh ). We found no intestinal trehalase activity in birds, trehalase was absent from the proteome of their intestinal BBM, and the gene coding for trehalase was absent in their genomes. Surveys of available transcriptomes support the hypothesis that Treh is absent in birds. The trehalase gene was found in the same conserved syntenic block within the genome of all vertebrates surveyed except birds. Our analysis suggests that Treh was lost in an inversion followed by a reinsertion of a large gene block. This event appears to have taken place after the split between crocodiles and birds and dinosaurs. Birds are unable to digest a common dietary sugar like trehalose because their ancestor lost the trehalase gene. The loss of this gene seems to represent an ecological cost, as insectivorous birds seem to be unable to digest a carbohydrate present in their prey. We also speculate that the paucity of mycophagy in birds is due to the presence of large amounts of this sugar in fungal tissues.

Trehalase (Treh) hydrolyzes trehalose to generate glucose and it plays important role in many physiological processes. Acetyl-CoA, the precursor of sex pheromone biosynthesis in the pheromone gland (PG) of Helicoverpa armigera, originates from glucose during glycolysis. However, the function of Treh in sex pheromone biosynthesis remains elusive. In the present study, H. armigera was used as a model to investigate the function of two Trehs (Treh1 and Treh2) in sex pheromone biosynthesis. Results demonstrated that knockdown of HaTreh1 or HaTreh2 in female PGs led to significant decreases in Z11-16:Ald production, female ability to attract males, and successful mating proportions. Pheromone biosynthesis activating neuropeptide (PBAN) treatment triggered HaTreh1 and HaTreh2 activities in the isolated PGs and Sf9 cells. However, the activities of HaTreh1 and HaTreh2 triggered by PBAN were offset by H-89, the specific inhibitor of protein kinase A (PKA). Furthermore, the H-89 treatment significantly decreased the phosphorylation level of Trhe2, which was induced by PBAN. In addition, sugar feeding (5% sugar) increased the enzyme activities of Treh1 and Treh2. In summary, our findings confirmed that PBAN activates Treh1/2 activities by recruiting cAMP/PKA signalling, promotes glycolysis to ensure the supply of acetyl-CoA, and ultimately facilitates sex pheromone biosynthesis and mating behaviour., (© 2022 Royal Entomological Society.)

Trehalose-6-phosphate (T6P) is an intermediate of trehalose biosynthesis that plays an essential role in plant metabolism and development. Here, we comprehensively analyzed sequences from enzymes of trehalose metabolism in sugarcane, one of the main crops used for bioenergy production. We identified protein domains, phylogeny, and in silico expression levels for all classes of enzymes. However, post-translational modifications and residues involved in catalysis and substrate binding were analyzed only in trehalose-6-phosphate synthase (TPS) sequences. We retrieved 71 putative full-length TPS, 93 trehalose-6-phosphate phosphatase (TPP), and 3 trehalase (TRE) of sugarcane, showing all their conserved domains, respectively. Putative TPS (Classes I and II) and TPP sugarcane sequences were categorized into well-known groups reported in the literature. We measured the expression levels of the sequences from one sugarcane leaf transcriptomic dataset. Furthermore, TPS Class I has specific N-glycosylation sites inserted in conserved motifs and carries catalytic and binding residues in its TPS domain. Some of these residues are mutated in TPS Class II members, which implies loss of enzyme activity. Our approach retrieved many homo(eo)logous sequences for genes involved in trehalose metabolism, paving the way to discover the role of T6P signaling in sugarcane., (© 2022. The Author(s).)

Key Message: Alternative translation initiation of the unique Arabidopsis trehalase gene allows for the production of two isoforms with different subcellular localization, providing enzyme access to both intra- and extra-cellular trehalose. The trehalose-hydrolyzing enzyme trehalase mediates drought stress tolerance in Arabidopsis thaliana by controlling ABA-induced stomatal closure. We now report the existence of two trehalase isoforms, produced from a single transcript by alternative translation initiation. The longer full-length N-glycosylated isoform (AtTRE1L) localizes in the plasma membrane with the catalytic domain in the apoplast. The shorter isoform (AtTRE1S) lacks the transmembrane domain and localizes in the cytoplasm and nucleus. The two isoforms can physically interact and this interaction affects localization of AtTRE1S. Consistent with their role in plant drought stress tolerance, both isoforms are activated by AtCPK10, a stress-induced calcium-dependent guard cell protein kinase. Transgenic plants expressing either isoform indicate that both can mediate ABA-induced stomatal closure in response to drought stress but that the short (cytoplasmic/nuclear) isoform, enriched in those conditions, is significantly more effective., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Insect trehalases are glycoside hydrolases essential for trehalose metabolism and stress resistance. We here report the extraction and purification of Acyrthosiphon pisum soluble trehalase (ApTreh-1), its biochemical and structural characterization, as well as the determination of its kinetic properties. The protein has been purified by ammonium sulphate precipitation, first followed by an anion-exchange and then by an affinity chromatography. The SDS-PAGE shows a main band at 70 kDa containing two isoforms of ApTreh-1 (X1 and X2), identified by mass spectrometry and slightly contrasting in the C-terminal region. A phylogenetic tree, a multiple sequence alignment, as well as a modelled 3D-structure were constructed and they all reveal the ApTreh-1 similarity to other insect trehalases, i.e. the two signature motifs 179 PGGRFRELYYWDTY 192 and 479 QWDFPNAWPP 489 , a glycine-rich region 549 GGGGEY 554 , and the catalytic residues Asp336 and Glu538. The optimum enzyme activity occurs at 45 °C and pH 5.0, with K m and V max values of ~ 71 mM and ~ 126 µmol/min/mg, respectively. The present structural and functional characterization of soluble A. pisum trehalase enters the development of new strategies to control the aphids pest without significant risk for non-target organisms and human health., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In agriculture, Trehalase is considered the main target of the biological fungicide validamycin A, and the toxicology mechanism of validamycin A is unknown. 14-3-3 proteins, highly conserved proteins, participate in diverse cellular processes, including enzyme activation, protein localization, and acting as a molecular chaperone. In Saccharomyces cerevisiae , the 14-3-3 protein Bmh1could interact with Nth1 to respond to specific external stimuli. Here, we characterized FgNth, FgBmh1, and FgBmh2 in Fusarium graminearum. ΔFgNth, ΔFgBmh1, and ΔFgBmh2 displayed great growth defects and their peripheral tips hyphae generated more branches when compared with wild-type (WT) PH-1. When exposed to validamycin A as well as high osmotic and high temperature stresses, ΔFgNth, ΔFgBmh1, and ΔFgBmh2 showed more tolerance than WT. Both ΔFgNth and ΔFgBmh1 displayed reduced deoxynivalenol production but opposite for ΔFgBmh2, and all three deletion mutants showed reduced virulence on wheat coleoptiles. In addition, coimmunoprecipitation (Co-IP) experiments suggested that FgBmh1 and FgBmh2 both interact with FgNth, but no interaction was detected between FgBmh1 and FgBmh2 in our experiments. Further, validamycin A enhances the interaction between FgBmh1 and FgNth in a positive correlation under concentrations of 1 to 100 μg/ml. In addition, both high osmotic and high temperature stresses promote the interaction between FgBmh1 and FgNth. Co-IP assay also showed that neither FgBmh1 nor FgBmh2 could interact with FgPbs2, a MAPKK kinase in the high-osmolarity glycerol pathway. However, FgBmh2 but not FgBmh1 binds to the heat shock protein FgHsp70 in F. graminearum . Taken together, our results demonstrate that FgNth and FgBmh proteins are involved in growth and responses to external stresses and virulence; and validamycin enhanced the interaction between FgNth and FgBmh1in F. graminearum .

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.

Trehalose serves multifarious roles in growth and development of insects. In this study, we demonstrated that the high trehalose diet increased the glucose content, and high glucose diet increased the glucose content but decreased the trehalose content of Acyrthosiphon pisum. RNA interference (RNAi) of trehalose-6-phosphate synthase gene (ApTPS) decreased while RNAi of trehalase gene (ApTRE) increased the trehalose and glucose contents. In the electrical penetration graph experiment, RNAi of ApTPS increased the percentage of E2 waveform and decreased the percentage of F and G waveforms. The high trehalose and glucose diets increased the percentage of E2 waveform of A. pisum red biotype. The correlation between feeding behavior and sugar contents indicated that the percentage of E1 and E2 waveforms were increased but np, C, F and G waveforms were decreased in low trehalose and glucose contents. The percentage of np, E1 and E2 waveforms were reduced but C, F and G waveforms were elevated in high trehalose and glucose contents. The results suggest that the A. pisum with high trehalose and glucose contents spent less feeding time during non-probing phase and phloem feeding phase, but had an increased feeding time during probing phase, stylet work phase and xylem feeding phase., (© 2021. The Author(s).)

Bovine Leukemia Virus (BLV) is an oncogenic virus which is the etiological agent of a neoplastic disease in infected cattle called enzootic bovine leukemia (EBL). The most common and sensitive diagnostic methods for EBL like enzyme-linked immunosorbent assay (ELISA) is time-consuming and requires manual handling which makes it unsuitable as an on-farm diagnostic test. Hence, there is a need for an alternative test with rapid detection and reduced manual labour. We have previously reported the use of E. coli periplasmic trehalase (TreA) in a split enzyme sensor diagnostic technology to detect immunoglobulins and antigen-specific antibodies. In the current study, a more sensitive detection was attempted by bacterial surface display of split TreA fragment by fusion with the autotransporter AIDA-I. The split TreA fragments fused to antigens require antigen-specific antibodies for complementation and to trigger trehalase activity. This surface complementation strategy was used to detect anti-BLV antibodies in clinical serum by incorporating the antigenic BLV capsid protein in the fusion proteins. To validate this assay, a panel of serum samples obtained from BLV positive and negative cattle were tested in comparison with ELISA results. Evaluation of this panel resulted in positive detection of all true positive samples. We further demonstrated that this assay can be enhanced by pre-adsorption of clinical serum samples using E. coli cells to increase the specificity and help reduce nonspecific binding. In conclusion, the p24 antigen specific BLV assay is a potential tool for simple and rapid diagnosis of BLV infection, which is compatible with both lab-based and a more user friendly on-farm format., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Deoxynivalenol (DON) is a vital virulence factor of Fusarium graminearum, which causes Fusarium head blight (FHB). We recently found that validamycin A (VMA), an aminoglycoside antibiotic, can be used to control FHB and inhibit DON contamination, but its molecular mechanism is still unclear. In this study, we found that both neutral and acid trehalase (FgNTH and FgATH) are the targets of VMA in F. graminearum, and the deficiency of FgNTH and FgATH reduces the sensitivity to VMA by 2.12- and 1.79-fold, respectively, indicating that FgNTH is the main target of VMA. We found FgNTH is responsible for vegetative growth, FgATH is critical to sexual reproduction, and both of them play an important role in conidiation and virulence in F. graminearum. We found that FgNTH resided in the cytoplasm, affected the localization of FgATH, and positively regulated DON biosynthesis; however, FgATH resided in vacuole and negatively regulated DON biosynthesis. FgNTH interacted with FgPK (pyruvate kinase), a key enzyme in glycolysis, and the interaction was reduced by VMA; the deficiency of FgNTH affected the localization of FgPK under DON induction condition. Strains with a deficiency of FgNTH were more sensitive to demethylation inhibitor (DMI) fungicides. FgNTH regulated the expression level of FgCYP51A and FgCYP51B by interacting with FgCYP51B. Taken together, VMA inhibits DON biosynthesis by targeting FgNTH and reducing the interaction between FgNTH and FgPK, and synergizes with DMI fungicides against F. graminearum by decreasing FgCYP51A and FgCYP51B expression., (© 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The Asian citrus psyllid, Diaphorina citri is the principal vector of huanglongbing, which transmits Candidatus Liberibacter asiaticus. Trehalase is a key enzyme involved in trehalose hydrolysis and plays an important role in insect growth and development. The specific functions of this enzyme in D. citri have not been determined. In this study, three trehalase genes (DcTre1-1, DcTre1-2, and DcTre2) were identified based on the D. citri genome database. Bioinformatic analysis showed that DcTre1-1 and DcTre1-2 are related to soluble trehalase, whereas DcTre2 is associated with membrane-bound trehalase. Spatiotemporal expression analysis indicated that DcTre1-1 and DcTre1-2 had the highest expression levels in the head and wing, respectively, and DcTre2 had high expression levels in the fat body. Furthermore, DcTre1-1 and DcTre1-2 expression levels were induced by 20-hydroxyecdysone and juvenile hormone Ⅲ, but DcTre2 was unaffected. The expression levels of DcTre1-1, DcTre1-2, and DcTre2 were significantly upregulated, which resulted in high mortality after treatment with validamycin. Trehalase activities and glucose contents were downregulated, but the trehalose content increased after treatment with validamycin. In addition, the expression levels of chitin metabolism-related genes significantly decreased at 24 and 48 h after treatment with validamycin. Furthermore, silencing of DcTre1-1, DcTre1-2, and DcTre2 reduced the expression levels of chitin metabolism-related genes and led to a malformed phenotype of D. citri. These results indicate that D. citri trehalase plays an essential role in regulating chitin metabolism and provides a new target for control of D. citri., (© 2020 Institute of Zoology, Chinese Academy of Sciences.)

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 K m and V max 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 Ca 2+ 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., (Copyright © 2021. Published by Elsevier Inc.)

The expression of trehalase in the midgut of insects plays an important role in glucose supply to the hemolymph. Energy metabolism is usually regulated by the estrogen-related receptor (ERR). A decrease in ATP levels is caused by the ERR hindering glycolysis. However, the relationship between trehalose accumulation and ERR expression is still unclear. Here, we found that silkworm ERR (BmERR) is concentrated and BmERR expression is strongly correlated with trehalase in the midgut during the last instar silkworm larval stage. We cloned the promoter of the trehalase from Bombyx mori (BmTreh) and found that the ERR bound directly to the core response elements of the promoter. Cell level interference and the overexpression of ERR can reduce or enhance BmTreh transcription and promoter activity. Overexpressed transgenic BmERR can significantly increase the expression of BmTreh in the midgut of the last instar silkworm larvae, thereby hydrolyzing trehalose into glucose and releasing it into the hemolymph. Additionally, increased hemolymph glucose content reduces silkworm pupa weight but does not affect silk protein production from the silk gland. Our results suggest a novel function for BmERR through its involvement in BmTreh regulation and expand the understanding of ERR functions in insect trehalose metabolism.

Drought is one of the main abiotic factors that affect agricultural productivity, jeopardizing food security. Modern biotechnology is a useful tool for the generation of stress-tolerant crops, but its release and field-testing involves complex regulatory frameworks. However, gene editing technology mediated by the CRISPR/Cas9 system is a suitable strategy for plant breeding, which can lead to precise and specific modifications in the plant genome. The aim of the present work is to produce drought-tolerant plant varieties by modifying the trehalase gene. Furthermore, a new vector platform was developed to edit monocot and dicot genomes, by modifying vectors adding a streptomycin resistance marker for use with the hypervirulent Agrobacterium tumefaciens AGL1 strain. The gRNA design was based on the trehalase sequence in several species of the genus Selaginella that show drought tolerance. Arabidopsis thaliana carrying editions in the trehalase substrate-binding domain showed a higher tolerance to drought stress. In addition, a transient transformation system for gene editing in maize leaves was characterized.

Trehalose-6-phosphate synthase (TPS) and trehalase (TRE) directly regulate trehalose metabolism and indirectly regulate chitin metabolism in insects. Real-time quantitative PCR (RT-qPCR) and RNA interference (RNAi) were used to detect the expressions and functions of the ApTPS and ApTRE genes. Abnormal phenotypes were found after RNAi of ApTRE in the Acyrthosiphon pisum. The molting deformities were observed in two color morphs, while wing deformities were only observed in the red morphs. The RNAi of ApTPS significantly down-regulated the expression of chitin metabolism-related genes, UDP-N-acetyglucosamine pyrophosphorylase (ApUAP), chitin synthase 2 (Apchs-2), Chitinase 2, 5 (ApCht2, 5), endo-beta-N-acetylglucosaminidase (ApENGase) and chitin deacetylase (ApCDA) genes at 24 h and 48 h; The RNAi of ApTRE significantly down-regulated the expression of ApUAP, ApCht1, 2, 8 and ApCDA at 24 h and 48 h, and up-regulated the expression of glucose-6-phosphate isomerase (ApGPI) and Knickkopf protein (ApKNK) genes at 48 h. The RNAi of ApTRE and ApTPS not only altered the expression of chitin metabolism-related genes but also decreased the content of chitin. These results demonstrated that ApTPS and ApTRE can regulate the chitin metabolism, deepen our understanding of the biological functions, and provide a foundation for better understanding the molecular mechanism of insect metamorphosis.

Due to the low specificity and sensitivity of non-invasive clinical tests trehalose malabsorption remained out of sight of gastroenterologists. Therefore, the specialists regard this disorder as rare. Trehalose became widely used in the food industry as a harmless sucrose substitute, sweetener and stabilizer. After the discovery of the trehalase gene (rs2276064 TREH), it was found that the A*TREH allele is the determinant of the disaccharide absorption disorders, and the allele's carriership may be high in some groups. There is not enough information on the A*TREH frequency in the population of Russia. The aim of the study was to analyze the allele and genotype frequencies of the trehalase gene (rs2276064 TREH) in the main population groups of the Russian Federation and neighboring countries. Methods . DNA samples from 1146 unrelated subjects belonging to 21 population groups of Russia, Azerbaijan, Tajikistan and Mongolia were genotyped by the two following methods: 1) using the Infinium iSelect HD Custom Genotyping BeadChip (Illumina, USA) on the iScan platform; 2) by the real time polymer-chain reaction (PCR) method on the Bio-Rad CFX96 Touch amplifier. Results . It has been found that on the territory of the Russian Federation the frequency of the A*TREH allele increases from the west to the east. The frequencies are lowest in the groups of Russians and Finns of the Northwest (0.01-0.03), up to 0.07 in the populations of Central Russia and the Volga region, and even higher toward the Southern Urals (Bashkirs 0.15), in the Transurals and Southern Siberia (0.19 in the Altai people, 0.30 in the Tuvinians and Mongols). Up to 1% of the population of the European part of the Russian Federation have the AA*TREH genotype (i.e. trehalose intolerance in phenotype), and up to 15% (GA*TREH genotype) have a reduced ability to absorb the disaccharide. In the Asian part of the country (Siberia, Altai, Baikal) the genotypes carriers constitute up to 12 and 46% respectively. Conclusion . Trehalose malabsorbtion is an underappreciated problem of particular practical importance for regions with high concentrations of indigenous population (Yakutia-Sakha, Buryatia, Tyva, etc.). It would be feasible to consider food labelling of trehalose., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

Background: 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., Objective: This study aimed to characterise the function of the acid trehalase in the physiopathology of C. glabrata., Methods: Gene deletion was performed to obtain a mutant ath1Δ strain, and the ability of the ath1Δ strain to grow in trehalase, or the presence of trehalase activity in the ath1Δ yeast cells, was verified. We also tested the virulence of the ath1Δ strain in a murine model of infection., Findings: The ath1Δ mutant strain grows normally in the presence of glucose, but loses its ability to grow in trehalose. Due to the high acid trehalase activity present in wild-type cells, the cytoplasmic neutral trehalase activity is only detected in the ath1Δ strain. We also observed a significantly lower virulence of the ath1Δ strain in a murine model of infection with either normal or immunocompromised mice., Main Conclusions: The acid trehalase is involved in the hydrolysis of external trehalose by C. glabrata, and the enzyme also plays a major virulence role during infectivity.

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 treA mutant strain allowed for full utilization of trehalose. Biochemical characterization of C. japonicus GH37 activity determined that the 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 and more broadly expands our understanding of the strategies that saprophytic bacteria employ to capture diverse carbohydrates from the environment. 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.)

Meloidogyne incognita is a devastating plant parasitic nematode that causes root knot disease in a wide range of plants. In the present study, we investigated host-induced RNA interference (RNAi) gene silencing of chitin biosynthesis pathway genes (chitin synthase, glucose-6-phosphate isomerase, and trehalase) in transgenic tobacco plants. To develop an RNAi vector, ubiquitin (UBQ1) promoter was directly cloned, and to generate an RNAi construct, expression of three genes was suppressed using the GATEWAY system. Further, transgenic Nicotiana benthamiana lines expressing dsRNA for chitin synthase (CS), glucose-6-phosphate isomerase (GPI), and trehalase 1 (TH1) were generated. Quantitative PCR analysis confirmed endogenous mRNA expression of root knot nematode (RKN) and revealed that all three genes were more highly expressed in the female stage than in eggs and in the parasitic stage. In vivo, transformed roots were challenged with M . incognita . The number of eggs and root knots were significantly decreased by 60-90% in RNAi transgenic lines. As evident, root galls obtained from transgenic RNAi lines exhibited 0.01- to 0.70-fold downregulation of transcript levels of targeted genes compared with galls isolated from control plants. Furthermore, phenotypic characteristics such as female size and width were also marginally altered, while effect of egg mass per egg number in RNAi transgenic lines was reduced. These results indicate the relevance and significance of targeting chitin biosynthesis genes during the nematode lifespan. Overall, our results suggest that further developments in RNAi efficiency in commercially valued crops can be applied to employ RNAi against other plant parasitic nematodes.

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 characterize the trehalase gene in Thelazia callipaeda through screening the annotated data of the T. callipaeda genome, and to investigate the biological characteristics of the trehalase gene-coding protein., Methods: The trehalase gene was screened from the T. callipaeda genome and subjected to validation by using a PCR assay. The structural features of the coding protein were analyzed with bioinformatics tools, including hydrophobicity, transmembrane region, signal peptides, conserved domains, as well as the secondary and tertiary structures and the antigen epitope. Homology analysis of the amino acid sequences was performed, and the phylogenetic tree was built by the MEGA X software. In addition, the protein-protein interaction network was deduced from the STRING database., Results: The sequence of the trehalase gene with the complete CDS region was obtained from T. callipaeda genome, which had a length of 1 638 bp and encoded 545 amino acids. The encoded protein was predicted to have a molecular weight of 63 478.48 ku and be a secretory protein. The 5' domain of the encoded protein contained a signal peptide without transmembrane regions, and was predicted to contain 7 antigen epitopes. Based on the protein-protein interaction network of nematodes in the STRING database, the protein-protein interaction network of the trehalase gene of T. callipaeda was deduced, and 27 interactions covering 10 genes were identified., Conclusions: A trehalase gene is successfully identified in T. callipaeda genome and its coding protein receives a bioinformatics analysis, which provides insights into the research on the biological functions of the protein and the screening of vaccine candidates for thelaziasis callipaeda.

Trehalase catalyzes the hydrolysis of the non-reducing disaccharide trehalose. The highly active trehalase MthT from Myceliophthora thermophila was screened from the trehalase genes of six species of filamentous fungi. An ingenious multi-copy knock-in expression strategy mediated by the CRISPR/Cas9 tool and medium optimization were used to improve MthT production in Aspergillus niger, up to 1698.83 U/mL. The protein background was dramatically abated due to insertion. The recombinant MthT showed optimal activity at pH 5.5 and 60 °C, and exhibited prominent thermal stability between 50 and 60 °C under acid conditions (pH 4.5-6.5). The ethanol conversion rate (ethanol yield/total glucose) was significantly improved by addition of MthT (51.88%) compared with MthT absence (34.38%), using 30% starch saccharification liquid. The results of this study provided an effective strategy, established a convenient platform for heterologous expression in A. niger and showed a potential strategy to decrease production costs in industrial ethanol production.

Trehalase catalyzes the conversion of one molecule of trehalose into two glucose molecules. The trehalase TreM from thermophilic fungus Myceliophthora sepedonium was expressed in Aspergillus niger via traditional homologous recombination with trehalase activity of 406.44 U/mL. The multi-copy knock-in expression strategy mediated by the CRISPR/Cas9 tool was used to improve the production of the TreM trehalase in Aspergillus niger, which was up to 1943.06 U/mL with a low-background of secreted proteins, 4.8-fold than the transformant obtained via the traditional method. The highest recombinant trehalase activity of the shake fermentation supernatant achieved 4268.29 U/mL when 1.5% glucose was added. Activity assaying showed that the recombinant TreM possessed a specific activity of 679.09 U/mg after gel filtration chromatography purification. The recombinant TreM displayed optimal activity at pH 5.6 and 60 °C and exhibited prominent stability under the conditions of 45-50 °C and pH 4.0-7.5. The activity of recombinant TreM was strongly enhanced by Co 2+ (1, 5 mM), Cu 2+ (1 mM), Mn 2+ (1, 5 mM) and ATP (5 mM), and was greatly inhibited by Cu 2+ (10 mM), EDTA (10 mM) and SDS (10 mM)., (Copyright © 2019. Published by Elsevier Inc.)

Gene duplication provides a major source of new genes for evolutionary novelty and ecological adaptation. However, the maintenance of duplicated genes and their relevance to adaptive evolution has long been debated. Insect trehalase (Treh) plays key roles in energy metabolism, growth, and stress recovery. Here, we show that the duplication of Treh in Lepidoptera (butterflies and moths) is linked with their adaptation to various environmental stresses. Generally, two Treh genes are present in insects: Treh1 and Treh2. We report three distinct forms of Treh in lepidopteran insects, where Treh1 was duplicated into two gene clusters (Treh1a and Treh1b). These gene clusters differ in gene expression patterns, enzymatic properties, and subcellular localizations, suggesting that the enzymes probably underwent sub- and/or neofunctionalization in the lepidopteran insects. Interestingly, selective pressure analysis provided significant evidence of positive selection on duplicate Treh1b gene in lepidopteran insect lineages. Most positively selected sites were located in the alpha-helical region, and several sites were close to the trehalose binding and catalytic sites. Subcellular adaptation of duplicate Treh1b driven by positive selection appears to have occurred as a result of selected changes in specific sequences, allowing for rapid reprogramming of duplicated Treh during evolution. Our results suggest that gene duplication of Treh and subsequent functional diversification could increase the survival rate of lepidopteran insects through various regulations of intracellular trehalose levels, facilitating their adaptation to diverse habitats. This study provides evidence regarding the mechanism by which gene family expansion can contribute to species adaptation through gene duplication and subsequent functional diversification., (© 2019 John Wiley & Sons Ltd.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Trehalose plays a crucial role in response to freezing stress in baker's yeast. MAL62 , a gene involved in the adenosine diphosphoglucose-dependent trehalose synthesis pathway, can increase trehalose content. However, the difference between MAL62 -related trehalose synthesis and traditional uridine diphosphoglucose-dependent trehalose synthesis is not well-understood. MAL62 overexpression showed less effect in enhancing intracellular trehalose compared to TPS1 overexpression. However, MAL62 overexpression elicited trehalose synthesis before fermentation with enhanced maltose metabolism and had a similar effect on cell viability after freezing. Furthermore, MAL62 and TPS1 overexpression in the NTH1 deletion background further strengthened freezing tolerance and improved leavening ability. Our results suggest that the enhancement in freezing tolerance by MAL62 overexpression may involve multiple pathways rather than simply enhancing trehalose synthesis. The results reveal valuable insights into the relationship between maltose metabolism and freezing tolerance and may help to develop better yeast strains for enhancing fermentation characteristics of frozen dough.

Trehalose is a nonreducing disaccharide, and it plays an intracellular protective role in organisms under various stress conditions. In this study, the trehalose synthesis and its protective role in Pleurotus ostreatus were investigated. As a signal in metabolic regulation, reactive oxygen species (ROS) accumulated in the mycelia of P. ostreatus under heat stress (HS). Furthermore, mycelial growth was significantly inhibited, and the malondialdehyde (MDA) level significantly increased under HS. First, exogenous addition of H 2 O 2 inhibited mycelial growth and elevated the MDA level, while N-acetyl cysteine (NAC) and vitamin C (VC) reduced the MDA level and recovered mycelial growth under HS by scavenging ROS. These results indicated that the mycelial radial growth defect under HS might be partly caused by ROS accumulation. Second, adding NAC and VC to the media resulted in rescued trehalose accumulation, which indicated that ROS has an effect on inducing trehalose synthesis. Third, the mycelial growth was recovered by addition of trehalose to the media after HS, and the MDA level was reduced. This effect was further verified by the overexpression of genes for trehalose-6-phosphate synthase (TPS) and neutral trehalase (NTH), which led to increased and reduced trehalose content, respectively. In addition, adding validamycin A (NTH inhibitor) to the media promoted trehalose accumulation and the recovered mycelial growth after HS. In conclusion, trehalose production was partly induced by ROS accumulation in the mycelia under HS, and the accumulated trehalose could promote the recovery of growth after HS, partly by reducing the MDA level in the mycelia.

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.

A highly conservative insulin signaling pathway, stable work of which is indicated by carbohydrates metabolism, is also known to play an important role in the control of stress resistance. Here we demonstrate that exposure to heat stress leads to a rise in the levels of trehalose and glucose in females of Drosophila melanogaster, but does not affect the expression level of the trehalase (Treh) gene. We have shown that the rise in juvenile hormone (JH) and dopamine decreases levels of both carbohydrates under the normal conditions but brings them to values close to normal following the stress exposure. The data obtained suggest that (a) dopamine and JH involved in the neuroendocrine stress reaction in D. melanogaster also take part in the regulation of carbohydrates metabolism, tending to normalize it after stress; (b) the regulation of trehalose content under stress does not occur at the level of transcription of the degrading enzyme., (© 2019 Wiley Periodicals, Inc.)

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.