Hydrolysis Of Trehalose Research Articles

Evaluating cellular roles and phenotypes associated with trehalose degradation genes in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, 2 types of trehalase activities have been described. Neutral trehalases (Nth1 and Nth2) are considered to be the main proteins that catalyze intracellular trehalose mobilization. In addition to Nth1 and Nth2, studies have shown that acid trehalase Ath1 is required for extracellular trehalose degradation. Although both neutral and acid-type trehalases have been predominantly investigated in laboratory strains of S. cerevisiae, we sought to examine the phenotypic consequences of disrupting these genes in wild strains. In this study, we constructed mutants of the trehalose degradation pathway (NTH1, NTH2, and ATH1) in 5 diverse S. cerevisiae strains to examine whether published lab strain phenotypes are also exhibited by wild strains. For each mutant, we assessed a number of phenotypes for comparison to trehalose biosynthesis mutants, including trehalose production, glycogen production, cell size, acute thermotolerance, high-temperature growth, sporulation efficiency, and growth on a variety of carbon sources in rich and minimal medium. We found that all trehalase mutants including single deletion nth1Δ, nth2Δ, and ath1Δ, as well as double deletion nth1nth2Δ, accumulated higher intracellular trehalose levels compared to their isogenic wild-type cells. Also, nth1Δ and nth1Δnth2Δ mutants exhibited mild thermal sensitivity, suggesting a potential minor role for trehalose mobilization when cells recover from stress. In addition, we evaluated phenotypes more directly relevant to trehalose degradation, including both extracellular and intracellular trehalose utilization. We discovered that intracellular trehalose hydrolysis is critical for typical spore germination progression, highlighting a role for trehalose in cell cycle regulation, likely as a storage carbohydrate providing glycolytic fuel. Additionally, our work provides further evidence suggesting Ath1 is indispensable for extracellular trehalose utilization as a carbon source, even in the presence of AGT1.

Toxicological, biochemical, and in silico investigations of three trehalase inhibitors for new ways to control aphids.

Insect trehalases have been identified as promising new targets for pest control. These key enzymes are involved in trehalose hydrolysis and plays an important role in insect growth and development. In this contribution, plant and microbial compounds, namely validamycin A, amygdalin, and phloridzin, were evaluated for their effect, through trehalase inhibition, on Acyrthosiphon pisum aphid. The latter is part of the Aphididae family, main pests as phytovirus vectors and being very harmful for crops. Validamycin A was confirmed as an excellent trehalase inhibitor with an half maximal inhibitory concentration and inhibitor constant of 2.2 × 10-7 and 5 × 10-8 M, respectively, with a mortality rate of ~80% on a A. pisum population. Unlike validamycin A, the insect lethal efficacy of amygdalin and phloridzin did not correspond to their trehalase inhibition, probably due to their hydrolysis by insect β-glucosidases. Our docking studies showed that none of the three compounds can bind to the trehalase active site, unlike their hydrolyzed counterparts, that is,validoxylamine A, phloretin, and prunasin. Validoxylamine A would be by far the best trehalase binder, followed by phloretin and prunasin.

Functional characterization of Nosema bombycis (microsporidia) trehalase 3.

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 6h 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.

Validamycin Inhibits the Synthesis and Metabolism of Trehalose and Chitin in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel).

The oriental fruit fly, Bactrocera dorsalis (Hendel), is a notorious invasive pest that has raised concerns worldwide. Validamycin has been demonstrated to be a very strong inhibitor against trehalase in a variety of organisms. However, whether validamycin can inhibit trehalase activity to suppress trehalose hydrolysis and affect any other relevant physiological pathways in B. dorsalis remains unknown. In this study, the effects of validamycin injection on the synthesis and metabolism of trehalose and chitin were evaluated. The results show that validamycin injection significantly affected trehalase activity and caused trehalose accumulation. In addition, the downstream pathways of trehalose hydrolysis, including the synthesis and metabolism of chitin, were also remarkably affected as the expressions of the key genes in these pathways were significantly regulated and the chitin contents were changed accordingly. Intriguingly, the upstream trehalose synthesis was also affected by validamycin injection due to the variations in the expression levels of key genes, especially BdTPPC1. Moreover, BdTPPC1 was predicted to have a binding affinity to validamycin, and the subsequent in vitro recombinant enzyme activity assay verified the inhibitory effect of validamycin on BdTPPC1 activity for the first time. These findings collectively indicate that validamycin can be considered as a promising potential insecticide for the management of B. dorsalis.

Sulfur, sterol and trehalose metabolism in the deep-sea hydrocarbon seep tubeworm Lamellibrachia luymesi

BackgroundLamellibrachia luymesi dominates cold sulfide-hydrocarbon seeps and is known for its ability to consume bacteria for energy. The symbiotic relationship between tubeworms and bacteria with particular adaptations to chemosynthetic environments has received attention. However, metabolic studies have primarily focused on the mechanisms and pathways of the bacterial symbionts, while studies on the animal hosts are limited.ResultsHere, we sequenced the transcriptome of L. luymesi and generated a transcriptomic database containing 79,464 transcript sequences. Based on GO and KEGG annotations, we identified transcripts related to sulfur metabolism, sterol biosynthesis, trehalose synthesis, and hydrolysis. Our in-depth analysis identified sulfation pathways in L. luymesi, and sulfate activation might be an important detoxification pathway for promoting sulfur cycling, reducing byproducts of sulfide metabolism, and converting sulfur compounds to sulfur-containing organics, which are essential for symbiotic survival. Moreover, sulfide can serve directly as a sulfur source for cysteine synthesis in L. luymesi. The existence of two pathways for cysteine synthesis might ensure its participation in the formation of proteins, heavy metal detoxification, and the sulfide-binding function of haemoglobin. Furthermore, our data suggested that cold-seep tubeworm is capable of de novo sterol biosynthesis, as well as incorporation and transformation of cycloartenol and lanosterol into unconventional sterols, and the critical enzyme involved in this process might have properties similar to those in the enzymes from plants or fungi. Finally, trehalose synthesis in L. luymesi occurs via the trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) pathways. The TPP gene has not been identified, whereas the TPS gene encodes a protein harbouring conserved TPS/OtsA and TPP/OtsB domains. The presence of multiple trehalases that catalyse trehalose hydrolysis could indicate the different roles of trehalase in cold-seep tubeworms.ConclusionsWe elucidated several molecular pathways of sulfate activation, cysteine and cholesterol synthesis, and trehalose metabolism. Contrary to the previous analysis, two pathways for cysteine synthesis and the cycloartenol-C-24-methyltransferase gene were identified in animals for the first time. The present study provides new insights into particular adaptations to chemosynthetic environments in L. luymesi and can serve as the basis for future molecular studies on host-symbiont interactions and biological evolution.

Trehalose hydrolysis and transport–related genes promote Bombyx mori nucleopolyhedrovirus proliferation through the phosphoinositide 3-kinase–Akt signalling pathway in BmN cell

The reprogramming of host physiology has been considered an essential process for baculovirus propagation. Trehalose, the main sugar in insect blood, plays a crucial role as an instant energy source. Although the trehalose level is modulated following infection with Bombyx mori nucleopolyhedrovirus (BmNPV), the mechanism of trehalose metabolism in response to BmNPV infection is still unclear. In this study, we demonstrated that the trehalose level tended to be lower in BmNPV-infected hemolymph and higher in the midgut. The omics analysis revealed that two trehalose transporters, BmTret1-1 and BmTret1-2, and trehalase, BmTRE1 and BmTRE2, were differentially expressed in the midgut after BmNPV infection. BmTret1-1 and BmTret1-2 had the ability to transport trehalose into the cell and promoted cellular absorption of trehalose. Furthermore, the functions of BmTret1-1, BmTret1-2, BmTRE1 and BmTRE2 in BmNPV infection were analyzed. These genes were upregulated in the midgut after BmNPV infection. Virus amplification analysis revealed that these genes could promote BmNPV proliferation in BmN cells. In addition, these genes could promote the expression of BmPI3K, BmPDK1 and BmAkt and inhibit the expression of BmFoxO in the phosphoinositide 3-kinase (PI3K)–Akt signalling pathway. Similarly, the increased trehalose level in BmN cells could promote the expression of BmPI3K, BmPDK1 and BmAkt and inhibit the expression of BmFoxO. Taken together, BmNPV infection promote the expression of trehalose hydrolysis and transport–related genes. These changes affect the PI3K–Akt signalling pathway to facilitate BmNPV proliferation. These findings help clarify the relationship between trehalose metabolism and BmNPV infection.

Identification and Characterization of a Novel Cold-Adapted GH15 Family Trehalase from the Psychrotolerant Microbacterium phyllosphaerae LW106

Psychrophiles inhabiting various cold environments are regarded as having evolved diverse physiological and molecular strategies, such as the accumulation of trehalose to alleviate cold stress. To investigate the possible contributions of trehalose metabolism-related enzymes to cold-adaption in psychrotrophic bacteria and enrich the resource bank of trehalose hydrolysis enzymes, a novel cold-adapted GH15 GA-like trehalase (MpTre15A) from psychrotolerant Microbacteriumphyllosphaerae LW106 isolated from glacier sediments was cloned and characterized. The recombinant MpTre15A from M. phyllosphaerae LW106 was expressed and purified in Escherichia coli BL21(DE3). The purified MpTre15A functioned as a hexamer and displayed maximal activity at pH 5.0 and 50 °C. Substrate specificity assay proved MpTre15A only showed hydrolytic activity toward α,α-trehalose. Site-directed mutation verified the key catalytic sites of Glu392 and Glu557 in MpTre15A. The kcat and kcat/Km values of MpTre15A at 4 °C (104.50 s−1 and 1.6 s−1 mM−1, respectively) were comparable to those observed for thermophilic GH15 trehalases at 50 °C, revealing its typical cold-adaptability. MpTre15A showed a trehalose conversion rate of 100% and 99.4% after 10 min and 15 min of incubation at 50 °C and 37 °C, respectively. In conclusion, this novel cold-adapted α,α-trehalase MpTre15A showed potential application for developing therapeutic enzymes, enzyme-based biosensors, and enzyme additives in the fermentation industry.

Plant hormone signals regulate trehalose accumulation against osmotic stress in watermelon cells.

Trehalose, one of the most chemically stable sugars, can effectively improve the tolerance of various plants against abiotic stress by protecting and stabilizing protein and cell membranes. However, the signaling pathway in trehalose biosynthesis triggered by abiotic stresses is still unclear. In the study, it can be shown that exogenous trehalose can alleviate the inhibitory effect of osmotic stress on cell growth, suppress extracellular alkalization, ROS burst, and maintain the integrity of the microtubular cytoskeleton. Trehalose-6-phosphate synthase (TPS) is the key limiting enzyme for trehalose synthesis and is encoded by 7 ClTPS genes, located in 7 different chromosomes of the watermelon genome. Expression analysis by qRT-PCR indicated that osmotic stress could upregulate the expression of all the family members of ClTPS and promote the accumulation of trehalose in watermelon cells accordingly. Exogenous methyl jasmonate (MeJA), ethephon (ETH), abscisic acid (ABA), or salicylic acid (SA) induced trehalose accumulation, with MeJA being the most effective treatment. When fluridone (FL), an ABA biosynthesis inhibitor, was pre-perfused into the cells before osmotic stress, trehalose accumulation and packed cell volume were suppressed significantly, whereas inhibition of ethylene biosynthesis could even restore cell growth. Moreover, inhibition of trehalose hydrolysis could also increase the tolerance against osmotic stress. This study shows that trehalose biosynthesis is phytohormone-dependent and the hydrolysis of trehalose is involved in osmotic tolerance regulation.

The vital hormone 20-hydroxyecdysone controls ATP production by upregulating binding of trehalase 1 with ATP synthase subunit α in Helicoverpa armigera

Trehalose is the major “blood sugar” of insects and it plays a crucial role in energy supply and as a stress protectant. The hydrolysis of trehalose occurs only under the enzymatic control of trehalase (Treh), which plays important roles in growth and development, energy supply, chitin biosynthesis, and abiotic stress responses. Previous reports have revealed that the vital hormone 20-hydroxyecdysone (20E) regulates Treh, but the detailed mechanism underlying 20E regulating Treh remains unclear. In this study, we investigated the function of HaTreh1 in Helicoverpa armigera larvae. The results showed that the transcript levels and enzymatic activity of HaTreh1 were elevated during molting and metamorphosis stages in the epidermis, midgut, and fat body, and that 20E upregulated the transcript levels of HaTreh1 through the classical nuclear receptor complex EcR-B1/USP1. HaTreh1 is a mitochondria protein. We also found that knockdown of HaTreh1 in the fifth- or sixth-instar larvae resulted in weight loss and increased mortality. Yeast two-hybrid, coimmunoprecipitation, and glutathione-S-transferase (GST) pull-down experiments demonstrated that HaTreh1 bound with ATP synthase subunit alpha (HaATPs-α) and that this binding increased under 20E treatment. In addition, 20E enhanced the transcript level of HaATPs-α and ATP content. Finally, the knockdown of HaTreh1 or HaATPs-α decreased the induction effect of 20E on ATP content. Altogether, these findings demonstrate that 20E controls ATP production by up-regulating the binding of HaTreh1 to HaATPs-α in H. armigera.

Trehalose metabolism in the Chinese mitten crab Eriocheir sinensis: Molecular cloning of trehalase and its expression during temperature stress

Trehalose, a nonreducing disaccharide, serves as the principal hemolymph sugar involved in energy metabolism and protection against stress in crustaceans. While there have been recent advances in understanding of trehalose biosynthesis in crustaceans, the catabolism of trehalose remains poorly understood. Trehalase catalyzes the hydrolysis of trehalose into glucose and plays a crucial physiological role in most organisms. In this study, we isolated and characterized a trehalase gene, EsTreh, in the Chinese mitten crab Eriocheir sinensis. The full-length EsTreh cDNA sequence was 2488 bp with an open reading frame (ORF) of 1914 bp encoding a protein of 638 amino acids with a deduced molecular weight of 73.53 kDa and a pI of 6.10. The Es-Treh protein contains signature motifs 1 and 2. The transcript levels of EsTreh were significantly inhibited after 24 h of heat and cold stress in E. sinensis. The trehalose content was maintained during 12 h of cold and heat stress, but increased significantly after 24 h of stress. The glucose and lactic acid contents showed a trend of initial rapid increase followed by an extended decline. The glycogen content significantly decreased with the extension of thermal stress. Our study provided the first description of trehalase in crustaceans and demonstrated that trehalase contributes to the accumulation of trehalose during thermal stress. Our findings suggest that the glycogenolysis and gluconeogenesis pathways might be involved in the transformation of glycogen and lactic acid to trehalose.

Characterization and Functional Analysis of trehalase Related to Chitin Metabolism in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae).

Glyphodes pyloalis Walker (G. pyloalis) is a serious pest on mulberry. Due to the increasing pesticide resistance, the development of new and effective environmental methods to control G. pyloalis is needed. Trehalase is an essential enzyme in trehalose hydrolysis and energy supply, and it has been considered a promising target for insect pest control. However, the specific function of trehalase in G. pyloalis has not been reported. In this study, two trehalase genes (GpTre1 and GpTre2) were identified from our previous transcriptome database. The functions of the trehalase in chitin metabolism were studied by injecting larvae with dsRNAs and trehalase inhibitor, Validamycin A. The open reading frames (ORFs) of GpTre1 and GpTre2 were 1,704 bp and 1,869 bp, which encoded 567 and 622 amino acid residues, respectively. Both of GpTre1 and GpTre2 were mainly expressed in the head and midgut. The highest expression levels of them were in 5th instar during different development stages. Moreover, knockdown both of GpTre1 and GpTre2 by the dsRNAs led to significantly decreased expression of chitin metabolism pathway-related genes, including GpCHSA, GpCDA1, GpCDA2, GpCHT3a, GpCHT7, GpCHSB, GpCHT-h, GpCHT3b, GpPAGM, and GpUAP, and abnormal phenotypes. Furthermore, the trehalase inhibitor, Validamycin A, treatment increased the expressions of GpTre1 and GpTre2, increased content of trehalose, and decreased the levels of glycogen and glucose. Additionally, the inhibitor caused a significantly increased cumulative mortality of G. pyloalis larvae on the 2nd (16%) to 6th (41.3%) day, and decreased the rate of cumulative pupation (72.3%) compared with the control group (95.6%). After the activities of trehalase were suppressed, the expressions of 6 integument chitin metabolism-related genes decreased significantly at 24 h and increased at 48 h. The expressions of GpCHSB and GpCHT-h, involved in chitin metabolism pathway of peritrophic membrane in the midgut, increased at 24 h and 48 h, and there were no changes to GpCHT3b and GpPAGM. These results reveal that GpTre1 and GpTre2 play an essential role in the growth of G. pyloalis by affecting chitin metabolism, and this provides useful information for insect pest control in the future.

Integrated Analysis of the Transcriptome and Metabolome Revealed Candidate Genes Involved in GA3-Induced Dormancy Release in Leymus chinensis Seeds.

Leymus chinensis is a perennial forage grass that has good palatability, high yield and high feed value, but seed dormancy is a major problem limiting the widespread cultivation of L. chinensis. Here, we performed transcriptomic and metabolomic analysis of hulled and de-hulled seeds of L. chinensis treated with or without GA3 to investigate the changes in gene and metabolites associated with dormancy release induced by GA3. The germination test revealed that the optimum concentration of GA3 for disruption of L. chinensis seed dormancy was 577 μM. A total of 4327 and 11,919 differentially expressed genes (DEGs) and 871 and 650 differentially abundant metabolites were identified in de-hulled and hulled seeds treated with GA3, respectively, compared with seeds soaked in sterile water. Most of the DEGs were associated with starch and sucrose metabolism, protein processing in the endoplasmic reticulum, endocytosis and ribosomes. Furthermore, isoquinoline alkaloid biosynthesis, tyrosine metabolism, starch and sucrose metabolism, arginine and proline metabolism, and amino sugar and nucleotide sugar metabolism were significantly enriched pathways. Integrative analysis of the transcriptomic and metabolomic data revealed that starch and sucrose metabolism is one of the most important pathways that may play a key role in providing carbon skeletons and energy supply for the transition of L. chinensis seeds from a dormant state to germination by suppressing the expression of Cel61a, egID, cel1, tpsA, SPAC2E11.16c and TPP2, enhancing the expression of AMY1.1, AMY1.2, AMY1.6 and GLIP5, and inhibiting the synthesis of cellobiose, cellodextrin, and trehalose while promoting the hydrolysis of sucrose, starch, cellobiose, cellodextrin, and trehalose to glucose. This study identified several key genes and provided new insights into the molecular mechanism of seed dormancy release induced by GA3 in L. chinensis. These putative genes will be valuable resources for improving the seed germination rate in future breeding studies.

Trehalase inhibition by validamycin A may be a promising target to design new fungicides and insecticides.

The introduction of insecticides and fungicides in agriculture has improved crop yields and, consequently, the quality of life for many people, especially in what is widely considered as the 'first world'. However, the indiscriminate use of dangerous chemical insecticides has led to pest resistance, human and animal poisoning and environmental pollution. Biochemical and genetic evidence concludes that the non-reducing disaccharide trehalose plays an essential role in the pathobiology of many insects and fungi. Both organisms share identical pathway for trehalose biosynthesis (the TPS/TPP pathway), while a high degree of homology in their trehalose hydrolysis capacity (trehalase activities) has also been demonstrated. In the search for new, effective and environmentally sustainable compounds, a set of trehalase inhibitors has emerged as a potentially interesting antifungal and insecticidal target. In particular, the trehalose analogue, Validamycin A, which has a strong inhibitory effect on several trehalases, has been successfully introduced for the treatment of various diseases caused by insects and fungi. Herein, we review the main features of the specific interaction between Validamycin A and trehalase as well as the expected advantages of the applications based on trehalase inhibition as insecticides and fungicides. © 2021 Society of Chemical Industry.

Expression and characterization of a novel trehalase from Microvirga sp. strain MC18

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.

1H NMR‐based metabonomic evaluation of the pesticides camptothecin and matrine against larvae of Spodoptera litura

Camptothecin (CPT) and matrine (MAT) have potential as botanical pesticides against several pest species. However, the mechanisms of metabolic and physiological changes in pests induced by CPT and MAT are unknown. In this study, a toxicological test, an NMR-based metabolomic study, an enzymatic test, and an RT quantitative PCR (RT-qPCR) experiment were all conducted to examine the effect of CPT and MAT on Spodoptera litura. CPT (0.5-1%) exerted high toxicity against larvae of S. litura and caused growth stagnation and high mortality of larvae. A variety of metabolites were significantly influenced by 0.5% CPT, including several energy-related metabolites such as trehalose, lactate, succinate, citrate, malate, and fumarate. In contrast, MAT showed low toxicity against larvae and induced almost no changes in hemolymph metabolites of S. litura. Enzymatic tests showed that trehalase activity was significantly decreased in larvae after feeding with 0.5% CPT. RT-qPCR showed that the transcription levels of alanine aminotransferase, malate dehydrogenase, and isocitrate dehydrogenase were decreased while lactate dehydrogenase was increased in the 0.5% CPT-treated group. These data indicate that one of the important mechanisms of CPT against S. litura larvae is via the inhibition of trehalose hydrolysis and glycolysis. Our findings also suggest that CPT exhibits a stronger toxicological effect than MAT against S. litura, which provides basic information for the application of CPT in the control of S. litura or other lepidoptera pests.

Inhibition of trehalase affects the trehalose and chitin metabolism pathways in Diaphorina citri (Hemiptera: Psyllidae).

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.

Untargeted metabolomics as a hypothesis-generation tool in plant protection product discovery: Highlighting the potential of trehalose and glycerol metabolism of fungal conidiospores as novel targets.

The production of high quality and safe food represents a main priority for the agri-food sector in the effort to sustain the exponentially growing human population. Nonetheless, there are major challenges that require the discovery of new, alternative, and improved plant protection products (PPPs). Focusing on fungal plant pathogens, the dissection of mechanisms that are essential for their survival provides insights that could be exploited towards the achievement of the aforementioned aim. In this context, the germination of fungal spores, which are essential structures for their dispersal, survival, and pathogenesis, represents a target of high potential for PPPs. To the best of our knowledge, no PPPs that target the germination of fungal spores currently exist. Within this context, we have mined for changes in the metabolite profiles of the model fungus Aspergillus nidulans FGSCA4 conidiospores during germination, in an effort to discover key metabolites and reactions that could potentially become targets of PPPs. Untargeted GC/EI-TOF/MS metabolomics and multivariate analyses were employed to monitor time-resolved changes in the metabolomes of germinating A. nidulans conidiospores. Analyses revealed that trehalose hydrolysis plays a pivotal role in conidiospore germination and highlighted the osmoregulating role of the sugar alcohols, glycerol, and mannitol. The ineffectiveness to introduce active ingredients that exhibit new mode(s)-of-action as fungicides, dictates the urge for the discovery of PPPs, which could be exploited to combat major plant protection issues. Based on the crucial role of trehalose hydrolysis in conidiospore dormancy breakage, and the subsequent involvement of glycerol in their germination, it is plausible to suggest their biosynthesis pathways as potential novel targets for the next-generation antifungal PPPs. Our study confirmed the applicability of untargeted metabolomics as a hypothesis-generation tool in PPPs' research and discovery.

Partitioning sources of CO2 emission after soil wetting using high-resolution observations and minimal models

Wetting dry soil triggers a pulse of microbial respiration. Respiration pulses after wetting can be complex, evolving through several stages before subsiding—possibly reflecting contributions from multiple carbon (C) sources. We hypothesized that respiration after wetting combines C from cellular sources (notably trehalose, a cellular osmo-protectant) and soluble extracellular C made available by the physical effects of wetting, with cellular sources contributing early during the pulse and extracellular C dominating later. We tested this hypothesis by making measurements of soil respiration, microbial biomass, trehalose, and water extractable organic C after wetting dry soil in the laboratory. We then devised several alternative minimal models to evaluate the potential contributions of trehalose and extracellular C to the respiration pulse. We found that the chloroform-labile fraction of the microbial biomass was comprised of 50% trehalose in dry soil. Both trehalose and water extractable organic C pools declined rapidly after wetting; trehalose disappeared within 3 h of water addition. Chloroform-labile glucose increased 3 h after water addition and then declined, suggesting rapid hydrolysis of trehalose and consumption of the resulting glucose. Respiration dynamics after wetting could be best explained by models that included two C sources, combining rapid metabolism of trehalose with slower consumption of extracellular soluble C. These results suggest that pulses of respiration after wetting reflect both osmolyte consumption within the microbial biomass and enhanced extracellular C availability; hence, models of microbial drought response must account for both processes to fully resolve soil C dynamics.

The secreted acid trehalase encoded by the CgATH1 gene is involved in Candida glabrata virulence.

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.

Sanguinarine caused larval lethality and growth inhibition by suppressing energy metabolism in silkworms, Bombyx mori

Sanguinarine (Sang) is a natural alkaloid and distributed in several plants of Papaveraceae. The antitumor, antioxidant, antimicrobial and anti-inflammatory effects of Sang were extensively reported, but its speciality and mechanism against Lepidoptera insects were still unknown. In this study, detailed toxicological parameters of Sang against silkworms, Bombyx mori (B. mori), were determined by a toxicological test. Then, a nuclear magnetic resonance-based (NMR) metabolomics method was adopted to analyze the changes in hemolymph metabolites of silkworms after feeding Sang. The growth of fourth-instar larvae was significantly ceased by the oral administration of 0.05–0.3% Sang and vast deaths appeared in 0.3% Sang group on Day 4 and Day 5. The quantitative analysis of metabolites indicated that trehalose and citrate levels in hemolymph were increased after 24 h of feeding 0.3% Sang, whereas the concentrations of pyruvate, succinate, malate and fumarate were decreased. In addition, the enzymatic determination and reverse transcription quantitative PCR (RT-qPCR) showed that the trehalase (THL) activity and the transcriptional level of one gene coding THL were uniformly weakened by 0.3% Sang. One of the important mechanisms of Sang against silkworms might be interpreted as follows. Sang impaired trehalose hydrolysis, reduced THL activity and transcription, and led to the inhibition of energy metabolism, consequent antigrowth and high lethality in larvae of B. mori. Our findings offered new insights into the insecticidal effect of Sang from the perspective of energy metabolism and provided the basis for the application of Sang in the control of Lepidoptera pests.