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Epilepsy is a prevalent neurological disorder characterized by recurrent seizures. Validamycin A (VA) is an antibiotic fungicide that inhibits trehalase activity and is widely used for crop protection in agriculture. In this study, we identified a novel function of VA as a potential anti-seizure medication in a zebrafish epilepsy model. Electroencephalogram (EEG) analysis demonstrated that VA reduced pentylenetetrazol (PTZ)-induced seizures in the brains of larval and adult zebrafish. Moreover, VA reduced PTZ-induced irregular movement in a behavioral assessment of adult zebrafish. The developmental toxicity test showed no observable anatomical alteration when the zebrafish larvae were treated with VA up to 10 µM within the effective range. The median lethal dose of VA in adult zebrafish was > 14,000 mg/kg. These results imply that VA does not demonstrate observable toxicity in zebrafish at concentrations effective for generating anti-seizure activity in the EEG and alleviating abnormal behavior in the PTZ-induced epileptic model. Furthermore, the effectiveness of VA was comparable to that of valproic acid. These results indicate that VA may have a potentially safer anti-seizure profile than valproic acid, thus offering promising prospects for its application in agriculture and medicine.

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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 (FB1) 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 Trp285, Arg447, Asp452, and Phe665 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 FB1 biosynthesis. These findings present a novel target for the development of mycotoxin control agents.

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Agriculture commonly utilizes crop protection products to tackle infestations from fungi, parasites, insects, and weeds. Validamycin A, an inhibitor of trehalase, possesses antibiotic and antifungal attributes. Epidemiological evidence has led to concerns regarding a potential link between pesticide usage and neurodegenerative diseases. The fruit fly, Drosophila melanogaster, has been recognized as a reliable model for genetic research due to its significant genetic similarities with mammals. Here, we propose to use D. melanogaster as an effective in vivo model system to investigate the genotoxic risks associated with exposure to validamycin A. In this study, we performed a neurotoxic evaluation of validamycin A in D. melanogaster larvae. Several endpoints were evaluated, including toxicity, intracellular oxidative stress (reactive oxygen species), intestinal damage, larval behavior (crawling behavior, light/dark sensitivity assay, and temperature sensitivity assay), locomotor (climbing) behavior, and neurogenotoxic effects (impaired DNA via Comet assay, enhanced by Endo III and formamidopyrimidine DNA glycosylase [FPG]). The results showed that exposure to validamycin A, especially at higher doses (1 and 2.5 mM), induced DNA impairment in neuroblasts as observed by Comet assay. Both larvae and adults exhibited behavioral changes and produced reactive oxygen species. Most importantly, this research represents a pioneering effort to report neurogenotoxicity data specifically in Drosophila larval neuroblasts, thus underscoring the importance of this species as a testing model in exploring the biological impacts of validamycin A. The in vivo findings from the experiments are a valuable and novel addition to the existing validamycin A neurogenotoxicity database.

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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.

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The dissipation and residue of validamycin A in grapes were investigated under field conditions. An ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination of validamycin A in grapes was established and validated. Methanol and water (90/10, v/v) were used for validamycin A extraction and purification used MCX solid-phase extraction cartridges. The average recoveries of validamycin A in grapes at 0.01, 0.50, and 5.0 mg/kg levels were between 83.8 and 91.4%, with relative standard deviations of 2.3-3.0%. The half-lives of validamycin A in grape were 4.4-6.1 days. The terminal residues in grapes over a range of harvest times (7, 14, and 21 days) were no more than 0.73 mg/kg. According to Chinese consumption data, the risk quotient (RQ) of validamycin A was 3.22%, demonstrating a low risk to consumers. The current study may offer guidance for validamycin A use and could aid the government in determining the maximum residue level (MRL) values for validamycin A in grapes.

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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.

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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.

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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.

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Validamycin A (VMA) is an aminoglycoside antibiotic used to control rice sheath blight. Although it has been reported that VMA can induce the plant defense responses, the mechanism remains poorly understood. Here, we found that reactive oxygen species (ROS) bursts and callose deposition in Arabidopsis thaliana, rice (Oryza sativa L.), and wheat (Triticum aestivum L.) were induced by VMA and were most intense with 10 µg of VMA per milliliter at 24 h. Moreover, we showed that VMA induced resistance against Pseudomonas syringae, Botrytis cinerea, and Fusarium graminearum in Arabidopsis leaves, indicating that VMA induces broad-spectrum disease resistance in both dicots and monocots. In addition, VMA-mediated resistance against P. syringae was not induced in NahG transgenic plants, was partially decreased in npr1 mutants, and VMA-mediated resistance to B. cinerea was not induced in npr1, jar1, and ein2 mutants. These results strongly indicated that VMA triggers plant defense responses to both biotrophic and necrotrophic pathogens involved in salicylic acid (SA) and jasmonic acid/ethylene (JA/ET) signaling pathways and is dependent on NPR1. In addition, transcriptome analysis further revealed that VMA regulated the expression of genes involved in SA, JA/ET, abscisic acid (ABA), and auxin signal pathways. Taken together, VMA induces systemic resistance involving in SA and JA/ET signaling pathways and also exerts a positive influence on ABA and auxin signaling pathways. Our study highlights the creative application of VMA in triggering plant defense responses against plant pathogens, providing a valuable insight into applying VMA to enhance plant resistance and reduce the use of chemical pesticides.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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Trehalase catalyzes hydrolysis of trehalose and plays a crucial role in insect metabolism. In the present study, phylogenetic analysis and multiple sequence alignment suggested that H. armigera trehalase-1 (HaTre-1) is closely related to other soluble trehalases with conserved signature features and functional sites. We have expressed and purified recombinant HaTre-1 having Vmax ~0.16mM/min and KM ~1.34mM. Inhibition kinetics and Microscale thermophoresis illustrated competitive inhibition of HaTre-1 by Validamycin A having Ki ~3nM and KD ~542nM, respectively. Docking studies of HaTre-1 with Validamycin A indicated that it binds at the active site with multiple hydrogen bonds. Ingestion of Validamycin A resulted in impediment of H. armigera growth and developmental defects. Treated larvae showed concentration dependent decrease in fecundity. It also led to total inhibition of ex-vivo trehalase activity and down-regulation of gene expression of HaTre-1. Relatively high insect mortality was observed on tomato plants sprayed with combination of Validamycin A with Azadirachta indica (neem) and Pongamia pinnata (karanj) oil as compared to the individual treatments. This report has re-emphasized trehalase inhibition as a potential insecticidal strategy and also recommends Validamycin A as a prospective value-added ingredient to commercial bio-pesticide formulations.

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Two polar aminoglycosides, kasugamycin and validamycin-A, were determined in cereals (brown rice, wheat and corn) by high-performance liquid chromatography-tandem mass spectrometry. The analytes were extracted from samples using methanol and water (70:30, v/v) at pH 5.5, purified using both a hydrophilic-hydrophobic-balanced cartridge and a strong cation-exchange cartridge, and then analysed using multiple reaction monitoring in positive electrospray ionisation mode with a special ReproSil 100 C18 high-performance liquid chromatography column. This newly proposed method yielded good sensitivity and excellent chromatographic performance. The limits of quantification for kasugamycin and validamycin-A were 4.1 µg/kg and 1.0 µg/kg, respectively. The recoveries for both compounds at three fortification levels (4, 100 and 500 µg/kg for kasugamycin; 1, 10 and 100 µg/kg for validamycin-A) ranged from 75% to 110%, and the relative standard deviations were below 15%.

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Validamycin A (Val-A) is produced by Streptomyces as a secondary metabolite with wide agricultural applications of controlling rice sheath blight, false smut and damping-off diseases. The effect of alkaline pH shock on enhancing Val-A production and its mechanism were investigated. A higher yield of Val-A was achieved by NaOH shock once or several times together with faster protein synthesis and sugar consumption and alkaline pH shock can increase Val-A production by 27.43%. Transcription of genes related to amino acid metabolism, carbon metabolism and electron respiratory chain was significantly up-regulated, accompanied by the substantial increase of respiratory activity and glutamate concentration. Val-A production was promoted by a series of complex mechanisms and made a response to pH stress signal, which led to the enhancement of glutamate metabolism and respiration activity. The obtained information will facilitate future studies for antibiotic yield improvement and the deep revealment of molecular mechanism.

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Giant linear plasmids, which replicate independently of the chromosomes, widely exist in actinobacteria. Previous studies mostly focused on the replication and evolution of the linear plasmids or the secondary metabolite gene clusters and the resistance gene clusters therein. However, the relationships of the linear plasmids to the productivities of secondary metabolites have not been studied. In this work, we developed a method to eliminate the indigenous linear plasmid pSHJG1 in Streptomyces hygroscopicus var. jinggangensis, and validamycin A titer increased by 12.5% (from 19.16 ± 1.93 to 21.56 ± 2.25 g/L) in the high-yielding strain TL01 and 43.7% (from 4.67 ± 0.05 to 6.71 ± 0.21 g/L) in the wild-type strain 5008, whereas the cellular growth of the plasmid-cured mutant was reduced. Subsequently, the plasmid-cured mutant was complemented with three structure genes involved in cellular growth in pSHJG1 under the control of a strong PvalA promoter. Among them, the complementation of genes pSHJG1.069 and pSHJG1.072, encoding a putative hydrolase and putative P-loop ATPase, respectively, resulted in the restoration of cellular growth and validamycin A titer. Furthermore, the elimination of indigenous linear plasmid pHZ228 in the candicidin producer Streptomyces sp. FR008 also led to enhanced candicidin production and reduced cellular growth. Because of the wide distribution of indigenous linear plasmids in actinobacteria, the engineering strategy described here could be implemented in a variety of strains for the overproduction of various natural products.

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Strawberry bacterial angular leaf spot (ALS) disease, caused by Xanthomonas fragariae has become increasingly problematic in the strawberry agro-industry. ALS causes small angular water-soaked lesions to develop on the abaxial leaf surface. Studies reported optimum temperature conditions for X. fragariae are 20°C and the pathogen suffers mortality above 32°C. However, at the nursery stage, disease symptoms have been observed under high temperature conditions. In the present study, results showed X. fragariae transmission was via infected maternal plants, precipitation, and sprinkler irrigation systems. Systemic infections were detected using X. fragariae specific primers 245A/B and 295A/B, where 300-bp and 615-bp were respectively amplified. During the nursery stage (from May to August), the pathogen was PCR detected only in maternal plants, but not in soil or irrigation water through the nursery stage. During the cultivation period, from September to March, the pathogen was detected in maternal plants, progeny, and soil, but not in water. Additionally, un-infected plants, when planted with infected plants were positive for X. fragariae via PCR at the late cultivation stage. Chemical control for X. fragariae with oxolinic acid showed 87% control effects against the disease during the nursery period, in contrast to validamycin-A, which exhibited increased efficacy against the disease during the cultivation stage (control effect 95%). To our knowledge, this is the first epidemiological study of X. fragariae in Korean strawberry fields.

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Validamycin A (Val-A) synthesized by Streptomyces hygroscopicus 5008 is widely used as a high-efficient antibiotic to protect plants from sheath blight disease. A novel fermentation strategy was introduced to stimulate Val-A production by adding oxygen carriers. About 58 % increase in Val-A production was achieved using liquid paraffin. Further, biomass, carbon source, metabolic genes, and metabolic enzymes were studied. It was also found that the supplementation of liquid paraffin increased the medium dissolved oxygen and intracellular oxidative stress level. The expression of the global regulators afsR and soxR sensitive to ROS, ugp catalyzing synthesis of Val-A precursor, and Val-A structural genes was enhanced. The change of the activities of glucose-6-phosphate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase was observed, which reflected the redirection of carbon metabolic flux. Based on these results, liquid paraffin addition as an oxygen carrier could be a useful technique in industrial production of Val-A and our study revealed a redox-based secondary metabolic regulation in S. hygroscopicus 5008, which provided a new insight into the regulation of the biosynthesis of secondary metabolites.

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Validamycin A (Val-A), produced by Streptomyces hygroscopicus 5008 in industrial fermentation, is one of the most widely used anti-fungal agro-antibiotics in Asia and high performance liquid chromatography (HPLC) assay is usually used to determine the production of Val-A. A new approach to determine Val-A by spectrophotometer is developed. During the fermentation of S. hygroscopicus 5008, a pigment secretion was found along with the Val-A biosynthesis. There was a stable relationship between the concentration of Val-A and spectral absorption (SA) value of this pigment at 450 nm, even in different fermentation cultures or conditions. Using SA value as interior label, a rapid spectrophotometric method for determining Val-A production was established. In comparing Val-A productivity by HPLC method with that by SA method, the relative standard deviation (R.S.D.) was 0.007 (less than 0.05, no variation) and the conditional probability [Pr(T < t)] was 0.3491 (greater than 0.05, no difference) at the optimal time point of Val-A fermentation, which demonstrated SA method was as stable and accurate as standard HPLC method. It was applied successfully to finding positive strains with high Val-A productivity and short fermentation time. SA assay is an accurate and cost-effective method for measuring Val-A and screening high-producing strains, and this work provides a new insight for rapid quantitative analysis of antibiotics in fermentation of pigment-producing strains.

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For the first time, a rapid, sensitive and accurate liquid chromatography-atmospheric pressure chemical ionisation-tandem mass spectrometry (LC-APCI-MS/MS) method was developed for determination of validamycin A in agricultural food samples (rice, agaric, almond, cabbage, green onion, carrot, tomato, cucumber and spinach). The validamycin A residue was extracted with methanol-water (9/1, v/v) or methanol by vortex, and a HLB solid-phase extraction cartridge was used for cleaning up the extracts. LC-APCI-MS/MS data acquisition was carried out in multiple reaction monitoring (MRM) mode. A series of matrix-matched calibration solutions ranging from 2.5 to 50ngmL(-1) were used to record calibration curve. The limit of quantification (LOQ) was 10µgkg(-1). The average recoveries, measured at three concentrations levels (10.0, 50.0, 100.0µgkg(-1)) were in the range 83.5-109.6%. The proposed method offers the best sensitivity and specificity for the routine analysis of validamycin A in agricultural food samples.

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Validamycin A (VAL-A) is an important agricultural antibiotic produced by Streptomyces hygroscopicus 5008, which uses starch as carbon source occupying about 20% of total production cost. To reduce the medium cost, corncob hydrolysate - a hemicellulose hydrolysate was applied as a low-cost substrate to VAL-A fermentation. It was found that three major sugars in corncob hydrolysate including d-glucose, d-xylose and l-arabinose could all be utilized by S. hygroscopicus 5008 to produce VAL-A while d-xylose was the main contributor. A higher VAL-A production titer from d-xylose was achieved by using a genetically engineered strain TC03 derived from S. hygroscopicus 5008, which resulted in 1.27-fold improvement of VAL-A production from the medium containing 13% (v/v) corncob hydrolysate compared to that by its original strain. A medium cost analysis was done and compared with previous reports. This work indicates a great potential of the hemicellulose hydrolysate as substrate for antibiotic fermentation.

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Fusarium verticillioides is a pathogen of maize that causes root, stalk and ear rot and produces fumonisins, toxic secondary metabolites associated with disease in livestock and humans. Environmental stresses such as heat and drought influence disease severity and toxin production, but the effects of abiotic stress on compatible solute production by F. verticillioides have not been fully characterized. We found that decreasing the growth temperature leads to a long-term reduction in polyol levels, whereas increasing the temperature leads to a transient increase in polyols. The effects of temperature shifts on trehalose levels are opposite the effects on polyols and more dramatic. Treatment with validamycin A, a trehalose analog with antifungal activity, leads to a rapid reduction in trehalose levels, despite its known role as a trehalase inhibitor. Mutant strains lacking TPS1, which encodes a putative trehalose-6-phosphate synthase, have altered growth characteristics, do not produce detectable amounts of trehalose under any condition tested, and accumulate glycogen at levels significantly higher than wild-type F. verticillioides. TPS1 mutants also produce significantly less fumonisin than wild type and are also less pathogenic than wild type on maize. These data link trehalose biosynthesis, secondary metabolism, and disease, and suggest that trehalose metabolic pathways may be a viable target for the control of Fusarium diseases and fumonisin contamination of maize.

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