RESUMO
The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has been produced by transgenic crops to counter pest resistance to the widely used crystalline (Cry) insecticidal proteins from Bt. To proactively manage pest resistance, there is an urgent need to better understand the genetic basis of resistance to Vip3Aa, which has been largely unknown. We discovered that retrotransposon-mediated alternative splicing of a midgut-specific chitin synthase gene was associated with 5,560-fold resistance to Vip3Aa in a laboratory-selected strain of the fall armyworm, a globally important crop pest. The same mutation in this gene was also detected in a field population. Knockout of this gene via CRISPR/Cas9 caused high levels of resistance to Vip3Aa in fall armyworm and 2 other lepidopteran pests. The insights provided by these results could help to advance monitoring and management of pest resistance to Vip3Aa.
Assuntos
Bacillus thuringiensis , Proteínas de Bactérias , Quitina Sintase , Resistência a Inseticidas , Retroelementos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quitina Sintase/genética , Quitina Sintase/metabolismo , Retroelementos/genética , Bacillus thuringiensis/genética , Resistência a Inseticidas/genética , Sistemas CRISPR-Cas , Processamento Alternativo/genética , Processamento Alternativo/efeitos dos fármacos , Spodoptera/efeitos dos fármacos , Plantas Geneticamente Modificadas , Mariposas/efeitos dos fármacos , Mariposas/genéticaRESUMO
The brown planthopper (Nilaparvata lugens) is a major destructive rice pest in Asia. High levels of insecticide resistance have been frequently reported, and the G932C mutation in the chitin synthase 1 (CHS1) gene has been found to mediate buprofezin resistance. However, there has been no direct evidence to confirm the functional significance of the single G932C substitution mutation leading to buprofezin resistance in N. lugens. Here, we successfully constructed a knock-in homozygous strain (Nl-G932C) of N. lugens using CRISPR/Cas9 coupled with homology-directed repair (HDR). Compared with the background strain susceptible to buprofezin (Nl-SS), the knock-in strain (Nl-G932C) showed a 94.9-fold resistance to buprofezin. Furthermore, resistant strains (Nl-932C) isolated from the field exhibited a 2078.8-fold resistance to buprofezin, indicating that there are other mechanisms contributing to buprofezin resistance in the field. Inheritance analysis showed that the resistance trait is incomplete dominance. In addition, the Nl-G932C strain had a relative fitness of 0.33 with a substantially decreased survival rate, emergence rate, and fecundity. This study provided in vivo functional evidence for the causality of G932C substitution mutation of CHS1 with buprofezin resistance and valuable information for facilitating the development of resistance management strategies in N. lugens. This is the first example of using CRISPR/Cas9 gene-editing technology in a hemipteran insect to directly confirm the role of a candidate target site mutation in insecticide resistance.
Assuntos
Sistemas CRISPR-Cas , Quitina Sintase , Hemípteros , Resistência a Inseticidas , Inseticidas , Tiadiazinas , Animais , Hemípteros/genética , Resistência a Inseticidas/genética , Tiadiazinas/farmacologia , Quitina Sintase/genética , Inseticidas/farmacologia , Mutação , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Técnicas de Introdução de Genes , Feminino , MasculinoRESUMO
Lufenuron, a benzoylurea chitin synthesis inhibitor, is effective against many insect pests. However, the insecticidal activity of lufenuron has not been completely elucidated, nor has its disturbing effect on chitin synthesis genes. In this study, bioassay results demonstrated an outstanding toxicity of lufenuron against Helicoverpa armigera larvae. The treated larvae died from abortive molting and metamorphosis defects, and severe separation of epidermis and subcutaneous tissues was observed. Treatment of 3rd- and 4th-instar larvae with LC25 lufenuron significantly extended the duration of larval and pupal stage, reduced the rates of pupation and emergence, and adversely affected pupal weight. Besides, lufenuron can severely reduce chitin content in larval integument, and the lufenuron-treated larvae showed reduced trehalose content in their hemolymph. Further analysis using RNA sequencing revealed that five chitin synthesis genes were down-regulated, whereas the expressions of two chitin degradation genes were significantly enhanced. Knockdown of chitin synthase 1 (HaCHS1), uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (HaUAP), phosphoacetyl glucosamine mutase (HaPGM), and glucosamine 6-phosphate N-acetyl-transferase (HaGNPAT) in H. armigera led to significant increase in larval susceptibilities to LC25 lufenuron by 75.48%, 65.00%, 68.42% and 28.00%, respectively. Our findings therefore revealed the adverse effects of sublethal doses of lufenuron on the development of H. armigera larvae, elucidated the perturbations on chitin metabolism, and proved that the combination of RNAi and lufenuron would improve the control effect of this pest.
Assuntos
Benzamidas , Quitina , Inseticidas , Larva , Mariposas , Animais , Quitina/biossíntese , Benzamidas/farmacologia , Larva/efeitos dos fármacos , Inseticidas/farmacologia , Inseticidas/toxicidade , Mariposas/efeitos dos fármacos , Mariposas/metabolismo , Mariposas/crescimento & desenvolvimento , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Quitina Sintase/metabolismo , Quitina Sintase/genética , Helicoverpa armigera , FluorocarbonosRESUMO
Aspergillosis is one of the most common fungal infections that can threaten individuals with immune compromised condition. Due to the increasing resistance of pathogens to the existing antifungal drugs, it is difficult to tackle such disease conditions. Whereas, nikkomycin is an emerging safe and effective antifungal drug which causes fungal cell wall disruption by inhibiting chitin synthase. Hence, the study aims at the development of nikkomycin loaded PEG coated PLGA nanoparticles for its increased antifungal efficiency and inhibiting Aspergillus infections. The P-PLGA-Nik NPs were synthesized by w/o/w double emulsification method which resulted in a particle size of 208.3 ± 15â¯nm with a drug loading of 52.97â¯%. The NPs showed first order diffusion-controlled drug release which was sustained for 24â¯h. These nanoparticle's antifungal efficacy was tested using the CLSI - M61 guidelines and the MIC50 defined against Aspergillus flavus and Aspergillus fumigatus was found to be >32⯵g/ml which was similar to the nikkomycin MIC. The hyphal tip bursting showed the fungal cell wall disruption. The non-cytotoxic and non-haemolytic nature highlights the drug safety profile.
Assuntos
Antifúngicos , Aspergillus flavus , Aspergillus fumigatus , Quitina Sintase , Testes de Sensibilidade Microbiana , Nanopartículas , Polietilenoglicóis , Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/crescimento & desenvolvimento , Antifúngicos/farmacologia , Antifúngicos/química , Nanopartículas/química , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/crescimento & desenvolvimento , Quitina Sintase/antagonistas & inibidores , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Tamanho da Partícula , Preparações de Ação Retardada/química , Humanos , Parede Celular/efeitos dos fármacos , AminoglicosídeosRESUMO
In order to speculate the three-dimensional structure of the potential binding pocket of the chitin synthase inhibitor, a series of 2,4-diphenyloxazoline derivatives with different lengths of alkyl chains and heteroatoms were designed and synthesized by a homologous strategy. The bioassay results indicate that both the length of the alkyl chains and the type of substituents can affect the acaricidal activity against mite eggs. Compounds containing chloropropyl, alkoxyalkyl, and para-substituted phenoxyalkyl or phenylthioalkyl groups exhibit good activity, while those containing steric hindrance substituents or carbonyl substituents on the benzene ring exhibit reduced activity. Three-dimensional quantitative structure-activity relationship (3D-QSAR) study showed that there may be a narrow hydrophobic region deep in the pocket, and the steric effect plays a more important role than the electrostatic effect. The current work will provide assistance for future molecular design and target binding research.
Assuntos
Acaricidas , Relação Quantitativa Estrutura-Atividade , Acaricidas/química , Acaricidas/farmacologia , Animais , Ácaros/efeitos dos fármacos , Ácaros/química , Oxazóis/química , Oxazóis/farmacologia , Desenho de Fármacos , Estrutura Molecular , Quitina Sintase/química , Quitina Sintase/antagonistas & inibidores , Quitina Sintase/metabolismoRESUMO
The mycelium of the plant pathogenic fungus Fusarium graminearum exhibits distinct structures for vegetative growth, asexual sporulation, sexual development, virulence, and chlamydospore formation. These structures are vital for the survival and pathogenicity of the fungus, necessitating precise regulation based on environmental cues. Initially identified in Magnaporthe oryzae, the transcription factor Con7p regulates conidiation and infection-related morphogenesis, but not vegetative growth. We characterized the Con7p ortholog FgCon7, and deletion of FgCON7 resulted in severe defects in conidium production, virulence, sexual development, and vegetative growth. The mycelia of the deletion mutant transformed into chlamydospore-like structures with high chitin level accumulation. Notably, boosting FgABAA expression partially alleviated developmental issues in the FgCON7 deletion mutant. Chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) analysis confirmed a direct genetic link between FgABAA and FgCON7. Furthermore, the chitin synthase gene Fg6550 (FGSG_06550) showed significant upregulation in the FgCON7 deletion mutant, and altering FgCON7 expression affected cell wall integrity. Further research will focus on understanding the behavior of the chitin synthase gene and its regulation by FgCon7 in F. graminearum. This study contributes significantly to our understanding of the genetic pathways that regulate hyphal differentiation and conidiation in this plant pathogenic fungus. IMPORTANCE: The ascomycete fungus Fusarium graminearum is the primary cause of head blight disease in wheat and barley, as well as ear and stalk rot in maize. Given the importance of conidia and ascospores in the disease cycle of F. graminearum, precise spatiotemporal regulation of these biological processes is crucial. In this study, we characterized the Magnaporthe oryzae Con7p ortholog and discovered that FgCon7 significantly influences various crucial aspects of fungal development and pathogenicity. Notably, overexpression of FgABAA partially restored developmental defects in the FgCON7 deletion mutant. ChIP-qPCR analysis confirmed a direct genetic link between FgABAA and FgCON7. Furthermore, our research revealed a clear correlation between FgCon7 and chitin accumulation and the expression of chitin synthase genes. These findings offer valuable insights into the genetic mechanisms regulating conidiation and the significance of mycelial differentiation in this plant pathogenic fungus.
Assuntos
Proteínas Fúngicas , Fusarium , Regulação Fúngica da Expressão Gênica , Doenças das Plantas , Esporos Fúngicos , Fatores de Transcrição , Fusarium/genética , Fusarium/patogenicidade , Fusarium/crescimento & desenvolvimento , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Doenças das Plantas/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência , Quitina Sintase/genética , Quitina Sintase/metabolismo , Quitina/metabolismo , Deleção de GenesRESUMO
Chitin is an essential structural component of fungal cell walls composed of transmembrane proteins called chitin synthases (CHSs), which have a large range of reported effects in ascomycetes; however, are poorly understood in agaricomycetes. In this study, evolutionary and molecular genetic analyses of chs genes were conducted using genomic information from nine ascomycete and six basidiomycete species. The results support the existence of seven previously classified chs clades and the discovery of three novel basidiomycete-specific clades (BI-BIII). The agaricomycete fungus Pleurotus ostreatus was observed to have nine putative chs genes, four of which were basidiomycete-specific. Three of these basidiomycete specific genes were disrupted in the P. ostreatus 20b strain (ku80 disruptant) through homologous recombination and transformants were obtained (Δchsb2, Δchsb3, and Δchsb4). Despite numerous transformations Δchsb1 was unobtainable, suggesting disruption of this gene causes a crucial negative effect in P. ostreatus. Disruption of these chsb2-4 genes caused sparser mycelia with rougher surfaces and shorter aerial hyphae. They also caused increased sensitivity to cell wall and membrane stress, thinner cell walls, and overexpression of other chitin and glucan synthases. These genes have distinct roles in the structural formation of aerial hyphae and cell walls, which are important for understanding basidiomycete evolution in filamentous fungi.
Assuntos
Quitina Sintase , Quitina , Proteínas Fúngicas , Filogenia , Pleurotus , Quitina Sintase/genética , Pleurotus/genética , Pleurotus/enzimologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Quitina/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Evolução Molecular , Basidiomycota/genética , Basidiomycota/enzimologiaRESUMO
Soybean phytophthora blight is a severe menace to global agriculture, causing annual losses surpassing USD 1 billion. Present crop loss mitigation strategies primarily rely on chemical pesticides and disease-resistant breeding, frequently surpassed by the pathogens' quick adaptive evolution. In this urgent scenario, our research delves into innovative antimicrobial peptides characterized by low drug resistance and environmental friendliness. Inhibiting chitin synthase gene activity in Phytophthora sojae impairs vital functions such as growth and sporulation, presenting an effective method to reduce its pathogenic impact. In our study, we screened 16 previously tested peptides to evaluate their antimicrobial effects against Phytophthora using structure-guided drug design, which involves molecular docking, saturation mutagenesis, molecular dynamics, and toxicity prediction. The in silico analysis identified AMP_04 with potential inhibitory activity against Phytophthora sojae's chitin synthase. Through three rounds of saturation mutagenesis, we pin-pointed the most effective triple mutant, TP (D10K, G11I, S14L). Molecular dynamic simulations revealed TP's stability in the chitin synthase-TP complex and its transmembrane mechanism, employing an all-atom force field. Our findings demonstrate the efficacy of TP in occupying the substrate-binding pocket and translocation catalytic channel. Effective inhibition of the chitin synthase enzyme can be achieved. Specifically, the triple mutant demonstrates enhanced antimicrobial potency and decreased toxicity relative to the wild-type AMP_04, utilizing a mechanism akin to the barrel-stave model during membrane translocation. Collectively, our study provides a new strategy that could be used as a potent antimicrobial agent in combatting soybean blight, contributing to sustainable agricultural practices.
Assuntos
Anti-Infecciosos , Phytophthora , Glycine max/genética , Phytophthora/fisiologia , Quitina Sintase/genética , Peptídeos Antimicrobianos , Simulação de Acoplamento Molecular , Resistência à Doença , Melhoramento Vegetal , Doenças das Plantas/prevenção & controle , Doenças das Plantas/genéticaRESUMO
Spiders, the major predatory enemies of insect pests in fields, are vulnerable to insecticides. In this study, we observed that the recommended dose of buprofezin delayed the molting of the pond wolf spider Pardosa pseudoannulata, although it had no lethal effect on the spiders. Since buprofezin is an insect chitin biosynthesis inhibitor, we identified two chitin synthase genes (PpCHS1 and PpCHS2) in P. pseudoannulata. Tissue-specific expression profiling showed that PpCHS1 was most highly expressed in cuticle. In contrast, PpCHS2 showed highest mRNA levels in the midgut and fat body. RNAi knockdown of PpCHS1 significantly delayed the molting of 12-days old spiderlings, whereas no significant effect on the molting was observed in the PpCHS2-silencing spiderlings. The expression of PpCHS1 was significantly suppressed in the spiderlings treated with buprofezin, but rescued by exogenous ecdysteroid ponasterone A (PA). Consistent with this result, the molting delay caused by buprofezin was also rescued by PA. The results revealed that buprofezin delayed the molting of spiders by suppressing PpCHS1 expression, which will benefit the protection of P. pseudoannulate and related spider species.
Assuntos
Animais Peçonhentos , Quitina Sintase , Aranhas , Tiadiazinas , Animais , Quitina Sintase/genética , Quitina Sintase/metabolismo , Muda/genética , Insetos , Aranhas/genética , Aranhas/metabolismo , Quitina/metabolismoRESUMO
Due to the adverse environmental impacts of toxic heavy metal-based antifoulants, the screening of environmentally friendly antifoulants has become important for the development of marine antifouling technology. Compared with the traditional lengthy and costly screening method, computer-aided drug design (CADD) offers a promising and efficient solution that can accelerate the screening process of green antifoulants. In this study, we selected barnacle chitin synthase (CHS, an important enzyme for barnacle settlement and development) as the target protein for docking screening. Three CHS genes were identified in the barnacle Amphibalanus amphitrite, and their encoded proteins were found to share a conserved glycosyltransferase domain. Molecular docking of 31,561 marine natural products with AaCHSs revealed that zoanthamine alkaloids had the best binding affinity (-11.8 to -12.6â¯kcal/mol) to AaCHSs. Considering that the low abundance of zoanthamine alkaloids in marine organisms would limit their application as antifoulants, a marine fungal-derived natural product, mycoepoxydiene (MED), which has a similar chemical structure to zoanthamine alkaloids and the potential for large-scale production by fermentation, was selected and validated for stable binding to AaCHS2L2 using molecular docking and molecular dynamics simulations. Finally, the efficacy of MED in inhibiting cyprid settlement of A. amphitrite was confirmed by a bioassay that demonstrated an EC50 of 1.97⯵g/mL, suggesting its potential as an antifoulant candidate. Our research confirmed the reliability of using AaCHSs as antifouling targets and has provided insights for the efficient discovery of green antifoulants by CADD.
Assuntos
Alcaloides , Incrustação Biológica , Thoracica , Animais , Quitina Sintase/genética , Quitina Sintase/metabolismo , Simulação de Acoplamento Molecular , Reprodutibilidade dos Testes , Incrustação Biológica/prevenção & controle , Alcaloides/farmacologia , LarvaRESUMO
Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) analyzed to date oligomerize and form resistosomes upon activation to initiate immune responses. Some NLRs are encoded in tightly linked co-regulated head-to-head genes whose products function together as pairs. We uncover the oligomerization requirements for different Arabidopsis paired CHS3-CSA1 alleles. These pairs form resting-state heterodimers that oligomerize into complexes distinct from NLRs analyzed previously. Oligomerization requires both conserved and allele-specific features of the respective CHS3 and CSA1 Toll-like interleukin-1 receptor (TIR) domains. The receptor kinases BAK1 and BIRs inhibit CHS3-CSA1 pair oligomerization to maintain the CHS3-CSA1 heterodimer in an inactive state. Our study reveals that paired NLRs hetero-oligomerize and likely form a distinctive "dimer of heterodimers" and that structural heterogeneity is expected even among alleles of closely related paired NLRs.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Quitina Sintase , Proteínas NLR , Doenças das Plantas , Imunidade Vegetal , Receptores Imunológicos , Alelos , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Quitina Sintase/química , Quitina Sintase/genética , Quitina Sintase/metabolismo , Mutação , Proteínas NLR/química , Proteínas NLR/genética , Proteínas NLR/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Imunidade Vegetal/genética , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Multimerização ProteicaRESUMO
This study aimed to investigate the role of the yeast cell wall and membrane in enhancing osmotic tolerance by antioxidant dipeptides (ADs) including Ala-His (AH), Thr-Tyr (TY), and Phe-Cys (FC). Results revealed that ADs could improve the integrity of the cell wall by restructuring polysaccharide structures. Specifically, FC significantly (p < 0.05) reduced the leakage of nucleic acid and protein by 2.86% and 5.36%, respectively, compared to the control. In addition, membrane lipid composition played a crucial role in enhancing yeast tolerance by ADs, including the increase of cell membrane integrity and the decrease of permeability by regulating the ratio of unsaturated fatty acids. The up-regulation of gene expression associated with the cell wall integrity pathway (RLM1, SLT2, MNN9, FKS1, and CHS3) and fatty acid biosynthesis (ACC1, HFA1, OLE1, ERG1, and FAA1) further confirmed the positive impact of ADs on yeast tolerance against osmotic stress.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Antioxidantes/metabolismo , Pressão Osmótica , Parede Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Membrana Celular/metabolismo , Quitina Sintase/metabolismoRESUMO
Reuterin is a natural antifungal agent derived from certain strains of Limosilactobacillus reuteri. Our previous study revealed that 6 mM reuterin inhibited completely the conidial germination of aflatoxigenic Aspergillus flavus. This study investigated the potential molecular mechanism of reuterin in inhibiting A. flavus conidial germination, which was pre-assumed that it correlated to the inhibition of some essential enzyme activity involved in conidial germination, specifically 1,3-ß-glucan synthase, chitin synthase, and catalases (catalase, bifunctional catalase-peroxidase, and spore-specific catalase). The complex of 1,3-ß-glucan synthase and chitin synthase with reuterin had a lower binding affinity than that with the substrate. Conversely, the complex of catalases with reuterin had a higher binding affinity than that with the substrate. It was suggested that 1,3-ß-glucan synthase and chitin synthase tended to bind the substrate rather than bind reuterin. In contrast, catalases tended to bind reuterin rather than bind the substrate. Therefore, reuterin could be a potential inhibitor of catalases but may not be an inhibitor of 1,3-ß-glucan synthase and chitin synthase. In this in silico study, we predicted that the potential molecular mechanism of reuterin in inhibiting A. flavus conidial germination was due to the inhibition of catalases activities by competitively binding to the enzymes active sites, thus resulting in the accumulation of reactive oxygen species in cells, leading to cells damage. PRACTICAL APPLICATION: This in silico study revealed that reuterin is a potential inhibitor of catalases in A. flavus, thereby interfering with the antioxidant system during conidial germination. This finding shows that reuterin can be used as an antifungal agent in food or agricultural products, inhibiting conidial germination completely.
Assuntos
Aspergillus flavus , Gliceraldeído/análogos & derivados , Propano , beta-Glucanas , Catalase/metabolismo , Esporos Fúngicos/metabolismo , Antifúngicos/química , Quitina Sintase/metabolismoRESUMO
Chitin, a natural polymer of N-acetylglucosamine chains, is a principal component of the apical extracellular matrix in arthropods. Chitin microfibrils serve as structural components of natural biocomposites present in the extracellular matrix of a variety of invertebrates including sponges, molluscs, nematodes, fungi and arthropods. In this review, we summarize the frontier advances of insect chitin synthesis. More specifically, we focus on the chitin synthase (CHS), which catalyzes the key biosynthesis step. CHS is also known as an attractive insecticidal target in that this enzyme is absent in mammals, birds or plants. As no insect chitin synthase structure have been reported so far, we review recent studies on glycosyltransferase domain structures derived from fungi and oomycetes, which are conserved in CHS from all species containing chitin. Auxiliary proteins, which coordinate with CHS in chitin biosynthesis and assembly, are also discussed.
Assuntos
Artrópodes , Quitina Sintase , Animais , Quitina Sintase/metabolismo , Insetos/genética , Insetos/metabolismo , Artrópodes/metabolismo , Invertebrados/metabolismo , Fungos , Quitina/metabolismo , Mamíferos/metabolismoRESUMO
Saprolegnia parasitica is an oomycete responsible for a fish disease called saprolegniosis, which poses an economic and environmental burden on aquaculture production. In Saprolegnia, CHS5 of S. parasitica (SpCHS5) contains an N-terminal domain, a catalytic domain of the glycosyltransferase -2 family containing a GT-A fold, and a C-terminal transmembrane domain. No three-dimensional structure of SpCHS5 is reported yet disclosing the structural details of this protein. We have developed a structural model of full-length SpCHS5 and validated it by molecular dynamics simulation technique. From the 1 microsecond simulations, we retrieved the stable RoseTTAFold model SpCHS5 protein to explain characteristics and structural features. Furthermore, from the analysis of the movement of chitin in the protein cavity, we assumed that ARG 482, GLN 527, PHE 529, PHE 530, LEU 540, SER 541, TYR 544, ASN 634, THR 641, TYR 645, THR 641, ASN 772 residues as a main cavity lining site. In SMD analysis, we investigated the opening of the transmembrane cavity required for chitin translocation. The pulling of chitin from the internal cavity to the extracellular region was observed through steered molecular dynamics simulations. A comparison of the initial and final structures of chitin complex showed that there's a transmembrane cavity opening in the simulations. Overall, this present work will help us understand the structural and functional basis of CHS5 and design inhibitors against SpCHS5.Communicated by Ramaswamy H. Sarma.
Assuntos
Saprolegnia , Animais , Saprolegnia/metabolismo , Fosfolipídeos , Quitina Sintase/metabolismoRESUMO
We applied a new RNA interference (RNAi) system using rolling circle transcription (RCT) technology to generate RNA microspheres (RMS) for targeting two key chitin synthetic pathway genes [chitin synthase A (CHSA), chitin synthase B (CHSB)] in the larvae of the oriental armyworm (Mythimna separate), a RNAi-unsusceptible agriculturally important lepidopteran pest. Feeding the third-instar larvae with the RMS-CHSA- or RMS-CHSB-treated corn leaf discs suppressed the expression of CHSA by 81.7% or CHSB by 88.1%, respectively, at 72 h. The silencing of CHSA consequently affected the larval development, including the reduced body weight (54.0%) and length (41.3%), as evaluated on the 7th day, and caused significant larval mortalities (51.1%) as evaluated on the 14th day. Similar results were obtained with the larvae fed RMS-CHSB. We also compared RNAi efficiencies among different strategies: 1) two multi-target RMS [i.e., RMS-(CHSA + CHSB), RMS-CHSA + RMS-CHSB], and 2) multi-target RMS and single-target RMS (i.e., either RMS-CHSA or RMS-CHSB) and found no significant differences in RNAi efficiency. By using Cy3-labeled RMS, we confirmed that RMS can be rapidly internalized into Sf9 cells (<6 h). The rapid cellular uptake of RMS accompanied with significant RNAi efficiency through larval feeding suggests that the RCT-based RNAi system can be readily applied to study the gene functions and further developed as bio-pesticides for insect pest management. Additionally, our new RNAi system takes the advantage of the microRNA (miRNA)-mediated RNAi pathway using miRNA duplexes generated in vivo from the RMS by the target insect. The system can be used for RNAi in a wide range of insect species, including lepidopteran insects which often exhibit extremely low RNAi efficiency using other RNAi approaches.
Assuntos
MicroRNAs , Mariposas , Animais , Interferência de RNA , Quitina Sintase/genética , Quitina Sintase/metabolismo , Microesferas , Mariposas/genética , Mariposas/metabolismo , Insetos/genética , Larva/metabolismo , RNA de Cadeia DuplaRESUMO
The chitin synthase Chs3 is a multipass membrane protein whose trafficking is tightly controlled. Accordingly, its exit from the endoplasmic reticulum (ER) depends on several complementary mechanisms that ensure its correct folding. Despite its potential failure on its exit, Chs3 is very stable in this compartment, which suggests its poor recognition by ER quality control mechanisms such as endoplasmic reticulum-associated degradation (ERAD). Here we show that proper N-glycosylation of its luminal domain is essential to prevent the aggregation of the protein and its subsequent recognition by the Hrd1-dependent ERAD-L machinery. In addition, the interaction of Chs3 with its chaperone Chs7 seems to mask additional cytosolic degrons, thereby avoiding their recognition by the ERAD-C pathway. On top of that, Chs3 molecules that are not degraded by conventional ERAD can move along the ER membrane to reach the inner nuclear membrane, where they are degraded by the inner nuclear membrane-associated degradation (INMAD) system, which contributes to the intracellular homeostasis of Chs3. These results indicate that Chs3 is an excellent model to study quality control mechanisms in the cell and reinforce its role as a paradigm in intracellular trafficking research.
Assuntos
Quitina Sintase , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Quitina Sintase/genética , Quitina Sintase/metabolismo , Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Dobramento de Proteína , Ubiquitina-Proteína Ligases/metabolismoRESUMO
A series of spiro[pyrrolidine-2,3'-quinoline]-2'-one derivatives were designed and synthesized for the discovery of novel antifungal drugs. The bioactivities of all derivatives were screened by evaluating their inhibitory effects against chitin synthase (CHS) and antimicrobial activities in vitro. Enzyme inhibition experiments showed that all the synthesized compounds inhibited the chitin synthase. Compounds 4d, 4k, 4n and 4o showed inhibitory effects against CHS with IC50 values which were close to that of the control drug (polyoxin B). The results of enzyme kinetics experiment showed that these compounds were non-competitive inhibitors of chitin synthase (Ki of compound 4o is 0.14 mM). Antimicrobial experiments showed that these compounds exhibited moderate to excellent antifungal activity against pathogenic fungal strains while the compounds showed little potency against bacteria. Among them, compounds 4d, 4f, 4k and 4n showed stronger antifungal activities against C. albicans than those of fluconazole and polyoxin B. Compounds 4f, 4n and 4o showed better antifungal activities against A. flavus than those of fluconazole and polyoxin B. Compound 4d showed similar activity to that of fluconazole and stronger activity than those of polyoxin B against C. neoformans and A. fumigatus. It is also showed that these compounds have the potency against drug-resistant fungal variants. The results of sorbitol protection assay and evaluation of antifungal activity against micafungin-resistant strains experiment further illustrated that these compounds inhibited the synthesis of chitin of fungal cell wall. Drug combination experiments showed that these compounds had synergistic or additive effects when combined with fluconazole or polyoxin B. The synergistic effects with polyoxin B further confirmed the compounds were non-competitive inhibitors of chitin synthase. Additionally, docking studies showed that these compounds had strong affinity with chitin synthase from C. albicans (CaChs2). These results indicate that the target of these synthesized compounds is chitin synthase, and these compounds had excellent antifungal activity while possessed the potency against drug-resistant fungal variants.
Assuntos
Cryptococcus neoformans , Quinolinas , Antifúngicos/farmacologia , Fluconazol , Quitina Sintase , Quitina , Candida albicans , PiperazinasRESUMO
Synthesis of chitin is a subject of great interest in the fields of physiology and immunology of crustaceans. Chitinous tissues include not only the carapace, but also an acellular membrane in the intestine called the peritrophic membrane (PM). Here, we describe the first report of chitin synthase (CHS) of a penaeid shrimp, kuruma shrimp Penaeus japonicus. Histological observations showed that fecal matter in the midgut of kuruma shrimp was wrapped with a PM, which physically separated it from the midgut epithelium. Subsequently, the chitin synthase transcript was amplified from the midgut of the shrimp. The chitin synthase gene of kuruma shrimp (MjCHS) encodes 1,523 amino acid residues. Structural prediction analysis showed that the N-terminal region of MjCHS protein included nine transmembrane helices, the middle region included the catalytic region with several conserved motifs which are found in CHSs from other arthropods, and the C-terminal region included seven transmembrane helices. Although insects have distinct exoskeletal and intestinal chitin synthases, the phylogenetic analysis suggested that crustaceans have a single CHS. MjCHS mRNA was constantly detected in the digestive tract, including the midgut and hepatopancreas of both juvenile and adult kuruma shrimp, suggesting a stable synthesis of chitin in those organs. In contrast, MjCHS mRNA was also detected in the hindgut and uropod of juvenile shrimp. After molting, the mRNA levels of MjCHS in the stomach and uropod were higher than other molting cycles. These results suggest that MjCHS contributes to chitin synthesis in both the digestive tract and the epidermis, providing fundamental insights into chitin synthesis of crustaceans.
Assuntos
Penaeidae , Animais , Penaeidae/genética , Penaeidae/metabolismo , Quitina Sintase/genética , Quitina Sintase/metabolismo , Filogenia , Trato Gastrointestinal , Quitina/metabolismo , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Mosquitoes are an important vector of viral transmission, and due to the complexity of the pathogens they transmit, vector control may be the most effective strategy to control mosquito-borne diseases. Chitin is required for insect growth and development and is absent in higher animals and plants, so regulating the chitin synthesis pathway can serve as a potentially effective means to control vector insects. Most of the current research on the chitin synthase (CHS) gene is focused on chitin synthase-1 (CHS-1), while relatively little is known about chitin synthase-2 (CHS-2). RESULTS: The CHS-2 gene of Ae. albopictus is highly conserved and closely related to that of Aedes aegypti. The expression of CHS-2 in the third-instar larvae and pupal stage of Ae. albopictus was relatively high, and CHS-2 expression in adult mosquitoes reached the highest value 24 h after blood-feeding. In the fourth-instar larvae of Ae. albopictus, CHS-2 expression was significantly higher in the midgut than in the epidermis. Silencing CHS-2 in Ae. albopictus larvae had no effect on larval survival and emergence. The expression of four genes related to chitin synthesis enzymes was significantly upregulated, the expression level of three genes was unchanged, and only the expression level of GFAT was significantly downregulated. The expression of chitin metabolism-related genes was also upregulated after silencing. The level of chitin in the midgut of Ae. albopictus larvae was significantly decreased, while the chitinase activity was unchanged. The epithelium of the midgut showed vacuolization, cell invagination and partial cell rupture, and the structure of the peritrophic membrane was destroyed or even absent. METHODS: The expression of CHS-2 in different developmental stages and tissues of Aedes albopictus was detected by real-time fluorescence quantitative PCR (qPCR). After silencing CHS-2 of the fourth-instar larvae of Ae. albopictus by RNA interference (RNAi), the expression levels of genes related to chitin metabolism, chitin content and chitinase activity in the larvae were detected. The structure of peritrophic membrane in the midgut of the fourth-instar larvae after silencing was observed by paraffin section and hematoxylin-eosin (HE) staining. CONCLUSION: CHS-2 can affect midgut chitin synthesis and breakdown by regulating chitin metabolic pathway-related genes and is involved in the formation of the midgut peritrophic membrane in Ae. albopictus, playing an important role in growth and development. It may be a potential target for enhancing other control methods.