RESUMO
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
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
The introduction of active groups of natural products into the framework of pesticide molecules is an effective approach for discovering active lead compounds, and thus has been widely used in the development of new agrochemicals. In this work, a novel series of 1,2,3,4-tetrahydroquinoline derivatives containing a pyrimidine ether scaffold were designed and synthesized by the active substructure splicing method. The new compounds showed good antifungal activities against several fungi. Especially, compound 4fh displayed excellent in vitro activity against Valsa mali and Sclerotinia sclerotiorum with EC50 values of 0.71 and 2.47 µg/mL, respectively. 4fh had slightly stronger inhibitory activity (68.08% at 50 µM) against chitin synthase (CHS) than that of polyoxin D (63.84% at 50 µM) and exhibited obvious curative and protective effects on S. sclerotiorum in vivo. Thus, 4fh can be considered as a new candidate fungicide as a chitin synthase inhibitor. An accurate and reliable three-dimensional quantitative structure-activity relationship (3D-QSAR) model presented a useful direction for the further excogitation of more highly active fungicides. Molecular docking revealed that the conventional hydrogen bond mainly affected the binding affinity of 4fh with chitin synthase. The present results will provide a guidance to discover potential CHS-based fungicides for plant disease control in agriculture.
Assuntos
Quitina Sintase , Fungicidas Industriais , Antifúngicos/química , Quitina , Quitina Sintase/genética , Quitina Sintase/metabolismo , Éter , Fungicidas Industriais/química , Simulação de Acoplamento Molecular , Pirimidinas/farmacologia , Quinolinas , Relação Estrutura-AtividadeRESUMO
The fungal cell wall consists of proteins and polysaccharides, formed by the co-ordinated activity of enzymes, such as chitin or glucan synthases. These enzymes are delivered via secretory vesicles to the hyphal tip. In the ascomycete Neurospora crassa, chitin synthases and ß(1,3)-glucan synthase are transported in different vesicles, whereas they co-travel along microtubules in the basidiomycete Ustilago maydis. This suggests fundamental differences in wall synthesis between taxa. Here, we visualize the class V chitin synthase ZtChs5 and the ß(1,3)-glucan synthase ZtGcs1 in the ascomycete Zymoseptoria tritici. Live cell imaging demonstrate that both enzymes co-locate to the apical plasma membrane, but are not concentrated in the Spitzenkörper. Delivery involves co-transport along microtubules of the chitin and glucan synthase. Live cell imaging and electron microscopy suggest that both cell wall synthases locate in the same vesicle. Thus, microtubule-dependent co-delivery of cell wall synthases in the same vesicle is found in asco- and basidiomycetes.
Assuntos
Ascomicetos/enzimologia , Quitina Sintase/metabolismo , Glucosiltransferases/metabolismo , Vesículas Secretórias/fisiologia , Ascomicetos/genética , Basidiomycota/metabolismo , Quitina Sintase/genética , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Glucosiltransferases/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Eletrônica , Neurospora crassa/metabolismo , Vesículas Secretórias/ultraestruturaRESUMO
Chitin, a homopolymer of ß-(1,4) linked N-acetylglucosamine (GlcNAc), is a major component of cyst wall in the protozoan parasites Entamoeba histolytica (Eh) and Entamoeba invadens (Ei). The Entamoeba chitin synthase makes chitin at the vesicular membrane rather than the plasma membrane in fungi, even though the chemistry of chitin synthesis is most likely the same. However, the role of nucleotide sugar transporter(s) (NSTs) that are involved in chitin synthesis in Entamoeba are not yet established. In this study, we have identified the putative UDP-GlcNAc transporter (EiNst5) of Ei by BLASTP analysis using the amino acid sequence of EhNst3, the UDP-GlcNAc transporter of Eh. Heterologous expression of both EhNst3 and EiNst5 was found to complement the function of Yea4p (UDP-GlcNAc transporter of S. cerevisiae) in YEA4 null mutant and increased the cell wall chitin content. Like Yea4p in S. cerevisiae, Myc-epitope tagged EhNst3 and EiNst5 were localized to the endoplasmic reticulum in Δyea4 cells. The EiNST5 transcript was up-regulated during the in vitro encystation and oxidative stress in E. invadens. Similar up-regulation was also seen for EhNST3 under oxidative stress in E. histolytica. Down-regulation of EiNst5 expression using gene-specific dsRNA significantly reduced cyst formation during in vitro encystation in E. invadens. Our observations suggest for the first time the involvement of EhNst3 and EiNst5 in chitin synthesis and so in encystation of Entamoeba.
Assuntos
Quitina/biossíntese , Entamoeba histolytica/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Encistamento de Parasitas , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Quitina Sintase/metabolismo , Resposta ao Choque Térmico/fisiologia , Membranas Intracelulares/metabolismo , Proteínas de Membrana Transportadoras/genética , Estresse Oxidativo/fisiologia , Proteínas de Protozoários/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The alarm is rang for friendly fire; Saccharomyces cerevisiae (S. cerevisiae) newfound as a fungal pathogen with an individual feature. S. cerevisiae has food safety and is not capable of producing infection but, when the host defenses are weakened, there is room for opportunistic S. cerevisiae strains to cause a health issues. Fungal diseases are challenging to treat because, unlike bacteria, the fungal are eukaryotes. Antibiotics only target prokaryotic cells, whereas compounds that kill fungi also harm the mammalian host. Small differences between mammalian and fungal cells regarding genes and proteins sequence and function make finding a drug target more challenging. Recently, Chitin synthase has been considered as a promising target for antifungal drug development as it is absent in mammals. In S. cerevisiae, CHS3, a class IV chitin synthase, produces 90% of the chitin and essential for cell growth. CHS3 from the trans-Golgi network to the plasma membrane requires assembly of the exomer complex (including proteins cargo such as CHS5, CHS6, Bach1, and Arf1). In this work, we performed SELEX (Systematic Evolution of Ligands by EXponential enrichment) as high throughput virtual screening of the RCSB data bank to find an aptamer as potential inhibit of the class IV chitin synthase of S. cerevisiae. Among all the candidates, G-rich VEGF (GVEGF) aptamer (PDB code: 2M53) containing locked sugar parts was observed as potential inhibitor of the assembly of CHS5-CHS6 exomer complex a subsequently block the chitin biosynthesis pathway as an effective anti-fungal. It was suggested from the simulation that an assembly of exomer core should begin CHS5-CHS6, not from CHS5-Bach1. It is notable that secondary structures of CHS6 and Bach1 was observed very similar, but they have only 25% identity at the amino acid sequence that exhibited different features in exomer assembly.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Quitina Sintase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Multimerização Proteica/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator A de Crescimento do Endotélio Vascular/química , Proteínas Adaptadoras de Transporte Vesicular/química , Sequência de Aminoácidos , Antifúngicos/metabolismo , Aptâmeros de Nucleotídeos/genética , Sítios de Ligação , Quitina Sintase/química , Quadruplex G , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Membrana/química , Simulação de Acoplamento Molecular , Ligação Proteica , Técnica de Seleção de Aptâmeros , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Alinhamento de SequênciaRESUMO
Fe3O4 nanoparticles, one kind of magnetic nanomaterials (NMs), are widely used in drug delivery, biological imaging, sensors, catalysts and pollution management. However, its toxicity to biological systems and related toxicity mechanisms remain to be explored. In this study, we investigate the effect of as-synthesized Fe3O4 nanoparticles on growth of Saccharomyces cerevisiae, an important model fungus. Growth inhibition assays showed that Fe3O4 nanoparticles remarkably inhibited yeast growth. Interestingly, this inhibitory effect was not attributed to the well-known plasma membrane damage, cell wall damage and ROS accumulation. Further investigations revealed that the nanoparticles strongly impaired mitochondrial functions, resulting in abnormal mitochondrial morphology, decreased mitochondrial membrane potential (MMP) and attenuated ATP production. Most importantly, the respiratory chain complex â £, rather than other respiratory chain complexes and ATP synthases, was found to be the main target of the nanoparticles. This study uncovers a novel ROS-independent toxicity mechanism of Fe3O4 nanoparticles to eukaryotic cells.
Assuntos
Óxido Ferroso-Férrico/química , Nanopartículas de Magnetita/toxicidade , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Quitina/metabolismo , Quitina Sintase/metabolismo , Glucosiltransferases/metabolismo , Nanopartículas de Magnetita/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Difração de Raios XRESUMO
Fungal cells are surrounded by an extracellular cell wall. This complex matrix of proteins and polysaccharides protects against adverse stresses and determines the shape of fungal cells. The polysaccharides of the fungal wall include 1,3-ß-glucan and chitin, which are synthesized by membrane-bound synthases at the growing cell tip. A hallmark of filamentous fungi is the class V chitin synthase, which carries a myosin-motor domain. In the corn smut fungus Ustilago maydis, the myosin-chitin synthase Mcs1 moves to the plasma membrane in secretory vesicles, being delivered by kinesin-1 and myosin-5. The myosin domain of Mcs1 enhances polar secretion by tethering vesicles at the site of exocytosis. It remains elusive, however, how other cell-wall-forming enzymes are delivered and how their activity is coordinated post secretion. Here, we show that the U. maydis class VII chitin synthase and 1,3-ß-glucan synthase travel in Mcs1-containing vesicles, and that their apical secretion depends on Mcs1. Once in the plasma membrane, anchorage requires enzyme activity, which suggests co-synthesis of chitin and 1,3-ß-glucan polysaccharides at sites of exocytosis. Thus, delivery of cell-wall-forming enzymes in Mcs1 vesicles ensures local foci of fungal cell wall formation.
Assuntos
Parede Celular/enzimologia , Parede Celular/fisiologia , Vesículas Secretórias/metabolismo , Ustilago/fisiologia , Membrana Celular/química , Membrana Celular/metabolismo , Quitina/metabolismo , Quitina Sintase/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glucosiltransferases/metabolismo , Miosinas/metabolismo , Vesículas Secretórias/química , Ustilago/enzimologia , Ustilago/crescimento & desenvolvimento , beta-Glucanas/metabolismoRESUMO
With the widespread use of avermectins (AVMs) for managing parasitic and agricultural pests, the resistance of worms and insects to AVMs has emerged as a serious threat to human health and agriculture worldwide. The reduced penetration of AVMs is one of the main reasons for the development of the resistance to the chemicals. However, the detailed molecular mechanisms remain elusive. Here, we use the larvae of Drosophila melanogaster as the model organism to explore the molecular mechanisms underlying the development of penetration resistance to AVMs. We clearly show that the chitin layer is thickened and the efflux transporter P-glycoprotein (P-gp) is overexpressed in the AVM-resistant larvae epidermis. We reveal that the activation of the transcription factor Relish by the over-activated epidermal growth factor receptor (EGFR)/AKT/ERK pathway induces the overexpression of the chitin synthases DmeCHS1/2 and P-gp in the resistant larvae. Interestingly, we discover for the first time, to the best of our knowledge, that AVM directly interacts with EGFR and leads to the activation of the EGFR/AKT/ERK pathway, which activates the transcription factor Relish and induces the overexpression of DmeCHS1/2 and P-gp. These findings provide new insights into the molecular mechanisms underlying the development of penetration resistance to drugs.
Assuntos
Drosophila melanogaster/metabolismo , Receptores ErbB/metabolismo , Resistência a Inseticidas/efeitos dos fármacos , Ivermectina/análogos & derivados , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Quitina Sintase/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ivermectina/metabolismo , Ivermectina/toxicidade , Larva/efeitos dos fármacos , Larva/enzimologia , Modelos Biológicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Curcumin, a yellow polyphenol compound, is known to possess antifungal activity for a range of pathogenic fungi. However, the fungicidal mechanism of curcumin (CUR) has not been identified. We have occasionally found that chitin redistributes to the cell wall outer layer of Sporothrix schenckii (S. schenckii) upon sublethal CUR treatment. Whether CUR can affect chitin synthesis via the protein kinase C (PKC) signaling pathway has not been investigated. This study describes a direct fungicidal activity of CUR against S. schenckii demonstrated by the results of a checkerboard microdilution assay and, for the first time, a synergistic effect of CUR with terbinafine (TRB). Furthermore, the results of real-time PCR showed that sublethal CUR upregulated the transcription of PKC, chitin synthase1 (CHS1), and chitin synthase3 (CHS3) in S. schenckii. The fluorescence staining results using wheat germ agglutinin-fluorescein isothiocyanate (WGA-FITC) and calcofluor white (CFW) consistently showed that chitin exposure and total chitin content were increased on the conidial cell wall of S. schenckii by sublethal CUR treatment. A histopathological analysis of mice infected with CUR-treated conidia showed dampened inflammation in the local lesion and a reduced fungal burden. The ELISA results showed proinflammatory cytokine secretion at an early stage from macrophages stimulated by the CUR-treated conidia. The present data led to the conclusion that CUR is a potential antifungal agent and that its fungicidal mechanism may involve chitin accumulation on the cell wall of S. schenckii, which is associated with decreased virulence in infected mice.
Assuntos
Antifúngicos/uso terapêutico , Quitina/metabolismo , Curcumina/uso terapêutico , Sporothrix/efeitos dos fármacos , Esporotricose/tratamento farmacológico , Animais , Células Cultivadas , Quitina Sintase/genética , Quitina Sintase/metabolismo , Sinergismo Farmacológico , Feminino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Naftalenos/uso terapêutico , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Sporothrix/patogenicidade , Sporothrix/fisiologia , Esporotricose/imunologia , Terbinafina , Regulação para Cima , VirulênciaRESUMO
Chitin, a biopolymer of N-acetylglucosamine, is abundant in invertebrates and fungi and is an important structural molecule [1, 2]. There has been a longstanding belief that vertebrates do not produce chitin; however, we have obtained compelling evidence to the contrary. Chitin synthase genes are present in numerous fishes and amphibians, and chitin is localized in situ to the lumen of the developing zebrafish gut, in epithelial cells of fish scales, and in at least three different cell types in larval salamander appendages. Chitin synthase gene knockdowns and various histochemical experiments in zebrafish further authenticated our results. Finally, a polysaccharide was extracted from scales of salmon that exhibited all the chemical hallmarks of chitin. Our data and analyses demonstrate the existence of endogenous chitin in vertebrates and suggest that it serves multiple roles in vertebrate biology.
Assuntos
Quitina Sintase/metabolismo , Quitina/metabolismo , Vertebrados/metabolismo , Anfíbios/metabolismo , Animais , Quitina/genética , Quitina Sintase/genética , Células Epiteliais/metabolismo , Peixes/metabolismo , Mucosa Intestinal/metabolismo , Larva/crescimento & desenvolvimento , Dados de Sequência Molecular , Vertebrados/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
Candida glabrata is one of the most common causes of candidemia, a life-threatening, systemic fungal infection, and is surpassed in frequency only by Candida albicans. Major factors contributing to the success of this opportunistic pathogen include its ability to readily acquire resistance to antifungals and to colonize and adapt to many different niches in the human body. Here we addressed the flexibility and adaptability of C. glabrata during interaction with macrophages with a serial passage approach. Continuous co-incubation of C. glabrata with a murine macrophage cell line for over six months resulted in a striking alteration in fungal morphology: The growth form changed from typical spherical yeasts to pseudohyphae-like structures - a phenotype which was stable over several generations without any selective pressure. Transmission electron microscopy and FACS analyses showed that the filamentous-like morphology was accompanied by changes in cell wall architecture. This altered growth form permitted faster escape from macrophages and increased damage of macrophages. In addition, the evolved strain (Evo) showed transiently increased virulence in a systemic mouse infection model, which correlated with increased organ-specific fungal burden and inflammatory response (TNFα and IL-6) in the brain. Similarly, the Evo mutant significantly increased TNFα production in the brain on day 2, which is mirrored in macrophages confronted with the Evo mutant, but not with the parental wild type. Whole genome sequencing of the Evo strain, genetic analyses, targeted gene disruption and a reverse microevolution experiment revealed a single nucleotide exchange in the chitin synthase-encoding CHS2 gene as the sole basis for this phenotypic alteration. A targeted CHS2 mutant with the same SNP showed similar phenotypes as the Evo strain under all experimental conditions tested. These results indicate that microevolutionary processes in host-simulative conditions can elicit adaptations of C. glabrata to distinct host niches and even lead to hypervirulent strains.
Assuntos
Adaptação Fisiológica , Candida glabrata/genética , Candidíase/microbiologia , Macrófagos/microbiologia , Polimorfismo de Nucleotídeo Único , Animais , Candida glabrata/crescimento & desenvolvimento , Candida glabrata/patogenicidade , Linhagem Celular , Quitina Sintase/genética , Quitina Sintase/metabolismo , Modelos Animais de Doenças , Feminino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Hifas , Camundongos , Mutação Puntual , Inoculações Seriadas , Organismos Livres de Patógenos Específicos , VirulênciaRESUMO
Chitin is an essential component of the fungal cell wall and a potential target in the development of new antifungal compounds, due to its presence in fungi and not in plants or vertebrates. Chitin synthase genes (chs) constitute a complex family in filamentous fungi and are involved in fungal development, morphogenesis, pathogenesis and virulence. In this study, additional chs genes in the citrus postharvest pathogen Penicillium digitatum have been identified. Comparative analyses included each PdChs in each one of the classes I to VII previously established, and support the grouping of these into three divisions. Disruption of the gene coding PdChsVII, which contains a short version of a myosin motor domain, has been achieved by using Agrobacterium tumefaciens-mediated transformation and revealed its role in the life cycle of the fungus. Disruption strains were viable but showed reduced growth and conidia production. Moreover, Pdchs mutants developed morphological defects as balloon-like enlarged cells and increased chitin content, indicative of an altered cell wall structure. Gene disruption also increased susceptibility to antifungal compounds such as calcofluor white (CFW), sodium dodecyl sulfate (SDS), hydroxide peroxide (H2O2) and commercial fungicides, but significantly no change was observed in the sensitivity to antifungal peptides. The PdchsVII mutants were able to infect citrus fruit and produced tissue maceration, although had reduced virulence and most importantly were greatly impaired in the production of visible mycelium and conidia on the fruit.
Assuntos
Quitina Sintase/metabolismo , Citrus/microbiologia , Proteínas Fúngicas/metabolismo , Miosinas/genética , Penicillium/fisiologia , Antifúngicos/farmacologia , Benzenossulfonatos/farmacologia , Parede Celular/metabolismo , Quitina Sintase/genética , Proteínas Fúngicas/genética , Fungicidas Industriais/farmacologia , Peróxido de Hidrogênio/farmacologia , Mutação , Penicillium/efeitos dos fármacos , Penicillium/patogenicidade , Filogenia , Doenças das Plantas/microbiologia , Estrutura Terciária de Proteína , Dodecilsulfato de Sódio/farmacologia , VirulênciaRESUMO
Chitin synthase is the key regulatory enzyme for chitin synthesis and excretion in insects, as well as a specific target of insecticides. The chitin synthase A gene (BmChsA) cloned from Bombyx mori, the model species of lepidopteran, is an epidermis-specific expressed gene during the molting stage. Knockdown BmChsA gene in 3rd instar larvae increased the number of non-molting and abnormal molting larvae. Exposure to nikkomycin Z, a chitin synthase inhibitor downregulated the expression of BmChsA and decreased the amount of epidermis chitin during the molting process. The thickness of the new epidermis and its dense structure varied greatly. The exogenous hormones significantly upregulated the expression of BmChsA with low levels of endogenous MH and high levels of endogenous JH immediately after molting. With low levels of endogenous hormones during the mulberry intake process, BmChsA was rarely upregulated by exogenous hormones. With high levels of endogenous MH and low levels of endogenous JH during the molting stage, we did not detect the upregulation of BmChsA by exogenous hormones. The expression of BmChsA was regulated by endocrine hormones, which directly affected the chitin synthesis-dependent epidermal regeneration and molting process.
Assuntos
Bombyx/genética , Quitina Sintase/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Muda/genética , Aminoglicosídeos/farmacologia , Animais , Bombyx/efeitos dos fármacos , Bombyx/enzimologia , Bombyx/crescimento & desenvolvimento , Quitina/biossíntese , Quitina Sintase/antagonistas & inibidores , Quitina Sintase/metabolismo , Ecdisona/análogos & derivados , Ecdisona/farmacologia , Inibidores Enzimáticos/farmacologia , Epiderme/efeitos dos fármacos , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Hormônios Juvenis/farmacologia , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Manduca/genética , Manduca/metabolismo , Metoprene/farmacologia , Muda/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Ferroxidases are essential components of the high-affinity reductive iron assimilation pathway in fungi. Two ferroxidase genes, FET3-1 and FET3-2, have been identified in the genome of the maize anthracnose fungus Colletotrichum graminicola. Complementation of growth defects of the ferroxidase-deficient Saccharomyces cerevisiae strain Δfet3fet4 showed that both Fet3-1 and Fet3-2 of C. graminicola represent functional ferroxidases. Expression of enhanced green fluorescent protein fusions in yeast and C. graminicola indicated that both ferroxidase proteins localize to the plasma membrane. Transcript abundance of FET3-1 increased dramatically under iron-limiting conditions but those of FET3-2 were hardly detectable. Δfet3-1 and Δfet3-2 single as well as Δfet3-1/2 double-deletion strains were generated. Under iron-sufficient or deficient conditions, vegetative growth rates of these strains did not significantly differ from that of the wild type but Δfet3-1 and Δfet3-1/2 strains showed increased sensitivity to reactive oxygen species. Furthermore, under iron-limiting conditions, appressoria of Δfet3-1 and Δfet3-1/2 strains showed significantly reduced transcript abundance of a class V chitin synthase and exhibited severe cell wall defects. Infection assays on intact and wounded maize leaves, quantitative data of infection structure differentiation, and infection stage-specific expression of FET3-1 showed that reductive iron assimilation is required for appressorial penetration, biotrophic development, and full virulence.
Assuntos
Parede Celular/metabolismo , Ceruloplasmina/genética , Colletotrichum/genética , Ferro/metabolismo , Doenças das Plantas/microbiologia , Zea mays/microbiologia , Sequência de Aminoácidos , Membrana Celular/enzimologia , Ceruloplasmina/metabolismo , Quitina Sintase/metabolismo , Colletotrichum/enzimologia , Colletotrichum/crescimento & desenvolvimento , Colletotrichum/patogenicidade , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Teste de Complementação Genética , Hifas , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Folhas de Planta/microbiologia , Espécies Reativas de Oxigênio/farmacologia , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Deleção de Sequência , VirulênciaRESUMO
Molluscs with various shell architectures evolved around 542-525 million years ago, as part of a larger phenomenon related to the diversification of metazoan phyla. Molluscs deposit minerals in a chitin matrix. The mollusc chitin is synthesized by transmembrane enzymes that contain several unique extracellular domains. Here we investigate the assembly mechanism of the chitin synthase Ar-CS1 via its extracellular domain ArCS1_E22. The corresponding transmembrane protein ArCS1_E22TM accumulates in membrane fractions of the expression host Dictyostelium discoideum. Soluble recombinant ArCS1_E22 proteins can be purified as monomers only at basic pH. According to confocal fluorescence microscopy experiments, immunolabeled ArCS1_E22 proteins adsorb preferably to aragonitic nacre platelets at pH 7.75. At pH 8.2 or pH 9.0 the fluorescence signal is less intense, indicating that protein-mineral interaction is reduced with increasing pH. Furthermore, ArCS1_E22 forms regular nanostructures on cationic substrates as revealed by atomic force microscopy (AFM) experiments on modified mica cleavage planes. These experiments suggest that the extracellular domain ArCS1_E22 is involved in regulating the multiple enzyme activities of Ar-CS1 such as chitin synthesis and myosin movements by interaction with mineral surfaces and eventually by protein assembly. The protein complexes could locally probe the status of mineralization according to pH unless ions and pCO2 are balanced with suitable buffer substances. Taking into account that the intact enzyme could act as a force sensor, the results presented here provide further evidence that shell formation is coordinated physiologically with precise adjustment of cellular activities to the structure, topography and stiffness at the mineralizing interface.
Assuntos
Exoesqueleto/crescimento & desenvolvimento , Exoesqueleto/metabolismo , Quitina Sintase/metabolismo , Quitina/metabolismo , Moluscos/metabolismo , Sequência de Aminoácidos , Animais , Carbonato de Cálcio/química , Quitina/biossíntese , Quitina/química , Dictyostelium/genética , Dictyostelium/metabolismo , Microscopia de Força Atômica , Dados de Sequência Molecular , Moluscos/crescimento & desenvolvimento , Miosinas/metabolismoRESUMO
Chitin is a major cell wall component of many filamentous fungi. Among the eight chitin synthase genes of Aspergillus nidulans, csmA and csmB encode a myosin motor-like domain (MMD) and a chitin synthase domain (CSD) at their N- and C-termini respectively. In our previous reports, we suggested that CsmA and CsmB play compensatory roles essential for polarized hyphal growth although their functions do not completely overlap, and that their MMDs are essential for their functions. In the present study, we constructed chimeric csm genes by swapping N-terminal MMD-encoding halves of csmA and csmB and studied them to identify functional differences in the MMDs. Expression of the chimeric gene encoding the MMD-including half of CsmA (MA) and the CSD-including half of CsmB thoroughly suppressed the phenotypic defects of the ΔcsmB mutant, whereas the chimeric gene encoding the MMD-including half of CsmB (MB) and the CSD-including half of CsmA did not fully suppress the defects of the ΔcsmA mutant, suggesting that MA suffices for the function of MB while MB is not functionally equivalent to MA.
Assuntos
Aspergillus nidulans/genética , Quitina Sintase/genética , Proteínas Fúngicas/genética , Proteínas Recombinantes de Fusão/genética , Aspergillus nidulans/enzimologia , Parede Celular/enzimologia , Parede Celular/genética , Quitina Sintase/química , Quitina Sintase/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Miosinas/química , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Aspergillus fumigatus has two chitin synthases (CSMA and CSMB) with a myosin motor-like domain (MMD) arranged in a head-to-head configuration. To understand the function of these chitin synthases, single and double csm mutant strains were constructed and analyzed. Although there was a slight reduction in mycelial growth of the mutants, the total chitin synthase activity and the cell wall chitin content were similar in the mycelium of all of the mutants and the parental strain. In the conidia, chitin content in the ΔcsmA strain cell wall was less than half the amount found in the parental strain. In contrast, the ΔcsmB mutant strain and, unexpectedly, the ΔcsmA/ΔcsmB mutant strain did not show any modification of chitin content in their conidial cell walls. In contrast to the hydrophobic conidia of the parental strain, conidia of all of the csm mutants were hydrophilic due to the presence of an amorphous material covering the hydrophobic surface-rodlet layer. The deletion of CSM genes also resulted in an increased susceptibility of resting and germinating conidia to echinocandins. These results show that the deletion of the CSMA and CSMB genes induced a significant disorganization of the cell wall structure, even though they contribute only weakly to the overall cell wall chitin synthesis.
Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Quitina Sintase/metabolismo , Equinocandinas/farmacologia , Miosinas/química , Aspergillus fumigatus/genética , Carboidratos/química , Parede Celular/química , Quitina Sintase/química , Quitina Sintase/genética , DNA Fúngico/genética , Farmacorresistência Fúngica/genética , Regulação Fúngica da Expressão Gênica , Glucosiltransferases/metabolismo , Testes de Sensibilidade Microbiana , Microscopia de Força Atômica , Mutação , Micélio/efeitos dos fármacos , Fenótipo , Polissacarídeos/química , Reação em Cadeia da Polimerase em Tempo Real , Esporos Fúngicos/químicaRESUMO
Pneumocystis pneumonia remains an important complication of immune suppression. The cell wall of Pneumocystis has been demonstrated to potently stimulate host inflammatory responses, with most studies focusing on ß-glucan components of the Pneumocystis cell wall. In the current study, we have elaborated the potential role of chitins and chitinases in Pneumocystis pneumonia. We demonstrated differential host mammalian chitinase expression during Pneumocystis pneumonia. We further characterized a chitin synthase gene in Pneumocystis carinii termed Pcchs5, a gene with considerable homolog to the fungal chitin biosynthesis protein Chs5. We also observed the impact of chitinase digestion on Pneumocystis-induced host inflammatory responses by measuring TNFα release and mammalian chitinase expression by cultured lung epithelial and macrophage cells stimulated with Pneumocystis cell wall isolates in the presence and absence of exogenous chitinase digestion. These findings provide evidence supporting a chitin biosynthetic pathway in Pneumocystis organisms and that chitinases modulate inflammatory responses in lung cells. We further demonstrate lung expression of chitinase molecules during Pneumocystis pneumonia.
Assuntos
Quitina Sintase/metabolismo , Quitina/metabolismo , Quitinases/metabolismo , Proteínas Fúngicas/metabolismo , Pulmão/enzimologia , Pneumocystis carinii/patogenicidade , Pneumonia por Pneumocystis/fisiopatologia , Sequência de Aminoácidos , Animais , Parede Celular/enzimologia , Células Cultivadas , Quitina Sintase/química , Quitina Sintase/genética , Quitinases/química , Quitinases/genética , Células Epiteliais/enzimologia , Feminino , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Pulmão/citologia , Macrófagos/enzimologia , Macrófagos/imunologia , Dados de Sequência Molecular , Pneumocystis carinii/enzimologia , Pneumocystis carinii/genética , Pneumonia por Pneumocystis/microbiologia , Ratos , Análise de Sequência de DNA , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Plant infection by pathogenic fungi requires polarized secretion of enzymes, but little is known about the delivery pathways. Here, we investigate the secretion of cell wall-forming chitin synthases (CHSs) in the corn pathogen Ustilago maydis. We show that peripheral filamentous actin (F-actin) and central microtubules (MTs) form independent tracks for CHSs delivery and both cooperate in cell morphogenesis. The enzyme Mcs1, a CHS that contains a myosin-17 motor domain, is travelling along both MTs and F-actin. This transport is independent of kinesin-3, but mediated by kinesin-1 and myosin-5. Arriving vesicles pause beneath the plasma membrane, but only ~15% of them get exocytosed and the majority is returned to the cell centre by the motor dynein. Successful exocytosis at the cell tip and, to a lesser extent at the lateral parts of the cell requires the motor domain of Mcs1, which captures and tethers the vesicles prior to secretion. Consistently, Mcs1-bound vesicles transiently bind F-actin but show no motility in vitro. Thus, kinesin-1, myosin-5 and dynein mediate bi-directional motility, whereas myosin-17 introduces a symmetry break that allows polarized secretion.