RESUMO
An aptamer targeting gliotoxin (GTX) was optimized to increase the binding affinity by approximately 20 times and achieve higher structural stability and targeting specificity. Molecular dynamics simulations were used to explore the molecular mechanism and key action sites underlying the recognition of GTX by the optimized aptamer. Subsequently, the optimized aptamer was split into two fragments and a convenient and rapid one-pot assay for GTX detection was successfully established using a target-driven split aptamer recognition and assembly strategy. The method exhibited a good linear range of 0.128 nM to 2 µM, a low detection limit of 0.07 nM, and excellent selectivity for GTX. Furthermore, the method had good accuracy and stability in real sample analysis. Therefore, the developed one-pot method provides a reliable, convenient, and cost-effective approach for the widespread application of GTX detection.
Assuntos
Aptâmeros de Nucleotídeos , Gliotoxina , Aptâmeros de Nucleotídeos/química , Gliotoxina/química , Gliotoxina/análise , Limite de Detecção , Contaminação de Alimentos/análise , Técnicas Biossensoriais/métodos , Simulação de Dinâmica Molecular , AnimaisRESUMO
Using CRISPR-Cas9 technology and a microhomology-mediated end-joining repair system, we substituted genes of the gliotoxin pathway in Aspergillus fumigatus with genes responsible for chetomin biosynthesis from Chaetomium cochliodes, leading to the production of three new epipolythiodioxopiperazines (ETPs). This work represents the first successful endeavor to produce ETPs in a non-native host. Additionally, the simultaneous disruption of five genes in a single transformation marks the most extensive gene knockout event in filamentous fungi to date.
Assuntos
Aspergillus fumigatus , Gliotoxina , Piperazinas , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/genética , Piperazinas/química , Piperazinas/metabolismo , Gliotoxina/biossíntese , Gliotoxina/química , Estrutura Molecular , Chaetomium/metabolismo , Chaetomium/química , Sistemas CRISPR-CasRESUMO
Gliotoxin can be developed as potent biopesticide. In this study, the positive transcriptional factor gliZ, glutathione-S transferase encoding gene gliG and gliN were firstly deleted by CRISPR/Cas9 system, which abolished the production of gliotoxin-like compounds in Dichotomomyces cejpii. CRISPR/dCas9 system targeting promoter of gliG was used to activate the biosynthetic genes in gli cluster. The overexpression of gliZ, gliN and gliG can significantly improve the yield of gliotoxin-like compunds. The gliotoxin yields was improved by 16.38 ± 1.36 fold, 18.98 ± 1.28 fold through gliZ overexpression and gliM deletion in D. cejpii FS110. In addtion, gliN was heterologously expressed in E. coli, the purified GliN can catalyze gliotoxin into methyl-gliotoxin. Furthermore, the binding sequences of GliZ in the promoters of gliG was determined by Dnase footprinting. This study firstly illustrated the transcriptional regulatory mechanism of DcGliZ for the gliotoxin biosynthesis in D. cejpii, and improved the yields of gliotoxins significantly in D. cejpii via biosynthetic approaches.
Assuntos
Gliotoxina , Gliotoxina/química , Gliotoxina/metabolismo , Vias Biossintéticas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fungos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Glutathione-S-transferases (GSTs) usually detoxify xenobiotics. The human pathogenic fungus Aspergillus fumigatus however uses the exceptional GST GliG to incorporate two sulfur atoms into its virulence factor gliotoxin. Because these sulfurs are essential for biological activity, glutathionylation is a key step of gliotoxin biosynthesis. Yet, the mechanism of carbon-sulfur linkage formation from a bis-hydroxylated precursor is unresolved. Here, we report structures of GliG with glutathione (GSH) and its reaction product cyclo[-l-Phe-l-Ser]-bis-glutathione, which has been purified from a genetically modified A. fumigatus strain. The structures argue for stepwise processing of first the Phe and second the Ser moiety. Enzyme-mediated dehydration of the substrate activates GSH and a helix dipole stabilizes the resulting anion via a water molecule for the nucleophilic attack. Activity assays with mutants validate the interactions of GliG with the ligands and enrich our knowledge about enzymatic C-S bond formation in gliotoxin and epipolythiodioxopiperazine (ETP) natural compounds in general.
Assuntos
Carbono/metabolismo , Gliotoxina/biossíntese , Enxofre/metabolismo , Aspergillus fumigatus/metabolismo , Carbono/química , Gliotoxina/química , Glutationa/química , Glutationa/metabolismo , Estrutura Molecular , Enxofre/químicaRESUMO
Six julichrome derivatives including a new monomeric julichrome named as julichrome Q10 (1), and previous reported julichrome Q6 (2), julichrome Q6.6 (4), julichrome Q3.5 (5), julichrome Q5.6 (6), julichrome Q2.3 (7), along with a diketopiperazine gliotoxin (3) were isolated from a soil derived strain Streptomyces sp. The structures of these compounds were identified by HR-ESI-MS, UV, IR and NMR methods. The isolated compounds were tested for their in vitro cytotoxicity against human hepatocarcinoma HepG-2 and SMMC-7721 cell lines, human breast cancer MCF-7 and MDA-MB-231 cell lines, and human normal heptical LO2 cell line. Gliotoxin (3) showed the most cytotoxic activity against the tested tumor cell lines, with IC50 values ranging from 0.11 to 1.45 µM. Julichrome Q6.6 (4) displayed selective cytotoxic activity against SMMC-7721, MCF-7 and MDA-MB-231 cell lines.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Gliotoxina/farmacologia , Streptomyces/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , China , Ensaios de Seleção de Medicamentos Antitumorais , Gliotoxina/química , Gliotoxina/isolamento & purificação , Células Hep G2 , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Microbiologia do Solo , Espectrometria de Massas por Ionização por Electrospray , Streptomyces/isolamento & purificaçãoRESUMO
Gliotoxin and related epidithiodiketopiperazines (ETP) from diverse fungi feature highly functionalized hydroindole scaffolds with an array of medicinally and ecologically relevant activities. Mutation analysis, heterologous reconstitution, and biotransformation experiments revealed that a cytochrome P450 monooxygenase (GliF) from the human-pathogenic fungus Aspergillus fumigatus plays a key role in the formation of the complex heterocycle. In vitro assays using a biosynthetic precursor from a blocked mutant showed that GliF is specific to ETPs and catalyzes an unprecedented heterocyclization reaction that cannot be emulated with current synthetic methods. In silico analyses indicate that this rare biotransformation takes place in related ETP biosynthetic pathways.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Gliotoxina/biossíntese , Biocatálise , Ciclização , Gliotoxina/química , Estrutura MolecularRESUMO
Dechdigliotoxins A-C (1-3), which represented the first examples of gliotoxin dimers with an unprecedented exocyclic disulfide linkage, were obtained from a deep-sea derived fungus Dichotomomyces cejpii FS110. The structures of these compounds were elucidated on the basis of spectroscopic analysis and the absolute configurations were unambiguously determined through quantum chemical calculations, as well as DP4+ probability simulations. The proposed biosynthetic pathway suggested 1-3 were generated from unusual L-Phe and D-Ser. All the isolates were evaluated for their cytotoxicity against four tumor cell lines.
Assuntos
Aspergillus/química , Gliotoxina/química , Linhagem Celular Tumoral , Gliotoxina/farmacologia , Células Hep G2 , Humanos , Células MCF-7RESUMO
Cyclization of linear dipeptidyl precursors derived from nonribosomal peptide synthetases (NRPSs) into 2,5-diketopiperazines (DKPs) is a crucial step in the biosynthesis of a large number of bioactive natural products. However, the mechanism of DKP formation in fungi has remained unclear, despite extensive studies of their biosyntheses. Here we show that DKP formation en route to the fungal virulence factor gliotoxin requires a seemingly extraneous couplet of condensation (C) and thiolation (T) domains in the NRPS GliP. In vivo truncation of GliP to remove the CT couplet or just the T domain abrogated production of gliotoxin and all other gli pathway metabolites. Point mutation of conserved active sites in the C and T domains diminished cyclization activity of GliP in vitro and abolished gliotoxin biosynthesis in vivo. Verified NRPSs of other fungal DKPs terminate with similar CT domain couplets, suggesting a conserved strategy for DKP biosynthesis by fungal NRPSs.
Assuntos
Aspergillus fumigatus/metabolismo , Dicetopiperazinas/metabolismo , Gliotoxina/biossíntese , Dicetopiperazinas/química , Regulação Fúngica da Expressão Gênica , Gliotoxina/química , Estrutura MolecularRESUMO
PURPOSE: The neutrophil-derived oxidant N-chlorotaurine (NCT) displays remarkable in vivo tolerability and efficacy against a range of pathogens. The aim of this study was to characterize the response of the pulmonary pathogen Aspergillus fumigatus to NCT. METHODOLOGY: The effect of NCT on the growth and viability of A. fumigatus was characterized. NCT-induced alteration of amino acids and gliotoxin from A. fumigatus mycelium was assessed. Label-free shotgun quantitative proteomic analysis was performed on A. fumigatus exposed to NCT for 24 h. RESULTS: Incubation of A. fumigatus with NCT at concentrations ranging from 6.8 to 55 mM decreased conidial growth and viability, and mycelium biomass relative to the controls. Exposure to NCT (13.77 mM) resulted in increased amino acids and gliotoxin levels from A. fumigatus mycelium. Exposure of A. fumigatus mycelium to NCT (6.8 mM) revealed an enrichment in proteins associated with the ribosome, transcription and translation and non-ribosomal peptide biosynthesis (e.g. Pes1, Pes3), which play an essential role in oxidative stress resistance in A. fumigatus. A decrease in the abundance of proteins associated with fumagillin and pseurotin biosynthesis highlighted the anti-virulence activity of NCT. CONCLUSION: These results indicate that NCT induces an oxidative stress response in A. fumigatus as evidenced by alterations in the proteome and inhibits conidial and mycelial growth. Clinical investigations of topical application of NCT to treat Aspergillus infections are encouraged.
Assuntos
Aspergillus fumigatus/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Taurina/análogos & derivados , Aminoácidos/química , Aminoácidos/efeitos dos fármacos , Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/metabolismo , Cromatografia Líquida de Alta Pressão , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Gliotoxina/análise , Gliotoxina/química , Humanos , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Permeabilidade/efeitos dos fármacos , Proteômica , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Esporos Fúngicos/efeitos dos fármacos , Taurina/farmacologiaRESUMO
Gliotoxin is an important epipolythiodioxopiperazine, which was biosynthesized by the gli gene cluster in Aspergillus genus. However, the regulatory mechanism of gliotoxin biosynthesis remains unclear. In this study, a novel Zn2Cys6 transcription factor DcGliZ that is responsible for the regulation of gliotoxin biosynthesis from the deep-sea-derived fungus Dichotomomyces cejpii was identified. DcGliZ was expressed in Escherichia coli and effectively purified from inclusion bodies by refolding. Using electrophoretic mobility shift assay, we demonstrated that purified DcGliZ can bind to gliG, gliM, and gliN promoter regions in the gli cluster. Furthermore, the binding kinetics and affinity of DcGliZ protein with different promoters were measured by surface plasmon resonance assays, and the results demonstrated the significant interaction of DcGliZ with the gliG, gliM, and gliN promoters. These new findings would lay the foundation for the elucidation of future gliotoxin biosynthetic regulation mechanisms in D. cejpii.
Assuntos
Fungos/genética , Gliotoxina/biossíntese , Família Multigênica/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Fungos/metabolismo , Gliotoxina/químicaRESUMO
Reduced-gliotoxin is a small molecule derived from the secondary metabolites of marine fungi; compared to other gliotoxin analogues, it exhibits potent anticancer effects. However, the molecular basis of the death of colorectal cancer (CRC) cells induced by reduced-gliotoxin is unclear. Thus, the aim of this study was to investigate the potency of reduced-gliotoxin against CRC cells and to elucidate the underlying mechanisms. Cell morphology, flow cytometric analysis and western bolt analysis were performed to examine the functions and mechanisms of cell death induced by reduced-gliotoxin. Our findings demonstrated that reduced-gliotoxin triggered rapid cell detachment and induced anoikis in CRC cells. Mechanistically, our data indicated that the anoikis induced by reduced-gliotoxin was associated with the disruption of integrin-associated cell detachment and multiple signaling pathways. Furthermore, reduced-gliotoxin induced the excessive production of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP), resulting in the activation of both endogenous and exogenous apoptotic pathways and eventually, in the apoptosis of CRC cells. The blockage of ROS generation with N-acetylcysteine (NAC) attenuated the anoikis induced by reduced-gliotoxin. Taken together, these results suggest that reduced-gliotoxin may prove to be a potential candidate in the treatment of CRC.
Assuntos
Anoikis/efeitos dos fármacos , Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Gliotoxina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/patologia , Gliotoxina/química , Gliotoxina/uso terapêutico , Células HCT116 , Células HT29 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
The formation of glutathione (GSH) conjugates, best known from the detoxification of xenobiotics, is a widespread strategy to incorporate sulfur into biomolecules. The biosynthesis of gliotoxin, a virulence factor of the human pathogenic fungus Aspergillus fumigatus, involves attachment of two GSH molecules and their sequential decomposition to yield two reactive thiol groups. The degradation of the GSH moieties requires the activity of the Cys-Gly carboxypeptidase GliJ, for which we describe the X-ray structure here. The enzyme forms a homodimer with each monomer comprising one active site. Two metal ions are present per proteolytic center, thus assigning GliJ to the diverse family of dinuclear metallohydrolases. Depending on availability, Zn2+, Fe2+, Fe3+, Mn2+, Cu2+, Co2+, or Ni2+ ions are accepted as cofactors. Despite this high metal promiscuity, a preference for zinc versus iron and manganese was noted. Mutagenesis experiments revealed details of metal coordination, and molecular modeling delivered insights into substrate recognition and processing by GliJ. The latter results suggest a reaction mechanism in which the two scissile peptide bonds of one gliotoxin precursor molecule are hydrolyzed sequentially and in a given order.
Assuntos
Carboxipeptidases/química , Carboxipeptidases/metabolismo , Gliotoxina/biossíntese , Metais/metabolismo , Modelos Moleculares , Fenômenos Bioquímicos , Domínio Catalítico , Cristalografia por Raios X , Gliotoxina/química , Metais/química , Estrutura Molecular , Dobramento de Proteína , Especificidade por SubstratoRESUMO
Eleven diketopiperazine and fumiquinazoline alkaloids (1-11) together with a tetracyclic triterpenoid helvolic acid (12) were obtained from the cultures of a deep-sea derived fungus Aspergillus sp. SCSIO Ind09F01. The structures of these compounds (1-12) were determined mainly by the extensive NMR, ESIMS spectra data and by comparison with previously described compounds. Besides, anti-tuberculosis, cytotoxic, antibacterial, COX-2 inhibitory and antiviral activities of these compounds were evaluated. Gliotoxin (3), 12,13-dihydroxy-fumitremorgin C (11) and helvolic acid (12) exhibited very strong anti-tuberculosis activity towards Mycobacterium tuberculosis with the prominent MIC50 values of <0.03, 2.41 and 0.894 µM, respectively, which was here reported for the first time. Meanwhile gliotoxin also displayed significant selective cytotoxicities against K562, A549 and Huh-7 cell lines with the IC50 values of 0.191, 0.015 and 95.4 µM, respectively.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Aspergillus/química , Alcaloides/química , Alcaloides/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Antivirais/química , Antivirais/farmacologia , Organismos Aquáticos , Avaliação Pré-Clínica de Medicamentos/métodos , Ácido Fusídico/análogos & derivados , Ácido Fusídico/química , Ácido Fusídico/farmacologia , Gliotoxina/química , Gliotoxina/farmacologia , Humanos , Células K562 , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacosRESUMO
Ergothioneine (EGT; 2-mercaptohistidine trimethylbetaine) is a trimethylated and sulphurised histidine derivative which exhibits antioxidant properties. Here we report that deletion of Aspergillus fumigatus egtA (AFUA_2G15650), which encodes a trimodular enzyme, abrogated EGT biosynthesis in this opportunistic pathogen. EGT biosynthetic deficiency in A. fumigatus significantly reduced resistance to elevated H2O2 and menadione, respectively, impaired gliotoxin production and resulted in attenuated conidiation. Quantitative proteomic analysis revealed substantial proteomic remodelling in ΔegtA compared to wild-type under both basal and ROS conditions, whereby the abundance of 290 proteins was altered. Specifically, the reciprocal differential abundance of cystathionine γ-synthase and ß-lyase, respectively, influenced cystathionine availability to effect EGT biosynthesis. A combined deficiency in EGT biosynthesis and the oxidative stress response regulator Yap1, which led to extreme oxidative stress susceptibility, decreased resistance to heavy metals and production of the extracellular siderophore triacetylfusarinine C and increased accumulation of the intracellular siderophore ferricrocin. EGT dissipated H2O2 in vitro, and elevated intracellular GSH levels accompanied abrogation of EGT biosynthesis. EGT deficiency only decreased resistance to high H2O2 levels which suggests functionality as an auxiliary antioxidant, required for growth at elevated oxidative stress conditions. Combined, these data reveal new interactions between cellular redox homeostasis, secondary metabolism and metal ion homeostasis.
Assuntos
Aspergillus fumigatus/metabolismo , Ergotioneína/biossíntese , Proteínas Fúngicas/metabolismo , Antioxidantes/química , Aspergillus fumigatus/genética , Carbono-Oxigênio Liases/metabolismo , Compostos Férricos/química , Ferricromo/análogos & derivados , Ferricromo/metabolismo , Proteínas Fúngicas/genética , Deleção de Genes , Teste de Complementação Genética , Gliotoxina/química , Glutationa/metabolismo , Histidina/química , Peróxido de Hidrogênio/química , Ácidos Hidroxâmicos/química , Liases/metabolismo , Metais Pesados/química , Oxirredução , Estresse Oxidativo , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismo , Sideróforos/química , Vitamina K 3/químicaRESUMO
Gliotoxin biosynthesis is encoded by the gli gene cluster in Aspergillus fumigatus. The biosynthesis of gliotoxin is influenced by a suite of transcriptionally-active regulatory proteins and a bis-thiomethyltransferase. A self-protection system against gliotoxin is present in A. fumigatus. Several additional metabolites are also produced via the gliotoxin biosynthetic pathway. Moreover, the biosynthesis of unrelated natural products appears to be influenced either by gliotoxin or by the activity of specific reactions within the biosynthetic pathway. The activity of gliotoxin against animal cells and fungi, often mediated by interference with redox homeostasis or protein modification, is revealing new metabolic interactions within eukaryotic systems. Nature has provided a most useful natural product with which to reveal some of its many molecular secrets.
Assuntos
Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidade , Gliotoxina/metabolismo , Animais , Aspergillus fumigatus/genética , Produtos Biológicos/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Gliotoxina/biossíntese , Gliotoxina/química , Gliotoxina/farmacologia , Metaboloma/fisiologia , Família MultigênicaRESUMO
The cytotoxic activities of extracts (50â µg/ml) from 48 fungal strains, recovered from sediments of Pecém's offshore port terminal (Northeast coast of Brazil), against HCT-116 colon cancer cell lines were investigated. The most promising extract was obtained from strain BRF082, identified as Dichotomomyces cejpii by phylogenetic analyses of partial RPB2 gene sequence. Thus, it was selected for bioassay-guided isolation of the cytotoxic compounds. Large-scale fermentation of BRF082 in potato dextrose broth, followed by chromatographic purification of the bioactive fractions from the liquid medium, yielded gliotoxin (4) and its derivatives acetylgliotoxin G (3), bis(dethio)bis(methylsulfanyl)gliotoxin (1), acetylgliotoxin (5), 6-acetylbis(dethio)bis(methylsulfanyl)gliotoxin (2), besides the quinazolinone alkaloid fiscalin B. All isolated compounds were tested for their cytotoxicities against the tumor cell lines HCT-116, revealing 4 and 3 as the most cytotoxic ones (IC50 0.41 and 1.06â µg/ml, resp.).
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Fungos/química , Sedimentos Geológicos/microbiologia , Antineoplásicos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Brasil , Neoplasias do Colo/tratamento farmacológico , Fungos/genética , Gliotoxina/análogos & derivados , Gliotoxina/química , Gliotoxina/isolamento & purificação , Gliotoxina/farmacologia , Células HCT116 , Humanos , Indóis/química , Indóis/isolamento & purificação , Indóis/farmacologia , Filogenia , Quinazolinas/química , Quinazolinas/isolamento & purificação , Quinazolinas/farmacologiaRESUMO
A linear ubiquitin chain, which consists of ubiquitin molecules linked via their N- and C-termini, is formed by a linear ubiquitin chain assembly complex (LUBAC) composed of HOIP, HOIL-1L, and SHARPIN, and conjugation of a linear ubiquitin chain on the NF-κB essential modulator (NEMO) is deeply involved in NF-κB activation induced by various signals. Since abnormal activation of NF-κB is associated with inflammatory disease and malignancy, we searched for an inhibitor of LUBAC by high-throughput screening (HTS) with a Tb(3+)-fluorescein FRET system. As a result, we found that the fungal metabolite gliotoxin inhibits LUBAC selectively by binding to the RING-IBR-RING domain of HOIP, the catalytic center of LUBAC. Gliotoxin has been well-known as an inhibitor of NF-κB activation, though its action mechanism has remained elusive. Here, we show that gliotoxin inhibits signal-induced NF-κB activation by selectively inhibiting LUBAC-mediated linear ubiquitin chain formation.
Assuntos
Gliotoxina/farmacologia , Quinase I-kappa B/antagonistas & inibidores , Imunossupressores/farmacologia , NF-kappa B/antagonistas & inibidores , Ubiquitina/antagonistas & inibidores , Relação Dose-Resposta a Droga , Fluoresceína/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Regulação da Expressão Gênica , Gliotoxina/química , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Imunossupressores/química , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , NF-kappa B/genética , NF-kappa B/imunologia , Transdução de Sinais , Térbio/química , Fatores de Transcrição , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina/genética , Ubiquitina/imunologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação/efeitos dos fármacos , Ubiquitinas/genética , Ubiquitinas/imunologiaRESUMO
The production of fungal metabolites can be remarkably influenced by various cultivation parameters. To explore the biosynthetic potentials of the marine fungus, Neosartorya pseudofischeri, which was isolated from the inner tissue of starfish Acanthaster planci, glycerol-peptone-yeast extract (GlyPY) and glucose-peptone-yeast extract (GluPY) media were used to culture this fungus. When cultured in GlyPY medium, this fungus produced two novel diketopiperazines, neosartins A and B (1 and 2), together with six biogenetically-related known diketopiperazines,1,2,3,4-tetrahydro-2, 3-dimethyl-1,4-dioxopyrazino[1,2-a]indole (3), 1,2,3,4-tetrahydro-2-methyl-3-methylen e-1,4-dioxopyrazino[1,2-a]indole (4), 1,2,3,4-tetrahydro-2-methyl-1,3,4-trioxopyrazino[1,2-a] indole (5), 6-acetylbis(methylthio)gliotoxin (10), bisdethiobis(methylthio)gliotoxin (11), didehydrobisdethiobis(methylthio)gliotoxin (12) and N-methyl-1H-indole-2-carboxamide (6). However, a novel tetracyclic-fused alkaloid, neosartin C (14), a meroterpenoid, pyripyropene A (15), gliotoxin (7) and five known gliotoxin analogues, acetylgliotoxin (8), reduced gliotoxin (9), 6-acetylbis(methylthio)gliotoxin (10), bisdethiobis(methylthio) gliotoxin (11) and bis-N-norgliovictin (13), were obtained when grown in glucose-containing medium (GluPY medium). This is the first report of compounds 3, 4, 6, 9, 10 and 12 as naturally occurring. Their structures were determined mainly by MS, 1D and 2D NMR data. The possible biosynthetic pathways of gliotoxin-related analogues and neosartin C were proposed. The antibacterial activity of compounds 2-14 and the cytotoxic activity of compounds 4, 5 and 7-13 were evaluated. Their structure-activity relationships are also preliminarily discussed.
Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Neosartorya/metabolismo , Estrelas-do-Mar/microbiologia , Alcaloides/química , Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Dicetopiperazinas/química , Dicetopiperazinas/isolamento & purificação , Dicetopiperazinas/farmacologia , Gliotoxina/química , Gliotoxina/isolamento & purificação , Gliotoxina/farmacologia , Humanos , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Neosartorya/isolamento & purificação , Metabolismo Secundário , Relação Estrutura-AtividadeRESUMO
Gliotoxin is a redox-active nonribosomal peptide produced by Aspergillus fumigatus. Like many other disulfide-containing epipolythiodioxopiperazines, a bis-thiomethylated form is also produced. In the case of gliotoxin, bisdethiobis(methylthio)gliotoxin (BmGT) is formed for unknown reasons by a cryptic enzyme. Here, we identify the S-adenosylmethionine-dependent gliotoxin bis-thiomethyltransferase (GtmA), which converts dithiogliotoxin to BmGT. This activity, which is induced by exogenous gliotoxin, is only detectable in protein lysates of A. fumigatus deficient in the gliotoxin oxidoreductase, gliT. Thus, GtmA is capable of substrate bis-thiomethylation. Deletion of gtmA completely abrogates BmGT formation and we now propose that the purpose of BmGT formation is primarily to attenuate gliotoxin biosynthesis. Phylogenetic analysis reveals 124 GtmA homologs within the Ascomycota phylum. GtmA is encoded outside the gliotoxin biosynthetic cluster and primarily serves to negatively regulate gliotoxin biosynthesis. This mechanism of postbiosynthetic regulation of nonribosomal peptide synthesis appears to be quite unusual.
Assuntos
Aspergillus fumigatus/metabolismo , Gliotoxina/biossíntese , Oxirredutases/metabolismo , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Aspergillus fumigatus/enzimologia , Gliotoxina/análogos & derivados , Gliotoxina/química , Metilação , Conformação Molecular , Oxirredutases/genética , Oxirredutases/isolamento & purificação , FilogeniaRESUMO
Gliotoxin (1), a virulence factor of the human pathogenic fungus Aspergillus fumigatus, is the prototype of epipoly(thiodioxopiperazine) (ETP) toxins. Here we report the discovery and functional analysis of two methyl transferases (MTs) that play crucial roles for ETP toxicity. Genome comparisons, knockouts, and in vitro enzyme studies identified a new S-adenosyl-l-methionine-dependent S-MT (TmtA) that is, surprisingly, encoded outside the gli gene cluster. We found that TmtA irreversibly inactivates ETP by S-alkylation and that this detoxification strategy appears to be not only limited to ETP producers. Furthermore, we unveiled that GliN functions as a freestanding amide N-MT. GliN-mediated amide methylation confers stability to ETP, damping the spontaneous formation of tri- and tetrasulfides. In addition, enzymatic N-alkylation constitutes the last step in gliotoxin biosynthesis and is a prerequisite for the cytotoxicity of the molecule. Thus, these specialized alkylating enzymes have dramatic and fully opposed effects: complete activation or inactivation of the toxin.