RESUMO
L-amino acid oxidase (ThLAAO) secreted by Trichoderma harzianum ETS323 is a ï¬avoenzyme with antimicrobial characteristics. In this study, we transformed the ThLAAO gene into tobacco to elucidate whether ThLAAO can activate defense mechanisms and confer resistance against phytopathogens. Transgenic tobacco overexpressing ThLAAO showed enhanced resistance against Sclerotinia sclerotiorum and Botrytis cinerea and activated the expression of defense-related genes and the genes involved in salicylic acid, jasmonic acid, and ethylene biosynthesis accompanied by substantial accumulation of H2O2 in chloroplasts, cytosol around chloroplasts, and cell membranes of transgenic tobacco. Scavenge of H2O2 with ascorbic acid abolished disease resistance against B. cinerea infection and decreased the expression of defense-related genes. ThLAAO-FITC application on tobacco protoplast or overexpression of ThLAAO-GFP in tobacco revealed the localization of ThLAAO in chloroplasts. Chlorophyll a/b binding protein (CAB) was isolated through ThLAAO-ConA affinity chromatography. The pull down assay results confirmed ThLAAO-CAB binding. Application of ThLAAO-Cy5.5 on cabbage roots promptly translocated to the leaves. Treatment of ThLAAO on cabbage roots induces systemic resistance against B. cinerea. Overall, these results demonstrate that ThLAAO may target chloroplast and activate defense mechanisms via H2O2 signaling to confer resistance against S. sclerotiorum and B. cinerea.
Assuntos
Ascomicetos , Botrytis , Resistência à Doença/genética , Proteínas Fúngicas/genética , Hypocreales/genética , L-Aminoácido Oxidase/genética , Nicotiana/imunologia , Doenças das Plantas/imunologia , Proteínas Fúngicas/fisiologia , Peróxido de Hidrogênio/metabolismo , Hypocreales/enzimologia , L-Aminoácido Oxidase/fisiologia , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Nicotiana/genética , Nicotiana/microbiologiaRESUMO
To investigate the molecular mechanism of Trichoderma L-amino acid oxidase (Th-LAAO) in protecting and in promoting growth of cabbage infected with Botrytis cinerea, a three-way interaction system was established. Cabbage leaves treated with purified Th-LAAO significantly constrained damaged leaf area caused by B. cinerea infection. In response to Th-LAAO treatment, the expression levels of genes involved in photosynthesis, such as ribulose-1,5-bisphosphate carboxylase oxygenase, Rubisco activase, and ATP synthase increased 2.54, 2.18, and 1.41 folds, respectively. The transcription levels of sucrose transport protein 1 increased 7.6 fold. As to the expression of defense-related genes, the transcription level of ascorbate peroxidase increased 1.46 fold. On the contrary, pathogenesis-related protein 1, chitinase, ß-1,3 glucanase, and glutathione S-transferase decreased significantly. Overall, the results indicated that Th-LAAO may stimulate CO2 fixation and sucrose transport and elicit host defense responses in cabbage against B. cinerea, and this elicitation of defense response is likely to contribute to induced systemic resistance of host plant.
Assuntos
Brassica , Resistência à Doença , L-Aminoácido Oxidase , Trichoderma , Botrytis/fisiologia , Brassica/efeitos dos fármacos , Brassica/genética , Brassica/microbiologia , Resistência à Doença/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , L-Aminoácido Oxidase/isolamento & purificação , L-Aminoácido Oxidase/farmacologia , Fotossíntese/efeitos dos fármacos , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/microbiologia , Trichoderma/química , Trichoderma/enzimologiaRESUMO
A new clade, Trichoderma formosa, secretes eliciting plant response-like 1 (Epl1), a small peptide elicitor that stimulates plant immunity. Nicotiana benthamiana pretreated with Epl1 for 3 days developed immunity against Tomato mosaic virus (ToMV) infection. The transcriptome profiles of T. formosa and N. benthamiana were obtained by deep sequencing; the transcript of Epl1 is 736 nt in length and encodes a 12-kDa peptide. Identifying critical genes in Epl1-mediated immunity was challenging due to high similarity between the transcriptome expression profiles of Epl1-treated and ToMV-infected N. benthamiana samples. Therefore, an efficient bioinformatics data mining approach was used for high-throughput transcriptomic assays in this study. We integrated gene-to-gene network analysis into the ContigViews transcriptome database, and genes related to jasmonic acid and ethylene signaling, salicylic acid signaling, leucine-rich repeats, transcription factors, and histone variants were hubs in the gene-to-gene networks. In this study, the Epl1 of T. formosa triggers plant immunity against various pathogen infections. Moreover, we demonstrated that high-throughput data mining and gene-to-gene network analysis can be used to identify critical candidate genes for further studies on the mechanisms of plant immunity.
Assuntos
Proteínas Fúngicas/farmacologia , Redes Reguladoras de Genes , Nicotiana/metabolismo , Doenças das Plantas/imunologia , Imunidade Vegetal/genética , Trichoderma/imunologia , Sequência de Bases , DNA Fúngico , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Regulação da Expressão Gênica de Plantas/imunologia , Imunidade Inata , Modelos Moleculares , Filogenia , Proteínas de Plantas/genética , Conformação Proteica , Nicotiana/genética , Nicotiana/imunologia , Trichoderma/genéticaRESUMO
To document the safety of pachybasin, a secondary metabolite of Trichoderma harzianum, for use as a bioagricultural agent, it was subjected to general toxicological testing in mice and developmental toxicity in zebrafish. With either 5 or 20 mg kg-1 pachybasin i.p. injection, mice behavioral responses such as motor coordination, spontaneous locomotor activity, or nociceptive pain were not influenced. In long-term effect (daily injection for 14 days), the physiological, hematological, liver, and kidney functions were not altered either. Evidence for the developmental toxicity of pachybasin (10-100 µM) in 72-h exposure period was shown in zebrafish larvae, based on developmental retardation, impairment of chorion, and increase of mortality. In summary, there are no significant general toxicities presented in the pachybasin-treated adult male mice. However, the embryo-toxicity in aquatic biota should be taken into consideration during bioagricultural agent application.
Assuntos
Antraquinonas/toxicidade , Fungicidas Industriais/toxicidade , Camundongos/crescimento & desenvolvimento , Trichoderma/química , Peixe-Zebra/crescimento & desenvolvimento , Animais , Antraquinonas/química , Antraquinonas/metabolismo , Fungicidas Industriais/metabolismo , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Masculino , Atividade Motora/efeitos dos fármacos , Metabolismo Secundário , Trichoderma/metabolismoRESUMO
Uncontrolled cell proliferation is a common feature of human cancer. Some of herbal extract or plant-derived medicine had been shown as an important source of effective anticancer agents. We previously reported that an n-BuOH-soluble fraction of Kalanchoe tubiflora has antiproliferative activity by inducing mitotic catastrophe. In this study, we showed that the H2 O-soluble fraction of Kalanchoe tubiflora (KT-W) caused cell cycle arrest, and senescence-inducing activities in A549 cells. We used 2 dimensional PAGE to analyze the protein expression levels after KT-W treatment, and identified that the energy metabolism-related proteins and senescence-related proteins were disturbed. In vivo experiments showed that the tumor growths in A549-xenografted nude mice were effectively inhibited by KT-W. Our findings implied that KT-W is a putative antitumor agent by inducing cell cycle arrest and senescence. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1663-1673, 2016.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Kalanchoe , Neoplasias Pulmonares/tratamento farmacológico , Extratos Vegetais/farmacologia , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , Fitoterapia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The iac locus is involved in indole-3-acetic acid (IAA) catabolism in Acinetobacter baumannii. Nine structural genes of iac are transcribed in the same direction, whereas iacR, which encodes a MarR-type transcriptional regulator, is transcribed in the opposite direction. The IacA protein, which is encoded by the second structural gene of the iac locus, is expressed in an IAA-dependent manner. Here, we characterized gene expression from this locus in wild type A. baumannii and in an iacR mutant; this revealed that the iacH promoter is negatively regulated by IacR. The transcriptional site of iacH was determined by using 5' rapid amplification of cDNA ends; one IacR-binding site was identified between positions -35 and +28 of the iacH promoter. Sequence analysis and an electrophoretic mobility shift assay indicated that recombinant IacR binds specifically to a sequence with dyad symmetry in the iacR-iacH overlapping promoters in the absence of IAA. In addition, a two-plasmid expression system in Escherichia coli showed that IAA probably serves as a ligand that binds to IacR and releases it from the iacH promoter, thereby allowing RNA polymerase to transcribe iac. Thus, iac is expressed in order to promote IAA degradation, whereas free IacR is required for iac repression. We conclude that IacR serves as a key regulator of IAA degradation in A. baumannii in the rhizosphere. These results provide new insights into the possible role of A. baumannii in the environment.
Assuntos
Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Ácidos Indolacéticos/metabolismo , Óperon , Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Dados de Sequência Molecular , Regiões Promotoras GenéticasRESUMO
Brassica oleracea deoxycytidine deaminase (BoDCD), a deoxycytidine deaminase (DCD, EC 3.5.4.14) enzyme, is known to play an important role in the Trichoderma harzianum ETS 323 mediated resistance mechanism in young leaves of B. oleracea var. capitata during Rhizoctonia solani infection. BoDCD potentially neutralizes cytotoxic products of host lipoxygenase activity, and thereby BoDCD restricts the hypersensitivity-related programmed cell death induced in plants during the initial stages of infection. To determine the biochemical characteristics and to partially elucidate the designated functional properties of BoDCD, the enzyme was cloned into an Escherichia coli expression system, and its potential to neutralize the toxic analogues of 2'-deoxycytidine (dC) was examined. BoDCD transformants of E. coli cells were found to be resistant to 2'-deoxycytidine analogues at all of the concentrations tested. The BoDCD enzyme was also overexpressed as a histidine-tagged protein and purified using nickel chelating affinity chromatography. The molecular weight of BoDCD was determined to be 20.8 kDa as visualized by SDS-PAGE. The substrate specificity and other kinetic properties show that BoDCD is more active in neutralizing cytotoxic cytosine ß-d-arabinofuranoside than in deaminating 2'-deoxycytinde to 2'-deoxyuridine in nucleic acids or in metabolizing cytidine to uridine. The optimal temperature and pH of the enzyme were 27 °C and 7.5. The Km and Vmax values of BoDCD were, respectively, 91.3 µM and 1.475 mM for its natural substrate 2'-deoxycytidine and 63 µM and 2.072 mM for cytosine ß-d-arabinofuranoside. The phenomenon of neutralization of cytotoxic dC analogues by BoDCD is discussed in detail on the basis of enzyme biochemical properties.
Assuntos
Brassica/enzimologia , Nucleosídeo Desaminases/química , Proteínas de Plantas/química , Brassica/química , Brassica/genética , Citidina/metabolismo , Citidina Desaminase , Citosina/metabolismo , Cinética , Peso Molecular , Nucleosídeo Desaminases/genética , Nucleosídeo Desaminases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especificidade por Substrato , Uridina/metabolismoRESUMO
Acute myeloid leukemia (AML), a very rare type of cancer, generally affects patients over 50 years old. While clinical drugs to treat advanced stages of AML exist, the disease becomes increasingly resistant to therapies. Euphorbia formosana Hayata (EF) is a native Taiwanese medicinal plant used to treat rheumatism, liver cirrhosis, herpes zoster, scabies, and photoaging, along with tumor suppression. However, the mechanisms by which it suppresses tumors have not been explored. Here, we provide molecular evidence that a hot-water extract of Euphorbia formosana (EFW) selectively inhibited the growth of human leukemic cancer cells more than other solid human cancer cell lines. Most importantly, the plant extract had limited toxicity toward healthy peripheral blood mononuclear cells (PBMCs). After THP-1 leukemic cells were treated with 50-100 µg/mL EFW for one day, the S phase DNA content of the cells increased, while treatment with 200-400 µg/mL caused the cells to accumulate in the G0/G1 phase. Notably, EFW did not affect A-549 lung cancer cells. The effectiveness of EFW against THP-1 cells may be through caspase-dependent apoptosis in leukemic cells, which is mediated through the Fas and mitochondrial pathways. The potent antileukemic activity of EFW in vitro warrants further investigation of this plant to treat leukemias and other malignancies.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Euphorbia/química , Leucemia/patologia , Mitocôndrias/metabolismo , Extratos Vegetais/farmacologia , Receptor fas/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia/enzimologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Raízes de Plantas/química , Transdução de Sinais/efeitos dos fármacos , ÁguaRESUMO
Plant interactions with microbial biocontrol agents are used as experimental models to understand resistance-related molecular adaptations of plants. In a hydroponic three-way interaction study, a novel Trichoderma harzianum ETS 323 mediated mechanism was found to induce resistance to Rhizoctonia solani infection in Brassica oleracea var. capitata plantlets. The R. solani challenge on leaves initiate an increase in lipoxygenase activity and associated hypersensitive tissue damage with characteristic "programmed cell death" that facilitate the infection. However, B. oleracea plantlets whose roots were briefly (6 h) colonized by T. harzianum ETS 323 developed resistance to R. solani infection through a significant reduction of the host hypersensitive tissue damage. The resistance developed in the distal leaf tissue was associated with the expression of a H(2)O(2)-inducible glutathione S-transferase (BoGST), which scavenges cytotoxic reactive electrophiles, and of a deoxycytidine deaminase (BoDCD), which modulates the host molecular expression and potentially neutralizes the DNA adducts and maintains DNA integrity. The cDNAs of BoGST and BoDCD were cloned and sequenced; their expressions were verified by reverse-transcription polymerase chain reaction analysis and were found to be transcriptionally activated during the three-way interaction.
Assuntos
Brassica/enzimologia , Glutationa Transferase/genética , Nucleosídeo Desaminases/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Rhizoctonia/fisiologia , Trichoderma/fisiologia , Brassica/genética , Brassica/imunologia , Brassica/microbiologia , Citidina Desaminase , Regulação da Expressão Gênica de Plantas , Glutationa Transferase/imunologia , Nucleosídeo Desaminases/imunologia , Doenças das Plantas/genética , Proteínas de Plantas/imunologia , Rhizoctonia/imunologiaRESUMO
ABSTRACT Previous studies have shown that the extracellular proteins of Trichoderma harzianum ETS 323 grown in the presence of deactivated Botrytis cinerea in culture include a putative l-amino acid oxidase and have suggested the involvement of this enzyme in the antagonistic mechanism. Here, we hypothesized that the mycoparasitic process of Trichoderma spp. against B. cinerea involves two steps; that is, an initial hyphal coiling stage and a subsequent hyphal coiling stage, with different coiling rates. The two-step antagonism of T. harzianum ETS 323 against B. cinerea during the mycoparasitic process in culture was evaluated using a biexponential equation. In addition, an l-amino acid oxidase (Th-l-AAO) was identified from T. harzianum ETS 323. The secretion of Th-l-AAO was increased when T. harzianum ETS 323 was grown with deactivated hyphae of B. cinerea. Moreover, in vitro assays indicated that Th-l-AAO effectively inhibited B. cinerea hyphal growth, caused cytosolic vacuolization in the hyphae, and led to hyphal lysis. Th-l-AAO also showed disease control against the development of B. cinerea on postharvest apple fruit and tobacco leaves. Furthermore, an apoptosis-like response, including the generation of reactive oxygen species, was observed in B. cinerea after treatment with Th-l-AAO, suggesting that Th-l-AAO triggers programmed cell death in B. cinerea. This may be associated with the two-step antagonism of T. harzianum ETS 323 against B. cinerea.
Assuntos
Antibiose/fisiologia , Botrytis/fisiologia , Malus/microbiologia , Nicotiana/microbiologia , Doenças das Plantas/microbiologia , Trichoderma/fisiologia , Apoptose , Agentes de Controle Biológico , Botrytis/citologia , Botrytis/crescimento & desenvolvimento , Dano ao DNA , Frutas/microbiologia , Proteínas Fúngicas/metabolismo , Hifas/crescimento & desenvolvimento , Hifas/fisiologia , Cinética , L-Aminoácido Oxidase/isolamento & purificação , L-Aminoácido Oxidase/metabolismo , Peroxidação de Lipídeos , Potencial da Membrana Mitocondrial , Folhas de Planta/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Trichoderma/enzimologiaRESUMO
Our aim was to determine the effects of two secondary metabolites secreted by Trichoderma harzianum, pachybasin and emodin, on the mycoparasitic coiling behavior and cAMP content of T. harzianum. The number of T. harzianum coils around Nylon 66 fiber was increased in the presence of R. solani. The number of T. harzianum coils around R. solani hyphae and Nylon 66 fiber were significantly increased in the presence of pachybasin and emodin. The cAMP level in T. harzianum was significantly increased by close contact with R. solani and much higer cAMP level in the presence of exogenous pachybasin and emodin. A cAMP inhibitor diminished the effect of pachybasin and emodin on T. harzianum coiling around Nylon 66 fiber. The results suggest that pachybasin and emodin mediate the increase in the number of Trichoderma mycoparasitic coils via cAMP signaling. This is the first report to suggest that pachybasin and emodin play roles in the biocontrol mechanism of Trichoderma.
Assuntos
Antraquinonas/farmacologia , Emodina/farmacologia , Trichoderma/fisiologia , Antraquinonas/metabolismo , AMP Cíclico/análise , AMP Cíclico/antagonistas & inibidores , Emodina/metabolismo , Micélio/efeitos dos fármacos , Micélio/fisiologia , Trichoderma/efeitos dos fármacosRESUMO
The monomeric L-amino acid oxidase (mTh-LAAO) of Trichoderma harzianum ETS 323 has been suggested to antagonize Rhizoctonia solani by an unknown mechanism. Here, the mTh-LAAO-treated R. solani exhibited hyphal lysis and apoptotic characteristics such as DNA fragmentation, reactive oxygen species (ROS) accumulation, lipid peroxidation, and mitochondrial membrane potential depolarization. This hyphal lysis was suppressed by the mitochondria-dependent apoptosis inhibitor oligomycin while accompanied by reduction of ROS accumulation. This result suggested that mitochondria-mediated apoptosis in R. solani was involved in mTh-LAAO-induced growth inhibition, which was supported by the evidence of cytocheome c release and activation of caspases 9 and 3. Furthermore, the data indicated that the mTh-LAAO-induced fungal cell death was also closely interrelated with the interaction of mTh-LAAO with R. solani hyphal cell wall proteins. These results illuminate the biological function and mechanism underlying the antagonistic action of T. harzianum mTh-LAAO against fungal pathogens.
Assuntos
Antibiose , Proteínas Fúngicas/metabolismo , L-Aminoácido Oxidase/metabolismo , Mitocôndrias/metabolismo , Rhizoctonia/fisiologia , Trichoderma/enzimologia , Trichoderma/fisiologia , Proteínas Fúngicas/química , L-Aminoácido Oxidase/química , Doenças das Plantas/microbiologia , Rhizoctonia/crescimento & desenvolvimento , Trichoderma/químicaRESUMO
As opposed to single-cell yeast and mammalian cell lines, apoptosis has not been greatly investigated in filamentous fungi because antibodies to the relevant fungal apoptosis-related proteins are not available commercially and because multicellular organisms cannot be studied using flow cytometry. Here we demonstrate how antibodies from a nonfungal source could be used to investigate this pathway. We show that apoptosis in the filamentous fungus Botrytis cinerea is triggered by the mitochondria-mediated caspase pathway, with release of the apoptotic factors cytochrome c, caspase 3, and caspase 9, on treatment with Trichoderma harzianum-derived L-amino acid oxidase.
Assuntos
Apoptose/fisiologia , Botrytis/citologia , L-Aminoácido Oxidase/farmacologia , Mitocôndrias/metabolismo , Botrytis/efeitos dos fármacos , Botrytis/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Citocromos c/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/farmacologia , Trichoderma/enzimologiaRESUMO
Although L-amino oxidase (LAAO; EC 1.4.3.2) has been reported to be a potent antibacterial agent, the mechanism responsible for its antibacterial activity has not been identified. The present study aimed to identify the mechanism responsible for the antibacterial activity of Th-LAAO, an LAAO recently isolated from the extracellular proteins of Trichoderma harzianum ETS 323, at the same time as elucidating the nature of this enzyme. The results obtained indicate that the enzyme activity and structure of Th-LAAO are stable at pH 6-8 and less stable at both pH 4-5.5 and pH 9. At pH 7.0, the optimum temperature for Th-LAAO was found to be 40 °C, comprising the temperature at which enzymatic activity is greatest, with enzymatic activity deceasing with further increases in temperature as a result of thermal denaturation of the enzyme, leading to partial denaturation at 50 °C. The results obtained by confocal microscopy and flow cytometry indicate that Th-LAAO interacts with bacteria to cause membrane permeabilization, and this interaction may be promoted by the amphipathic sequence in Th-LAAO and other cytotoxic LAAOs located at the N-terminus. The findings of increased exogenous H(2) O(2) production and reactive oxidative species accumulation in Th-LAAO-treated bacteria indicate that reactive oxidative species accumulation may trigger forms of cell damage, including lipid peroxidation and DNA strand breakage that results in bacterial growth inhibition. Taken together, the results indicate that the processes of bacterial interaction, membrane permeabilization and H(2)O(2) production are involved in the mechanism responsible for the antibacterial activity of Th-LAAO.
Assuntos
Antibacterianos/metabolismo , Permeabilidade da Membrana Celular , Proteínas Fúngicas/metabolismo , Peróxido de Hidrogênio/metabolismo , L-Aminoácido Oxidase/metabolismo , Trichoderma/enzimologia , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Antibiose , Biocatálise , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sequência Consenso , Fragmentação do DNA , Estabilidade Enzimática , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacologia , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Cinética , L-Aminoácido Oxidase/química , L-Aminoácido Oxidase/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Dados de Sequência Molecular , Desnaturação Proteica , Espécies Reativas de Oxigênio/metabolismo , Alinhamento de Sequência , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , TemperaturaRESUMO
L-amino acid oxidases (L-AAOs) have been isolated from many organisms, such as snake, and are known to have antibacterial activity. To the best of the authors' knowledge, this is the first report of the cloning of cDNA encoding a novel Trichoderma harzianum ETS 323 L-amino acid oxidase (Th-L-AAO). The protein was overexpressed in Escherichia coli and purified to homogeneity. Comparisons of its deduced amino acid sequence with the sequence of other L-AAOs revealed the similarity to be between 9 and 24%. The molecular mass of the purified protein was 52 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme substrate specificity was highest for L-phenylalanine, and its optimal pH and temperature for activity were 7 and 40 °C, respectively; exogenous metal ions had no significant effect on activity. Circular dichroism spectroscopy indicated that the secondary structure of Th-L-AAO is composed of 17% α-helices, 28% ß-sheets, and 55% random coils. The bacterially expressed Th-L-AAO also mediated antibacterial activity against both gram-positive and gram-negative food spoilage microorganisms. Furthermore, a three-dimensional protein structure was created to provide more information about the structural composition of Th-L-AAO, suggesting that the N-terminal sequence of Th-L-AAO may have contributed to the antibacterial activity of this protein.
Assuntos
Clonagem Molecular , L-Aminoácido Oxidase/genética , L-Aminoácido Oxidase/metabolismo , Trichoderma/enzimologia , Sequência de Aminoácidos , Antibacterianos , DNA Complementar/genética , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , L-Aminoácido Oxidase/química , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
Trichoderma spp. are used as biocontrol agents against phytopathogens such as Rhizoctonia solani, but their biocontrol mechanisms are poorly understood. A novel L-amino oxidase (Th-LAAO) was identified from the extracellular proteins of Trichoderma harzianum ETS 323. Here, we show a FAD-binding glycoprotein with the best substrate specificity constant for L-phenylalanine. Although the amino acid sequence of Th-LAAO revealed limited homology (16-24%) to other LAAO members, a highly conserved FAD-binding motif was identified in the N-terminus. Th-LAAO was shown to be a homodimeric protein, but the monomeric form was predominant when grown in the presence of deactivated Rhizoctonia solani. Furthermore, in vitro assays demonstrated that Th-LAAO had an antagonistic effect against Rhizoctonia solani and a stimulatory one on hyphal density and sporulation in T. harzianum ETS 323. These findings further our understanding of T. harzianum as a biocontrol agent and provide insight into the biological function of l-amino acid oxidase.
Assuntos
Antibiose , Proteínas Fúngicas/metabolismo , L-Aminoácido Oxidase/metabolismo , Rhizoctonia/fisiologia , Trichoderma/enzimologia , Trichoderma/fisiologia , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , L-Aminoácido Oxidase/química , L-Aminoácido Oxidase/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Especificidade por Substrato , Trichoderma/química , Trichoderma/genéticaRESUMO
Trichoderma harzianum ETS 323 secretes two glucanases, a 23.5 kDa endoglucanase (EG Th1) and a 61 kDa exoglucanase (ExG Th1). They were identified by their hydrolysis products and were purified to homogeneity. The optimal temperature and pH for both EG Th1 (7.3-fold purification, 5.0% yield) and ExG Th1 (33.7-fold purification, 0.15% yield) were 50 °C and pH 4.5, respectively. The kinetic parameters of EG Th1 (K(m) = 23 mg mL(-1), V(max) = 294 µM min(-1), specific activity = 7.4 U mg(-1)) and ExG Th1 (K(m) = 85 mg mL(-1), V(max) = 385 µM min(-1), specific activity = 24.6 U mg(-1)) toward carboxymethyl cellulose were determined. Both enzymes favored CMC and maintained 100% activity for 10 days at 38 °C. KCl, MgCl(2), HgCl(2), and FeCl(3) showed approximately 30% inhibition against EG Th1 but not ExG Th1. They catalyzed transglycosylation of glucose in the presence of cellobiose, but ExG Th1 exhibited better activity and higher product diversity.
Assuntos
Glicosídeo Hidrolases/isolamento & purificação , Trichoderma/enzimologia , Celobiose/farmacologia , Glucose/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Especificidade por SubstratoRESUMO
BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is characterized by dense infiltrates of and defective apoptosis by mucosal cell populations. Some probiotics inhibit monocytes' expansion, although mechanisms remain unknown. Supernatants of Lactobacillus strains were investigated for inducing apoptosis of monocytes. METHODS: Secreted factors produced by Lactobacillus strains were tested on human lymphocytes, monocytes and a human monocytic leukemia-cell line (THP-1). Cell death mechanisms were investigated by a variety of methods. Lipopolysaccharide (LPS)-induced proinflammatory cytokines (IL-1beta, IL-6, IL-8, TNF-alpha) and anti-inflammatory TGF-beta1 were determined. RESULTS: Soluble factor(s) from Lactobacillus casei rhamnosus strain supernatants (LcrS) effectively induced apoptosis of immune cells. These were mainly soluble proteins (MW 5-30 kDa; LcrS(5-30)). For immune cells, but not human colonic epithelial carcinoma cells (HT-29), pretreatment with LcrS(5-30) significantly promoted apoptosis via a mitochondrial pathway. LcrS(5-30) suppressed pro-inflammatory cytokines and induced anti-inflammatory TGF-beta1. CONCLUSIONS: Probiotic Lcr produced heat-stable molecules (MW range 5-30 kDa) that promoted immune cell apoptosis without affecting intestinal epithelial cells. LcrS(5-30) triggered apoptosis by a mitochondrial pathway, but not via TGF-beta signaling pathway. LcrS(5-30) also inhibited LPS-induced inflammatory cytokines by immune cells. Thus, LcrS(5-30) promotes apoptosis of immune cells, and suggests probiotics-based regimens for prevention of IBD.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Bactérias/imunologia , Citocinas/imunologia , Lacticaseibacillus casei/imunologia , Lacticaseibacillus rhamnosus/imunologia , Monócitos/efeitos dos fármacos , Análise de Variância , Proteínas de Bactérias/farmacologia , Western Blotting , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Humanos , Marcação In Situ das Extremidades Cortadas , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Monócitos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Fungal mycelia mass and pigments are major obstacles to investigating the secretion of bioactive substances such as enzyme activities using a plate assay. In this study, we applied a cellophane membrane and demonstrated that it can block mycelia mass and conidia (especially pigmented spores that would likely interfere with any subsequent color development-based activity detection) while allowing secreting enzymes to pass through. Visual observation after lifting the cellophane membrane and the collected mycelia and conidia indicated that the bioactivities on specific plates were improved significantly, although some fungal growth hurdle was noted. This proved to be true whether the assays were color development based or not.
Assuntos
Celofane/química , Celulase/metabolismo , Fosfatos de Cálcio/química , Micélio/enzimologia , Esporos Fúngicos/enzimologia , Trichoderma/enzimologiaRESUMO
The biocontrol fungal species of Trichoderma, which colonizes plant roots, are well-known for their potential to control plant pathogens. Six anthraquinones, of which four have been identified for the first time from Trichoderma and two have already been reported in other strains, were purified from Trichoderma harzianum strain Th-R16 to evaluate their biological activities. The structures of the compounds were determined by one- and two-dimensional NMR. The compounds were shown to exhibit stronger antifungal activity than antibacterial activity. Low yield compounds, like 1,5-dihydroxy-3-hydroxymethyl-9,10-anthraquinone, were found to be more active against fungal pathogens than pachybasin and crysophanol, which were found to be the major extracellular metabolites. Test anthraquinones with higher oxidation numbers had better antifungal activity, and their activities were concentration-dependent.