RESUMO
Biological invasions are now seen as one of the main threats to the Antarctic ecosystem. An example of such an invasion is the recent colonization of the H. Arctowski Polish Antarctic Station area by the non-native grass Poa annua. This site was previously occupied only by native plants like the Antarctic hair grass Deschampsia antarctica. To adapt successfully to new conditions, plants interact with soil microorganisms, including fungi. The aim of this study was to determine how the newly introduced grass P. annua established an interaction with fungi compared to resident grass D. antarctica. We found that fungal diversity in D. antarctica roots was significantly higher compared with P. annua roots. D. antarctica managed a biodiverse microbiome because of its ability to recruit fungal biocontrol agents from the soil, thus maintaining a beneficial nature of the endophyte community. P. annua relied on a set of specific fungal taxa, which likely modulated its cold response, increasing its competitiveness in Antarctic conditions. Cultivated endophytic fungi displayed strong chitinolysis, pointing towards their role as phytopathogenic fungi, nematode, and insect antagonists. This is the first study to compare the root mycobiomes of both grass species by direct culture-independent techniques as well as culture-based methods.
Assuntos
Ecossistema , Endófitos , Fungos , Espécies Introduzidas , Poaceae , Regiões Antárticas , Poaceae/microbiologia , Fungos/classificação , Fungos/fisiologia , Endófitos/fisiologia , Raízes de Plantas/microbiologia , Microbiologia do Solo , Micobioma , Poa/microbiologia , BiodiversidadeRESUMO
Some Trichoderma spp. exhibit natural abilities to reduce fungal diseases of plants through their mycoparasitic and antagonistic properties. In this study, we created new Trichoderma atroviride strains with elevated antifungal activity. This effect was achieved by improving the activity of cis-prenyltransferase, the main enzyme in dolichol synthesis, by expressing the RER2 gene from Saccharomyces cerevisiae. Since dolichyl phosphate is the carrier of carbohydrate residues during protein glycosylation, activation of its synthesis enhanced the activities of dolichyl-dependent enzymes, DPM synthase and N-acetylglucosamine transferase, as well as stimulated glycosylation of secretory proteins. Cellulases secreted by the transformants revealed significantly higher levels or activities compared to the control strain. Consequently, the resulting Trichoderma strains were more effective against the plant pathogens Pythium ultimum.
RESUMO
Mucoromycotina are often considered mainly in pathogenic context but their biology remains understudied. We describe the genomes of six Mucoromycotina fungi representing distant saprotrophic lineages within the subphylum (i.e., Umbelopsidales and Mucorales). We selected two Umbelopsis isolates from soil (i.e., U. isabellina, U. vinacea), two soil-derived Mucor isolates (i.e., M. circinatus, M. plumbeus), and two Mucorales representatives with extended proteolytic activity (i.e., Thamnidium elegans and Mucor saturninus). We complement computational genome annotation with experimental characteristics of their digestive capabilities, cell wall carbohydrate composition, and extensive total lipid profiles. These traits inferred from genome composition, e.g., in terms of identified encoded enzymes, are in accordance with experimental results. Finally, we link the presence of associated bacteria with observed characteristics. Thamnidium elegans genome harbors an additional, complete genome of an associated bacterium classified to Paenibacillus sp. This fungus displays multiple altered traits compared to the remaining isolates, regardless of their evolutionary distance. For instance, it has expanded carbon assimilation capabilities, e.g., efficiently degrades carboxylic acids, and has a higher diacylglycerol:triacylglycerol ratio and skewed phospholipid composition which suggests a more rigid cellular membrane. The bacterium can complement the host enzymatic capabilities, alter the fungal metabolism, cell membrane composition but does not change the composition of the cell wall of the fungus. Comparison of early-diverging Umbelopsidales with evolutionary younger Mucorales points at several subtle differences particularly in their carbon source preferences and encoded carbohydrate repertoire. Nevertheless, all tested Mucoromycotina share features including the ability to produce 18:3 gamma-linoleic acid, use TAG as the storage lipid and have fucose as a cell wall component.
RESUMO
Protein glycosylation requires dolichyl phosphate as a carbohydrate carrier. Dolichols are α-saturated polyprenols, and their saturation in S. cerevisiae is catalyzed by polyprenyl reductase Dfg10 together with some other unknown enzymes. The aim of this study was to identify such enzymes in Candida. The Dfg10 polyprenyl reductase from S. cerevisiae comprises a C-terminal 3-oxo-5-alpha-steroid 4-dehydrogenase domain. Alignment analysis revealed such a domain in two ORFs (orf19.209 and orf19.3293) from C. albicans, which were similar, respectively, to Dfg10 polyprenyl reductase and Tsc13 enoyl-transferase from S. cerevisiae. Deletion of orf19.209 in Candida impaired saturation of polyprenols. The Tsc13 homologue turned out not to be capable of saturating polyprenols, but limiting its expression reduce the cellular level of dolichols and polyprenols. This reduction was not due to a decreased expression of genes encoding cis-prenyltransferases from the dolichol branch but to a lower expression of genes encoding enzymes of the early stages of the mevalonate pathway. Despite the resulting lower consumption of acetyl-CoA, the sole precursor of the mevalonate pathway, it was not redirected towards fatty acid synthesis or elongation. Lowering the expression of TSC13 decreased the expression of the ACC1 gene encoding acetyl-CoA carboxylase, the key regulatory enzyme of fatty acid synthesis and elongation.
Assuntos
Candida albicans/metabolismo , Dolicóis/biossíntese , Ácidos Graxos/metabolismo , Acetilcoenzima A/metabolismo , Sequência de Aminoácidos , Candida albicans/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Humanos , Hifas/crescimento & desenvolvimento , Ácido Mevalônico/metabolismo , Mutação/genética , Filogenia , Poliprenois/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
In a wide range of organisms, dolichyl phosphate mannose (DPM) synthase is a complex of tree proteins Dpm1, Dpm2, and Dpm3. However, in the yeast Saccharomyces cerevisiae, it is believed to be a single Dpm1 protein. The function of Dpm3 is performed in S. cerevisiae by the C-terminal transmembrane domain of the catalytic subunit Dpm1. Until present, the regulatory Dpm2 protein has not been found in S. cerevisiae. In this study, we show that, in fact, the Yil102c-A protein interacts directly with Dpm1 in S. cerevisiae and influences its DPM synthase activity. Deletion of the YIL102c-A gene is lethal, and this phenotype is reversed by the dpm2 gene from Trichoderma reesei. Functional analysis of Yil102c-A revealed that it also interacts with glucosylphosphatidylinositol-N-acetylglucosaminyl transferase (GPI-GnT), similar to DPM2 in human cells. Taken together, these results show that Yil102c-A is a functional homolog of DPMII from T. reesei and DPM2 from humans.
Assuntos
Proteínas Fúngicas/genética , Manosiltransferases/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos/genética , Fosfatos de Dolicol/metabolismo , Proteínas Fúngicas/metabolismo , Glicosilação , Humanos , Manose/metabolismo , Manosiltransferases/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Trichoderma/genéticaRESUMO
Tuber species may be regarded as complex microhabitats hosting diverse microorganisms inside their fruiting bodies. Here, we investigated the structure of microbial communities inhabiting the gleba of wild growing (in stands) T. aestivum, using Illumina sequencing and culture-based methods. The two methods used in combination allowed to extract more information on complex microbiota of Tuber aestivum gleba. Analysis of the V3-V4 region of 16S rDNA identified nine phyla of bacteria present in the gleba of T. aestivum ascomata, mostly Proteobacteria from the family Bradyrhizobiaceae. Our results ideally match the earlier data for other Tuber species where the family Bradyrhizobiaceae was the most represented. The ITS1 region of fungal rDNA represented six alien fungal species belonging to three phyla. To complement the metagenomic analysis, cultivable fungi and bacteria were obtained from the gleba of the same T. aestivum fruiting bodies. The identified fungi mostly belong to the phylum Basidiomycota and same to Ascomycota. Analysis of cultivable bacteria revealed that all the specimens were colonized by different strains of Bacillus. Fungal community inhabiting T. aestivum fruiting bodies was never shown before.
Assuntos
Ascomicetos/fisiologia , Bacillus/isolamento & purificação , Basidiomycota/isolamento & purificação , Bradyrhizobiaceae/isolamento & purificação , Carpóforos/fisiologia , Bacillus/classificação , Bacillus/genética , Basidiomycota/classificação , Basidiomycota/genética , Bradyrhizobiaceae/classificação , Bradyrhizobiaceae/genética , DNA Ribossômico/genética , Sequenciamento de Nucleotídeos em Larga Escala , MicrobiotaRESUMO
Some Trichoderma spp. have an ability to inhibit proliferation of fungal plant pathogens in the soil. Numerous compounds with a proven antifungal activity are synthesized via the terpene pathway. Here, we stimulated the activity of the mevalonate pathway in T. atroviride P1 by expressing the Saccharomyces cerevisiae ERG20 gene coding for farnesyl pyrophosphate (FPP) synthase, a key enzyme of this pathway. ERG20-expressing Trichoderma strains showed higher activities of FPP synthase and squalene synthase, the principal recipient of FPP in the mevalonate pathway. We also observed activation of dolichyl phosphate mannose (DPM) synthase, an enzyme in protein glycosylation, and significantly increased O- and N-glycosylation of secreted proteins. The hyper-glycosylation of secretory hydrolases could explain their increased activity observed in the ERG20 transformants. Analysis of the antifungal properties of the new strains revealed that the hydrolases secreted by the transformants inhibited growth of a plant pathogen, Pythium ultimum more efficiently compared to the control strain. Consequently, the biocontrol activity of the transgenic strains, determined as their ability to protect bean seeds and seedlings against harmful action of P. ultimum, was also improved substantially.
Assuntos
Hypocreales/metabolismo , Ácido Mevalônico/metabolismo , Antifúngicos/metabolismo , Fabaceae/microbiologia , Regulação Fúngica da Expressão Gênica/genética , Geraniltranstransferase/genética , Geraniltranstransferase/metabolismo , Glicosilação , Hypocreales/genética , Manosiltransferases/genética , Pythium/crescimento & desenvolvimento , Esteróis/metabolismo , Trichoderma/genéticaRESUMO
The essential role of dolichyl phosphate (DolP) as a carbohydrate carrier during protein N-glycosylation is well established. The cellular pool of DolP is derived from de novo synthesis in the dolichol branch of the mevalonate pathway and from recycling of DolPP after each cycle of N-glycosylation, when the oligosaccharide is transferred from the lipid carrier to the protein and DolPP is released and then dephosphorylated. In Saccharomyces cerevisiae, the dephosphorylation of DolPP is known to be catalyzed by the Cwh8p protein. To establish the role of the Cwh8p orthologue in another distantly related yeast species, Candida albicans, we studied its mutant devoid of the CaCWH8 gene. A double Cacwh8∆/Cacwh8∆ strain was constructed by the URA-blaster method. As in S. cerevisiae, the mutant was impaired in DolPP recycling. This defect, however, was accompanied by an elevation of cis-prenyltransferase activity and higher de novo production of dolichols. Despite these compensatory changes, protein glycosylation, cell wall integrity, filamentous growth, and biofilm formation were impaired in the mutant. These results suggest that the defects are not due to the lack of DolP for the protein N-glycosylation but rather that the activity of oligosacharyltransferase could be inhibited by the excess DolPP accumulating in the mutant.
Assuntos
Candida albicans/metabolismo , Dolicóis/biossíntese , Proteínas Fúngicas/genética , Oligossacarídeos de Poli-Isoprenil Fosfato/metabolismo , Processamento de Proteína Pós-Traducional , Pirofosfatases/genética , Candida albicans/crescimento & desenvolvimento , Parede Celular/metabolismo , Dolicóis/genética , Proteínas Fúngicas/metabolismo , Glicosilação , Morfogênese , Pirofosfatases/metabolismoRESUMO
Mono-saturated polyprenols (dolichols) have been found in almost all Eukaryotic cells, however, dolichols containing additional saturated bonds at the ω-end, have been identified in A. fumigatus and A. niger. Here we confirm using an LC-ESI-QTOF-MS analysis, that poly-saturated dolichols are abundant in other filamentous fungi, Trichoderma reesei, A. nidulans and Neurospora crassa, while the yeast Saccharomyces cerevisiae only contains the typical mono-saturated dolichols. We also show, using differential scanning calorimetry (DSC) and fluorescence anisotropy of 1,6-diphenyl-l,3,5-hexatriene (DPH) that the structure of dolichols modulates the properties of membranes and affects the functioning of dolichyl diphosphate mannose synthase (DPMS). The activity of this enzyme from T. reesei and S. cerevisiae was strongly affected by the structure of dolichols. Additionally, the structure of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) model membranes was more strongly disturbed by the poly-saturated dolichols from Trichoderma than by the mono-saturated dolichols from yeast. By comparing the lipidome of filamentous fungi with that from S. cerevisiae, we revealed significant differences in the PC/PE ratio and fatty acids composition. Filamentous fungi differ from S. cerevisiae in the lipid composition of their membranes and the structure of dolichols. The structure of dolichols profoundly affects the functioning of dolichol-dependent enzyme, DPMS.
Assuntos
Dolicóis/metabolismo , Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Glicosiltransferases/metabolismo , Aspergillus niger/química , Aspergillus niger/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Dolicóis/análise , Fungos/química , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Modelos Moleculares , Neurospora crassa/química , Neurospora crassa/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Trichoderma/química , Trichoderma/metabolismoRESUMO
AstA (alternative sulfate transporter) belongs to a large, but poorly characterized, Dal5 family of allantoate permeases of the Major Facilitator Superfamily. The astA gene has been cloned from an IAM 2006 Japanese strain of Aspergillus nidulans by complementation of a sulfate permease-deficient mutant. In this study we show that conserved lysine residues in Central Cytoplasmic Loop (CCL) of the AstA protein may participate in anion selectivity, and control kinetic properties of the AstA transporter. A three-dimensional model containing four clustered lysine residues was created, showing a novel substrate-interacting structure in Major Facilitator Superfamily transporters. The assimilation constant (Kτ) of wild type AstA protein is 85 µM, while Vmax/mg of DW of AstA is twice that of the main sulfate transporter SB per mg of dry weight (DW) of mycelium (1.53 vs. 0.85 nmol/min, respectively). Amino acid substitutions in CCL did not abolish sulfate uptake, but affected its kinetic parameters. Mutants affected in the lysine residues forming the postulated sulfate-interacting pocket in AstA were able to grow and uptake sulfate, indicating that CCL is not crucial for sulfate transportation. However, these mutants exhibited altered values of Kτ and Vmax, suggesting that CCL is involved in control of the transporter activity.
Assuntos
Proteínas de Transporte de Ânions/química , Aspergillus nidulans/enzimologia , Proteínas Fúngicas/química , Sulfatos/metabolismo , Substituição de Aminoácidos , Proteínas de Transporte de Ânions/genética , Transporte Biológico , Cristalografia , Citoplasma/enzimologia , Proteínas Fúngicas/genética , Cinética , Lisina/química , Lisina/genética , Modelos Moleculares , Mutação , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
BACKGROUND: The pathogenic potential of Candida albicans depends on adhesion to the host cells mediated by highly glycosylated adhesins, hyphae formation and growth of biofilm. These factors require effective N-glycosylation of proteins. Here, we present consequences of up- and down-regulation of the newly identified ALG13 gene encoding N-acetylglucosaminyl transferase, a potential member of the Alg7p/Alg13p/Alg14p complex catalyzing the first two initial reactions in the N-glycosylation process. METHODS: We constructed C. albicans strain alg13∆::hisG/TRp-ALG13 with one allele of ALG13 disrupted and the other under the control of a regulatable promoter, TRp. Gene expression and enzyme activity were measured using RT-qPCR and radioactive substrate. Cell wall composition was estimated by HPLC DIONEX. Protein glycosylation status was analyzed by electrophoresis of HexNAcase, a model N-glycosylated protein in C. albicans. RESULTS: Both decreased and elevated expression of ALG13 changed expression of all members of the complex and resulted in a decreased activity of Alg7p and Alg13p and under-glycosylation of HexNAcase. The alg13 strain was also defective in hyphae formation and growth of biofilm. These defects could result from altered expression of genes encoding adhesins and from changes in the carbohydrate content of the cell wall of the mutant. GENERAL SIGNIFICANCE: This work confirms the important role of protein N-glycosylation in the pathogenic potential of C. albicans.
Assuntos
Candida albicans/genética , Candida albicans/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Alelos , Sequência de Aminoácidos , Biofilmes/crescimento & desenvolvimento , Candida albicans/enzimologia , Carboidratos/genética , Parede Celular/genética , Parede Celular/metabolismo , Regulação para Baixo/genética , Expressão Gênica/genética , Glicosilação , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Regiões Promotoras Genéticas/genética , Alinhamento de SequênciaRESUMO
The cell wall is one of the major keys to fungal identity. Fungi use their cell wall to sense the environment, and localize nutrients and competing microorganism. Pathogenic species additionally modify their cell walls to hide from a host's immune system. With the growing number of fungal infections and alarming shortage of available drugs, we are in need of new approaches to fight pathogens. The cell wall seems to be a natural target, since animal host cells are devoid of it. The current knowledge about fungal cell wall components is often limited, and there is huge diversity both in structure and composition between species. In order to compare the distribution of diverse proteins involved in cell wall biosynthesis and maintenance, we performed sequence homology searches against 24 fungal proteomes from distinct taxonomic groups, all reported as human pathogens. This approach led to identification of 4014 cell wall proteins (CWPs), and enabled us to speculate about cell wall composition in recently sequenced pathogenic fungi with limited experimental information. We found large expansions of several CWP families, in particular taxa, and a number of new CWPs possibly involved in evading host immune recognition. Here, we present a comprehensive evolutionary history of fungal CWP families in the context of the fungal tree of life.
RESUMO
The mevalonate pathway is the most diverse metabolic route resulting in the biosynthesis of at least 30,000 isoprenoid compounds, many of which, such as sterols or dolichols, are indispensable for living cells. In the filamentous fungus Trichoderma of major biotechnological interest isoprenoid metabolites are also involved in the biocontrol processes giving the mevalonate pathway an additional significance. On the other hand, little is known about genes coding for enzymes of the mevalonate pathway in Trichoderma. Here, we present cloning and functional analysis of the erg20 gene from Trichoderma reesei coding for farnesyl pyrophosphate (FPP) synthase (EC 2.5.1.10), an enzyme located at the branching point of the mevalonate pathway. Expression of the gene in a thermosensitive erg20-2 mutant of Saccharomyces cerevisiae impaired in the FPP synthase activity suppressed the thermosensitive phenotype. The same gene overexpressed in T. reesei significantly enhanced the FPP synthase activity and also stimulated the activity of cis-prenyltransferase, an enzyme of the dolichyl branch of the mevalonate pathway. Unexpectedly, the activity of squalene synthase from the other, sterol branch, was significantly decreased without, however, affecting ergosterol level.
Assuntos
Farnesil-Difosfato Farnesiltransferase/genética , Expressão Gênica , Geraniltranstransferase/genética , Ácido Mevalônico/metabolismo , Transferases/metabolismo , Trichoderma/enzimologia , Sequência de Aminoácidos , Ergosterol/metabolismo , Geraniltranstransferase/biossíntese , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Trichoderma/genéticaRESUMO
Antagonism of Trichoderma spp. against phytopathogenic fungi is widely exploited for biocontrol of plant diseases. A crucial role in the biocontrol mechanism is attributed to cell-wall-degrading enzymes secreted by Trichoderma spp. Therefore, more efficient production and secretion of the enzymes should elevate the biocontrol abilities of Trichoderma spp. Because the majority of secretory hydrolases are glycoproteins, it has been postulated that the posttranslational modification of these proteins could constitute a bottleneck in their production and secretion. Our previous study showed that improvement of O-glycosylation elevated protein secretion by Trichoderma reesei. In this study, we enhanced the biocontrol abilities of T. atroviride P1 against plant pathogens by overexpressing the Saccharomyces cerevisiae DPM1 gene coding for dolichyl phosphate mannose (DPM) synthase, a key enzyme in the O-glycosylation pathway. The transformants we obtained showed doubled DPM synthase activity and, at the same time, significantly elevated cellulolytic activity. They also revealed an improved antifungal activity against the plant pathogen Pythium ultimum.
Assuntos
Manosiltransferases/metabolismo , Controle Biológico de Vetores , Doenças das Plantas/microbiologia , Trichoderma/enzimologia , Trichoderma/fisiologia , Parede Celular/metabolismo , Fabaceae/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Germinação , Manosiltransferases/genética , Organismos Geneticamente Modificados , Pythium/crescimento & desenvolvimento , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Plântula/crescimento & desenvolvimento , Plântula/microbiologia , Sementes/microbiologia , Sementes/fisiologia , Trichoderma/genética , Trichoderma/crescimento & desenvolvimentoRESUMO
In Trichoderma reesei, dolichyl phosphate mannose (dpm) synthase, a key enzyme in the O-glycosylation process, requires three proteins for full activity. In this study, the dpm2 and dpm3 genes coding for the DPMII and DPMIII subunits of T. reesei DPM synthase were cloned and functionally analyzed after expression in the Saccharomyces cerevisiae dpm1Δ [genotype (BY4743; his3Δ1; /leu2Δ0; lys2Δ0; /ura3Δ0; YPR183w::kanMX4] mutant. It was found that apart from the catalytic subunit DPMI, the DPMIII subunit is also essential to form an active DPM synthase in yeast. Additional expression of the DPMII protein, considered to be a regulatory subunit of DPM synthase, decreased the enzymatic activity. We also characterized S. cerevisiae strains expressing the dpm1, 2, 3 or dpm1, 3 genes and analyzed the consequences of dpm expression on protein O-glycosylation in vivo and on the cell wall composition.
Assuntos
Manosiltransferases/genética , Manosiltransferases/metabolismo , Mutação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trichoderma/enzimologia , Clonagem Molecular , Expressão Gênica , Genótipo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Trichoderma/genéticaRESUMO
In fungi, transfer of the first mannosyl residue to proteins during their O-glycosylation is catalyzed by protein O-mannosyltransferases. Integration of additional copies of the pmt1 gene into Trichoderma reesei genome unexpectedly resulted in the silencing of pmt1 expression. Strains carrying the additional copies of pmt1 gene exhibited lower total activity of protein O-mannosyltransferases, lower O- and N-glycosylation of secreted proteins and showed defects in their cell wall composition. Moreover, the strains grew slowly on solid medium and were hypersensitive to an antifungal reagent, Calcofluor white. These results indicate that protein O-mannosyltransferases are required for proper cell wall formation, and their decreased activity influences not only O- but also N-glycosylation.
Assuntos
Parede Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dosagem de Genes , Manosiltransferases/genética , Manosiltransferases/metabolismo , Trichoderma/enzimologia , Parede Celular/enzimologia , Parede Celular/genética , Glicosilação , Trichoderma/genética , Trichoderma/metabolismoRESUMO
Sorbitol is often used at 1 mol/liter as an osmotic stabilizer for cultivation of fungi with a fragile cell wall phenotype. On the other hand, at this concentration sorbitol causes an osmotic stress in fungal cells resulting in intensive production of intracellular glycerol. The highly increased consumption of glucose for glycerol synthesis may lead to changes in processes requiring carbohydrate residues. This study provides new information on the consequences of osmotic stress to the cell wall composition, protein production and glycosylation, and cell morphology of Trichoderma reesei. We observed that high osmolarity conditions enhanced biomass production and strongly limited synthesis of cell wall glucans and chitin. Moreover, in these conditions the amount of secreted protein decreased nearly ten-fold and expression of cbh1 and cbh2 genes coding for cellobiohydrolase I and cellobiohydrolase II, the main secretory proteins in T. reesei, was inhibited resulting in a lack of the proteins in the cell and cultivation medium. The activity of DPM synthase, enzyme engaged in both N- and O-glycosylation pathways, was reduced two-fold, suggesting an overall inhibition of protein glycosylation. However, the two modes of glycosylation were affected divergently: O-glycosylation of secreted proteins decreased in the early stages of growth while N-glycosylation significantly increased in the stationary phase.
Assuntos
Parede Celular/metabolismo , Proteínas Fúngicas/metabolismo , Sorbitol/metabolismo , Trichoderma/metabolismo , Parede Celular/química , Parede Celular/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Glicosilação , Concentração Osmolar , Trichoderma/química , Trichoderma/genéticaRESUMO
Due to its natural properties, Trichoderma reesei is commonly used in industry-scale production of secretory proteins. Since almost all secreted proteins are O-glycosylated, modulation of the activity of enzymes of the O-glycosylation pathway are likely to affect protein production and secretion or change the glycosylation pattern of the secreted proteins, altering their stability and biological activity. Understanding how the activation of different components of the O-glycosylation pathway influences the glycosylation pattern of proteins and their production and secretion could help in elucidating the mechanism of the regulation of these processes and should facilitate creation of engineered microorganisms producing high amounts of useful proteins. In this review we focus on data concerning Trichoderma, but also present some background information allowing comparison with other fungal species.
Assuntos
Proteínas Fúngicas/biossíntese , Trichoderma/metabolismo , Sequência de Carboidratos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Genes Fúngicos , Glicoproteínas/biossíntese , Glicoproteínas/química , Glicoproteínas/genética , Glicosilação , Dados de Sequência Molecular , Engenharia de Proteínas , Trichoderma/genéticaRESUMO
In fungi transfer of the first mannosyl residue to proteins during their O-glycosylation is catalyzed by protein O-mannosyltransferases encoded by pmt genes. Disruption of the pmt1 gene in Trichoderma caused a significant decrease in the total activity of protein O-mannosyltransferases. Moreover, disruption of the pmt1 gene also led to osmotic sensitivity of the strain, indicating an essential role of the PMTI protein activity for cell wall synthesis. At the same time, the strain was defective in septa formation, producing only half the number of septa per unit length of hypha compared with the wild type. Disruption of the pmt1 gene decreased protein secretion but had no effect on glycosylation of secreted proteins, which suggests that PMTI protein O-mannosyltranferase does not take part in glycosylation of these proteins.
Assuntos
Genes Fúngicos , Manosiltransferases/genética , Manosiltransferases/metabolismo , Trichoderma/enzimologia , Trichoderma/genética , Sequência de Bases , Parede Celular/química , DNA Fúngico/genética , Proteínas Fúngicas/metabolismo , Glicosilação , Mutação , Deleção de Sequência , Trichoderma/crescimento & desenvolvimentoRESUMO
Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increased amount of N-acetylglucosamine, suggesting an increase in chitin content. Calcofluor white staining followed by fluorescence microscopy indicated changes in chitin distribution. Moreover, we also observed a decreased concentration of mannose and alkali-soluble beta-(1,6) glucan. A comparison of protein secretion from protoplasts with that from mycelia showed that the cell wall created a barrier for secretion in the DPM1 transformants. We also discuss the relationships between the observed changes in the cell wall, increased protein glycosylation, and the greater secretory capacity of T. reesei strains expressing the yeast DPM1 gene.