Cloning and functional analysis of dpm2 and dpm3 genes from Trichoderma reesei expressed in Saccharomyces cerevisiae dpm1∆ mutant

Abstract 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

The role of Alg13 N-acetylglucosaminyl transferase in the expression of pathogenic features of Candida albicans.

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

Identification of bacteria and fungi inhabiting fruiting bodies of Burgundy truffle (Tuber aestivum Vittad.)

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

Internalization of the Aspergillus nidulans AstA Transporter into Mitochondria Depends on Growth Conditions, and Affects ATP Levels and Sulfite Oxidase Activity

The astA gene encoding an alternative sulfate transporter was originally cloned from the genome of the Japanese Aspergillus nidulans isolate as a suppressor of sulfate permease-deficient strains. Expression of the astA gene is under the control of the sulfur metabolite repression system. The encoded protein transports sulfate across the cell membrane. In this study we show that AstA, having orthologs in numerous pathogenic or endophytic fungi, has a second function and, depending on growth conditions, can be translocated into mitochondria. This effect is especially pronounced when an astA-overexpressing strain grows on solid medium at 37 °C. AstA is also recruited to the mitochondria in the presence of mitochondria-affecting compounds such as menadione or antimycin A, which are also detrimental to the growth of the astA-overexpressing strain. Disruption of the Hsp70–Porin1 mitochondrial import system either by methylene blue, an Hsp70 inhibitor, or by deletion of the porin1-encoding gene abolishes AstA translocation into the mitochondria. Furthermore, we observed altered ATP levels and sulfite oxidase activity in the astA-overexpressing strain in a manner dependent on sulfur sources. The presented data indicate that AstA is also involved in the mitochondrial sulfur metabolism in some fungi, and thereby indirectly manages redox potential and energy state

Candida albicans; exploring glycosylation pathway in the search of targets for antimicrobial agents and yeast to hyphae transition

Microbial cell wall is mostly synthesized by the glycosylated proteins with the distinct enzymatic activity. In this review we have concentrated on the description of the certain steps of glycosylation and their effect on the cell wall integrity and yeast to hyphae transition, the process enhancing the pathogenic properties of C.albicans. The glycoproteins play an invaluable role in C. albicans virulence and they modulate adhesive, invasive, morphogenetic and immune stimulating properties of the pathogen as well as its susceptibility to the antifungal agents. Therefore, understanding of C. albicans glycobiology might let us expand the arsenal in the war against fungal enemies. The early stages of N-, O-glycans and GPI-anchor synthesis requires dolichol - the lipid carrier of sugar residues. Diminished supply of dolichol causes series of defects in C. albicans cells, among which aberrant protein glycosylation is the most evident. Furthermore, the relations between the cell wall composition and integrity, resistance to some antifungal and cell wall disturbing agents and dolichol dependent glycosylation are observed. Moreover relevance of these reactions for the morphological differentiation of C.albicans is described

Molecular characterization of central cytoplasmic loop in Aspergillus nidulans AstA transporter

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

Yil102c-A is a Functional Homologue of the DPMII Subunit of Dolichyl Phosphate Mannose Synthase in Saccharomyces cerevisiae

: 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

Increased activity of the sterol branch of the mevalonate pathway elevates glycosylation of secretory proteins and improves antifungal properties of Trichoderma atroviride.

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

Expression of Saccharomyces cerevisiae RER2 Gene Encoding Cis-Prenyltransferase in Trichoderma atroviride Increases the Activity of Secretory Hydrolases and Enhances Antimicrobial Features

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 pro�tein 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

Inhibition of Dephosphorylation of Dolichyl Diphosphate Alters the Synthesis of Dolichol and Hinders Protein N-Glycosylation and Morphological Transitions in Candida albicans

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