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

The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast

Abstract Rot1 is an essential yeast protein originally shown to be implicated in such diverse processes such as β-1,6-glucan synthesis, actin cytoskeleton dynamics, or lysis of autophagic bodies. More recently also a role as a molecular chaperone has been discovered. Here we report that Rot1 interacts in a synthetic manner with Ost3, one of the nine subunits of the oligosaccharyltransferase complex, the key enzyme of N-glycosylation. Deletion of OST3 in the rot1-1 mutant causes a temperature sensitive phenotype as well as sensitivity towards compounds interfering with cell wall biogenesis such as Calcofluor White, caffeine, Congo Red and hygromycin B, whereas deletion of OST6, a functional homolog of OST3, has no effect. Oligosaccharyltransferase activity in vitro determined in membranes from rot1-1ost3∆ cells was found to be decreased to 45% compared to wild-type membranes, and model glycoproteins of N-glycosylation, like carboxypeptidase CPY, Gas1 or DPAP B, displayed an underglycosylation pattern. By affinity chromatography a physical interaction between Rot1 and Ost3 was demonstrated. Moreover, Rot1 was found to be involved also in the Omannosylation process, as glycosylation of distinct glycoproteins of this type were affected as well. Altogether the data extend the role of Rot1 as a chaperone required to ensure proper glycosylation

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

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

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

Synthesis of Dolichols in Candida albicans is Co-Regulated With Elongation of Fatty Acids

Abstract: Protein glycosylation requires dolichyl phosphate as a carbohydrate carrier. Dolichols are α-saturated polyprenols, and their saturation in S. cerevisiae is catalyzed by polyprenyl re-ductase Dfg10 together with some other unknown enzymes. The aim of this study was to identi-fy 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, respective-ly, 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 dol-ichols 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 en-zymes 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

The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast

Rot1 is an essential yeast protein originally shown to be implicated in such diverse processes such as β-1,6-glucan synthesis, actin cytoskeleton dynamics, or lysis of autophagic bodies. More recently also a role as a molecular chaperone has been discovered. Here we report that Rot1 interacts in a synthetic manner with Ost3, one of the nine subunits of the oligosaccharyltransferase complex, the key enzyme of N-glycosylation. Deletion of OST3 in the rot1-1 mutant causes a temperature sensitive phenotype as well as sensitivity towards compounds interfering with cell wall biogenesis such as Calcofluor White, caffeine, Congo Red and hygromycin B, whereas deletion of OST6, a functional homolog of OST3, has no effect. Oligosaccharyltransferase activity in vitro determined in membranes from rot1-1ost3Δ cells was found to be decreased to 45% compared to wild-type membranes, and model glycoproteins of N-glycosylation, like carboxypeptidase CPY, Gas1 or DPAP B, displayed an underglycosylation pattern. By affinity chromatography a physical interaction between Rot1 and Ost3 was demonstrated. Moreover, Rot1 was found to be involved also in the Omannosylation process, as glycosylation of distinct glycoproteins of this type were affected as well. Altogether the data extend the role of Rot1 as a chaperone required to ensure proper glycosylation. Keywords: ROT1/ N‐glycosylation/ O‐glycosylation/ oligosaccharyltransferase/ dolichol/ Saccharomyces cerevisiae Downloaded from http://glycob.oxfordjournals.org/ at Instytut Biochemii i Biofizyki PAN on April 17, 201

Impact of Yeast Glycosylation Pathway on Cell Integrity and Morphology, Glycosylation, Stefana Petrescu (Ed.), ISBN: 978-953-51-0771-2, InTech, Available from: http://www.intechopen.com/books/glycosylation/impact-of-yeast-glycosylation-pathway-on-cell-integrity-and-morphology

Protein glycosylation is a multi step reaction, well conserved in the eukaryotic cells. In N-glycosylation reactions dolichyl phosphate (DolP) serves as a lipid acceptor of sugar residues forming DolPPGlcNAc2Man9Glc3. Dolichyl phosphate mannose (DolPMan) is also a substrate for protein O-glycosylation, where it serves as a donor of the first mannose to be attached to hydroxyl groups of serine or treonine. DolPMan is also involved in the synthesis of the sugar part of glycosylphosphatidyl inositol anchor in yeast and other eukaryotes. Its remnant structure is responsible for the attachment of a large group of glycoproteins to the glucan polymers of the cell wall . Thus, a functional link could be predicted between the dolichol biosynthetic (mevalonate) pathway and subsequent N-glycosylation and O-mannosylation, cell wall assembly and/or fungus–host interaction. Moreover, on the basis of the data presented in this chapter, it can be assumed that the glycosylation pathway in yeast and fungi offers many levels of regulation, which might influence the final quality and quantity of cell wall glycoproteins and consequently cell surface immunogenicity. In this work we concentrate on early glycosylation defects, resulting from the impaired synthesis of dolichol (Dol) and dolichyl phosphate (DolP) or DolPP oligosaccharide (DolPPGlcNAc2Man9Glc3) assembly, and their effect on the cell integrity and morphology