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Laboratory of Fungal Glycobiology

Head: Professor Grażyna Palamarczyk

The overall focus of our work concentrates on basic studies concerning glycosylation and regulation of the cell wall glycoproteins. Two interrelated lines of research concern the role of protein glycosylation for the antifungal drug resistance and pathogenesis (lead by G.Palamarczyk) and the metabolic engineering of the  Trichoderma sp., with the aim to improve biotechnological properties of the strains (lead by J.Kruszewska).


Molecular mechanisms in protein glycosylation in the yeast: Saccharomyces cerevisiae and Candida albicans

Group leader:  Prof. Grażyna Palamarczyk
Staff: Dr. Anna Janik, Ph.D. students: M.Sc. Monika Pasikowska, M.Sc. Mateusz Juchimiuk

 

This project concerns the basic studies on glycosylation and regulation of the cell wall glycoproteins and their impact on the cell wall integrity. This area, although important, is still only in part understood even in geneticaly tractable microorganism such as S. cerevisiae, and, despite its potential importance in medical mycology, is unexplored in the human pathogenic fungi such as C. albicans.

The vast majority of eukaryotes (fungi, plants, animals) synthesise asparagine (Asn) linked N-glycans by means of a lipid precursor dolichyl-phosphate linked oligosaccharide. The isoprenoid lipid, dolichyl phosphate, is also involved in the O-mannosylation pathway which is essential for the cell wall integrity maintenance.

We have demonstrated that the glycosylation defect resulting from the synthesis of dolichyl phosphate results in the alterations of the S. cerevisiae cell wall structure and composition (Orłowski et al.2007).

Subsequently we would like to characterize the key steps in dolichol biosynthesis and dolichol dependent glycosylation and establish how alterations in these reactions affect the cell wall and pathogeneity of C. albicans.

Such information will inform the field of host/ fungal pathogen interactions and may be exploitable by the antifungal drug industry.

To this end we have isolated the genes involved in dolichol, and dolichyl phosphate formation and demonstrated that impaired dolichol synthesis affects formation of the hyphal (virulent) forms of  C. albicans  (Orłowski 2008, PhD Thesis). Subsequently we plan to construct a set of glycosylation mutants and test their sensitivity to the antifungal drugs as well as virulency.

All together, this project will increase our fundamental understanding of the molecular and biochemical regulation of cell wall glycoprotein assembly in C. albicans with the aim of better understanding of fungal pathogenicity and selection of targets for antifungal therapy.


Research grants:
2004-2005 "Euro Cell Wall, Fungal cell wall as a target for antimicrobial therapy", (5FP UE)
2005-2008 "The cell wall assembly in S. cerevisiae and C. albicans as a target for the antifungal drugs",
                   (Ministry of Science and Higher Education)

Selected publications:

  1. Kuranda K., François J., Palamarczyk G. The isoprenoid pathway and transcriptional response to its inhibitors in the yeast Saccharomyces cerevisiae. FEMS Yeast Res. (2009) - Epub ahead of print
  2.  Kuranda K., Grabinska K., Berges T., Karst F., Leberre V., Sokol S., François J., Palamarczyk G. The YTA7 gene is involved in the regulation of the isoprenoid pathway in the yeast Saccharomyces cerevisiae. FEMS Yeast Res. (2009) 9(3): 381-90
  3. Orłowski J., Machula K., Janik A., Zdebska E., Palamarczyk G. Dissecting the role of dolichol in cell wall assembly in the yeast mutants impaired in early glycosylation reactions. Yeast (2007) 24(4): 239-52
  4. Kuranda K., Leberre V., Sokol S., Palamarczyk G., François J. Investigating the caffeine effects in the yeast Saccharomyces cerevisiae brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. Mol Microbiol. (2006) 61(5): 1147-66
  5. Grabińska K., Sosińska G., Orłowski J., Swiezewska E., Berges T., Karst F., Palamarczyk G. Functional relationships between the Saccharomyces cerevisiae cis-prenyltransferases required for dolichol biosynthesis. Acta Biochim. Pol. (2005) 52(1): 221-32
  6.  Kamińska J., Kwapisz M., Grabińska K., Orłowski J., Boguta M., Palamarczyk G., Zoładek T. Rsp5 ubiquitin ligase affects isoprenoid pathway and cell wall organization in S. cerevisiae. Acta Biochim Pol. (2005) 52(1): 207-20
  7. Grabińska K., Palamarczyk G. Dolichol biosynthesis in the yeast Saccharomyces cerevisiae: an insight into the regulatory role of farnesyl diphosphate synthase. FEMS Yeast Res. (2002) 2(3): 259-65
  8. Janik A., Sosnowska M., Kruszewska J., Krotkiewski H., Lehle L., Palamarczyk G. Overexpression of GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae corrects defects in dolichol-linked saccharide formation and protein glycosylation. Biochim. Biophys. Acta. (2003) 1621(1): 22-30

 



Genetic engineering of fungi important in biotechnology and biocontrol

Group leader: Assoc. Prof. Joanna Kruszewska

Staff: Dr. Urszula Perlińska-Lenart  Senior Assistant, Dr. Wioletta Górka-Nieć Senior Assistant, M.Sc. Patrycja Zembek Ph.D. student, M.Sc. Sebastian Graczyk Ph.D. student

 

We have studied the influence of changes in the activity of enzymes engaged in the glycosylation pathways on protein production and secretion.

Expression of the yeast DPM1 gene (coding for DPM-synthase) in T. reesei increased the intensity of protein glycosylation and secretion, and caused ultrastructural changes in the fungal cell wall. We have undertaken 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 increase in chitin content and changes in chitin distribution. Moreover, we also observed a decreased concentration of mannose and alkali-soluble β-(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.

It was observed in S. cerevisiae that disruption of PMT genes, coding for protein O-mannosyltransferases catalyzing the transfer of the first mannosyl residue to proteins during their O-glycosylation, resulted in cell wall alterations.

Disruption of the pmt1 gene in Trichoderma caused a significant decrease in the total activity of protein O-mannosyltransferases and led to osmotic sensitivity of the strain, indicating an essential role of the PMTI protein activity for cell wall synthesis. 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.

To study influence of higher activity of protein O-mannosyltransferases on protein production and secretion we integrated an additional copy of the pmt1 gene into the Trichoderma genome. This integration unexpectedly resulted in silencing of pmt1 expression. Strains carrying the additional copy 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. At the same time, the strains grew slowly on solid medium and were hypersensitive to an antifungal reagent, Calcofluor white. These results indicate that protein O-manosyltransferases are required for proper cell wall formation, and their decreased activity influences not only O- but also N-glycosylation.

We also have cloned and analysed function of dpm2 and dpm3 genes coding for subunits of DPM synthase.  It was found that apart from the catalytic subunit DPMI, also the DPMIII subunit was 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 enzyme activity. We also characterized S. cerevisiae strains expressing dpm1,2,3 or dpm1,3 genes and analyzed the consequences of dpm expression on protein O- and N-glycosylation in vivo and on the cell wall composition.

 

Research grants:

2009-2012 "Engineering of Trichoderma strains with enhanced biocontrol capacities", (Structural Funds, POIG,
                    Ośrodek Przetwarzania Informacji)

2006-2009 "Up-regulation of biocontrol capacities of Trichoderma", (Ministry of Science and Higher Education)

2005-2008  part of the project “The functional genomics of the model microorganisms in molecular studies of inherited
                   human diseases and in mechanism of pathogenesis”, (Ministry of Science and Higher Education)

 

Selected publications:

  1. Górka-Nieć W., Pniewski M., Kania A., Perlińska-Lenart U., Palamarczyk G., Kruszewska J.S. Disruption of Trichoderma reesei gene encoding protein O-mannosyltransferase I results in a decrease of the enzyme activity and alteration of cell wall composition. Acta Biochim.Pol. (2008) 55: 251-260
  2. Kruszewska J.S., Perlińska-Lenart U., Górka-Nieć W., Orłowski J., Zembek P. Palamarczyk G. Alterations in protein secretion caused by metabolic engineering of glycosylation pathways in fungi. Acta Biochim.Pol. (2008) 55: 447-456
  3. Górka-Nieć W., Bańkowska R, Palamarczyk G., Krotkiewski H., Kruszewska J.S. Protein glycosylation in pmt mutants of Saccharomyces cerevisiae. Influence of heterologously expressed cellobiohydrolase II of Trichoderma reesei and elevated levels of GDP mannose and cis-prenyltransferase activity. Biochim. Biophys. Acta (2007) 1770: 774–780
  4. Perlinska-Lenart U., Bankowska R., Palamarczyk G., Kruszewska J.S. Overexpression of the Saccharomyces cerevisiae RER2 gene in Trichoderma reesei affects dolichol dependent enzymes and protein glycosylation. Fungal Genet. Biol. (2006) 43(6): 422-9
  5. Perlińska-Lenart U.,  Orłowski J., Laudy A.E., Zdebska E., Palamarczyk G., Kruszewska J.S Glycoprotein hypersecretion alters cell wall in Trichoderma reesei strains expressing the Saccharomyces cerevisiae dolichylphosphate mannose synthase gene. Appl. Environ. Microbiol. (2006) 72: 7778-7784
  6. Perlińska-Lenart U., Kurzątkowski W., Janas P., Kopińska A, Palamarczyk G., Kruszewska J.S Protein production and secretion in an Aspergillus nidulans mutant impaired in glycosylation. Acta Biochim. Pol. (2005) 52: 195-205