Protein glycosylation in fungi

1. Protein glycosylation in fungi David Singleton Biology YCP March 11, 2009

2. My Background • Contacts for mentoring and networking • Involvement in Society activities • http://www.microbiologycareers.org/ • http://www.ascb.org/newsfiles/jobhunt.pdf • http://sciencecareers.sciencemag.org/ • Choices for grad school/professional school/post doc

3. What kind of questions can we ask using microorganisms? • Model systems! • "From the elephant to butyric acid bacterium—it is all the same!“ Albert Kluyver, 1926 • Prokaryotic microorganisms; many similarities in biochemistry • Eukaryotic microorganisms; many similarities in cell biology and development

4. Why yeast?

5. Why yeast? • Initial screen: 23 complementation groups • Cloning and sequencing • Conserved pathways • Secretory pathway • Cell cycle • Signal transduction • Metabolism

6. Why yeast? • 1st sequenced eukaryote • Gene deletion project • Protein interaction web • Protein localization • Transcription profiling

7. C. albicans is a normal component of human microbial flora • Common organism on skin, mucous membranes, oral cavity, GI tract • Opportunistic pathogen • Many disease predispositions • 4th most common post-operative nosocomial blood borne infection

8. Surface hydrophobicity enables fungi to adhere to surfaces

9. Hydrophobicity is correlated with surface fibril length • Rapid high pressure freezing preserves morphology (K. Czymmek, U Del) • Fibril components: high molecular weight mannoproteins • Fibrils are longer and loosely packed on hydrophilic cells cell wall cytoplasm fibrils

10. Fungal N-glycosylation is a virulence factor • Post-translational addition of sugars • Acid-hydrolyzable phosphate linkage distinguishes acid-labile and acid-stable regions

11. Fungal N-glycosylation may be a regulator of hydrophobicity • Little difference in composition of proteins and carbohydrates between hydrophobic and hydrophilic cells • Most striking difference is in the acid-labile region • Increase in β-1,2-mannose polymer length in hydrophobic cells • Working model: proteins confer hydrophobic properties to cell surface, which are modulated by glycosylation

12. Construction of mnn4 serotype B deletion strain Wild-type yeast MNN4/MNN4 MPA sensitive MNN4 MNN4 MNN4 MNN4 MPA MPA Transform to MPAR MNN4/mnn4 MPA resistant Counterselect MPAS Loss of MNN4  derivative lacking acid-labile region  potentially always hydrophilic MNN4/mnn4 MPA sensitive Repeat!

13. Phenotypic analysis of mnn4 deletion strain B6 epitope B6.1 epitope

14. STEP 3: Label secondary branches with ANTS and separate by electrophoresis STEP 2: Cleave primary backbone • Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) • J. Masuoka; MSU Wichita Falls, TX STEP 1: Remove acid labile group

15. Summary of mnn4 mutant phenotype • Loss of detectable mannosylphosphate; no acid labile addition • Surprising increase in hydrophobicity • Perturbation of remaining acid-stable region in mutant • Change in in vivo fitness of derivative in co-infection model

16. Potential functions for Mnn4p • Catalytic: shares small region of glycosyltransferase homology • Predict Golgi localization, and raises potential for in vitro reconstitution • Regulatory: supported by genetic and mass screening studies • No localization prediction, but allows potential for overall control of cell surface properties

17. Plan to identify a function for MNN4 • Characterize interactions common between S. cerevisiaeand C. albicansMnn4p • Can begin to identify pathways • Identify suppressors of mnn4 mutation • Extends pathway delineation • Identify cellular site of action of Mnn4p • Indicates potential mechanism • Describe phylogenetic distribution of MNN4 genes • Why do fungi place mannosylphosphate on surfaces?

18. Protein Interaction Studies Gene “X” Mnn4p Gene “Y” Phosphate addition

19. Plan to identify a function for MNN4 • Characterize interactions common between S. cerevisiaeand C. albicansMnn4p • Can begin to identify pathways • Identify suppressors of mnn4 mutation • Extends pathway delineation • Identify cellular site of action of Mnn4p • Indicates potential mechanism • Describe phylogenetic distribution of MNN4 genes • Why do fungi place mannosylphosphate on surfaces?

20. Genetic Suppression Phosphate addition X Mnn4p Gene “X” Gene “Y” Second mutation allows recovery of phenotype First mutation (mnn4) blocks here

21. Plan to identify a function for MNN4 • Characterize interactions common between S. cerevisiaeand C. albicansMnn4p • Can begin to identify pathways • Identify suppressors of mnn4 mutation • Extends pathway delineation • Identify cellular site of action of Mnn4p • Indicates potential mechanism • Describe phylogenetic distribution of MNN4 genes • Why do fungi place mannosylphosphate on surfaces?

22. Localization using Yellow Fluorescent Protein Lee SA, Khalique Z, Gale CA, Wong B. Med Mycol. 2005 Aug;43(5):423-30.

23. Plan to identify a function for MNN4 • Characterize interactions common between S. cerevisiaeand C. albicansMnn4p • Can begin to identify pathways • Identify suppressors of mnn4 mutation • Extends pathway delineation • Identify cellular site of action of Mnn4p • Indicates potential mechanism • Describe phylogenetic distribution of MNN4 genes • Why do fungi place mannosylphosphate on surfaces?

24. MNN4-like genes are found in many fungal species

25. Summary • Cell surface hydrophobicity is an important mediator of adhesion in fungal cell virulence • Regulation of CSH phenotype is dependent on environmental conditions of cell • Understanding of Mnn4p function will allow us to understand how fungi can alter surface characteristics