1. 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. Initial screen: 23 complementation groups Cloning and sequencing Conserved pathways Secretory pathway Cell cycle Signal transduction Metabolism

6. Why yeast? 1 st 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 4 th most common post- operative nosocomial blood borne infection

8. Surface hydrophobicity enables fungi to adhere to surfaces Cell PopulationPhenotype 23ºCHydrophobic 37ºCHydrophilic HyphaeHydrophobic 37ºC shiftRapid shift to hydrophobic, then hydrophilic

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 cytoplasm cell wall 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 MNN4 MPAMNN4MPAMNN4 Wild-type yeast MNN4/MNN4 MPA sensitive Transform to MPA R MNN4/mnn4 MPA resistant Counterselect MPA S MNN4/mnn4 MPA sensitive Loss of MNN4  derivative lacking acid-labile region  potentially always hydrophilic Repeat!

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

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

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. cerevisiae and C. albicans Mnn4p 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 Mnn4p Gene “X” Gene “Y” Phosphate addition

19. Plan to identify a function for MNN4 Characterize interactions common between S. cerevisiae and C. albicans Mnn4p 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 Mnn4pGene “X”Gene “Y” Phosphate addition X First mutation (mnn4) blocks here Second mutation allows recovery of phenotype

21. Plan to identify a function for MNN4 Characterize interactions common between S. cerevisiae and C. albicans Mnn4p 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. cerevisiae and C. albicans Mnn4p 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