David SingletonBiology YCP

March 11, 2009

My BackgroundContacts for mentoring and networkingInvolvement in Society activities

http://www.microbiologycareers.org/http://www.ascb.org/newsfiles/jobhunt.pdfhttp://sciencecareers.sciencemag.org/

Choices for grad school/professional school/post doc

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, 1926Prokaryotic microorganisms; many

similarities in biochemistryEukaryotic microorganisms; many similarities

in cell biology and development

Why yeast?

Why yeast?Initial screen: 23

complementation groups

Cloning and sequencing

Conserved pathwaysSecretory pathwayCell cycleSignal transductionMetabolism

Why yeast?1st sequenced

eukaryoteGene deletion

projectProtein interaction

webProtein localizationTranscription

profiling

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

Surface hydrophobicity enables fungi to adhere to surfaces

Cell Population Phenotype

23ºC Hydrophobic

37ºC Hydrophilic

Hyphae Hydrophobic

37ºC shift Rapid shift to hydrophobic, then hydrophilic

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

Fungal N-glycosylation is a virulence factor

Post-translational addition of sugars

Acid-hydrolyzable phosphate linkage distinguishes acid-labile and acid-stable regions

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

Construction of mnn4 serotype B deletion strain

MNN4MNN4

MPA

MNN4MPA

MNN4

Wild-type yeastMNN4/MNN4MPA sensitive

Transform to MPAR

MNN4/mnn4MPA resistant

Counterselect MPAS

MNN4/mnn4MPA sensitive

Loss of MNN4 derivative lacking acid-labile region potentially always hydrophilic Repeat!

B6.1 epitope

B6 epitope

Phenotypic analysis of mnn4 deletion strain

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

Summary of mnn4 mutant phenotype

Loss of detectable mannosylphosphate; no acid labile addition

Surprising increase in hydrophobicityPerturbation of remaining acid-stable region in mutantChange in in vivo fitness of derivative in co-infection

model

Potential functions for Mnn4pCatalytic: shares small region of

glycosyltransferase homologyPredict Golgi localization, and

raises potential for in vitro reconstitution

Regulatory: supported by genetic and mass screening studiesNo localization prediction, but

allows potential for overall control of cell surface properties

Plan to identify a function for MNN4Characterize interactions common between S. cerevisiae

and C. albicans Mnn4pCan begin to identify pathways

Identify suppressors of mnn4 mutationExtends pathway delineation

Identify cellular site of action of Mnn4pIndicates potential mechanism

Describe phylogenetic distribution of MNN4 genesWhy do fungi place mannosylphosphate on

surfaces?

Protein Interaction Studies

Mnn4p

Gene “X”

Gene “Y”

Phosphate addition

Plan to identify a function for MNN4Characterize interactions common between S. cerevisiae

and C. albicans Mnn4pCan begin to identify pathways

Identify suppressors of mnn4 mutationExtends pathway delineation

Identify cellular site of action of Mnn4pIndicates potential mechanism

Describe phylogenetic distribution of MNN4 genesWhy do fungi place mannosylphosphate on

surfaces?

Genetic Suppression

Mnn4p Gene “X” Gene “Y”

Phosphate additionX

First mutation (mnn4) blocks here

Second mutation allows recovery of

phenotype

Plan to identify a function for MNN4Characterize interactions common between S. cerevisiae

and C. albicans Mnn4pCan begin to identify pathways

Identify suppressors of mnn4 mutationExtends pathway delineation

Identify cellular site of action of Mnn4pIndicates potential mechanism

Describe phylogenetic distribution of MNN4 genesWhy do fungi place mannosylphosphate on

surfaces?

Localization using Yellow Fluorescent Protein

Lee SA, Khalique Z, Gale CA, Wong B.Med Mycol. 2005 Aug;43(5):423-30.

Plan to identify a function for MNN4Characterize interactions common between S. cerevisiae

and C. albicans Mnn4pCan begin to identify pathways

Identify suppressors of mnn4 mutationExtends pathway delineation

Identify cellular site of action of Mnn4pIndicates potential mechanism

Describe phylogenetic distribution of MNN4 genesWhy do fungi place mannosylphosphate on

surfaces?

MNN4-like genes are found in many fungal species

SummaryCell surface hydrophobicity is an important mediator

of adhesion in fungal cell virulenceRegulation of CSH phenotype is dependent on

environmental conditions of cellUnderstanding of Mnn4p function will allow us to

understand how fungi can alter surface characteristics