Post on 07-Jan-2016
description
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