Transcript Protein glycosylation in pathogenic and non

David Singleton
Biology YCP
March 11, 2009
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
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
Why yeast?
Why yeast?
 Initial screen: 23
complementation groups
 Cloning and sequencing
 Conserved pathways
 Secretory pathway
 Cell cycle
 Signal transduction
 Metabolism
Why yeast?
 1st sequenced eukaryote
 Gene deletion project
 Protein interaction web
 Protein localization
 Transcription 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
cytoplasm
mannoproteins
• Fibrils are longer and
loosely packed on
hydrophilic cells
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
MNN4
MNN4
Wild-type yeast
MNN4/MNN4
MPA sensitive
MPA
Transform to MPAR
MNN4
MPA
MNN4/mnn4
MPA resistant
Counterselect MPAS
Loss of MNN4 
derivative lacking acid-labile
region 
potentially always hydrophilic
MNN4
MNN4/mnn4
MPA sensitive
Repeat!
Phenotypic analysis of mnn4
deletion strain
B6 epitope
B6.1 epitope
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
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
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
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?
Protein Interaction Studies
Gene “X”
Mnn4p
Gene “Y”
Phosphate addition
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?
Genetic Suppression
Mnn4p
X
Gene “X”
First mutation (mnn4)
blocks here
Gene “Y”
Second mutation
allows recovery of
phenotype
Phosphate
addition
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?
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 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?
MNN4-like genes are found in
many fungal species
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