Download - Wig-Kukreja PhD presentation
Transcription factors involved in iron and copper homeostasis in
Candida albicans
Gunjan Mukesh Wig
Supervisor: Professor Annette Cashmore
Introduction
• Obligate commensal eukaryote
• Gastro-intestinal tract & mouth
• Most common opportunistic pathogen
• Candidiasis
• Life-threatening infections in immuno-compromised hosts
• Candidaemia- 49% mortality rate
• Mammalian host’s defence mechanism– restriction
• Defects in Cu uptake system- impairment of Fe uptake
• Essential for virulence in mouse models
• Cu and Fe play major role in virulence
Hyphal growth obtained in serum at 37˚
Budding yeast and pseudo-hyphal growth
Why use Sacharomyces cerevsiae?
• Candida is a diploid that lacks a complete sexual cycle
• Reverse genetic-approaches are used to study
• Well-established model organism
• S. cerevisiae -Budding yeast used in baking & brewing
• Same Family- Saccharomycetaceae
• Considerable homology
• Regulation differs
Sacharomyces cerevsiae
Mac1pCu+
Cu+
Ctr1p Cu+
Ctr3p Cu+
Cu2+
Fe2+
Fe3+
Aft2p
Aft1p
Cu+
Ftr1p
Fet3p
Cu+
Fe3+
Fe2+
Cu+
Cu+
Cu+
Candida albicans• CaMac1p – functional
homologue of ScMac1p
• Major Cu-responsive transcription factor
• Iron uptake related genes regulator –SFU1, SEF1?
CaMAC1p
Cu2+
Cu2+
CaCTR1p Cu+
CaFtr1
p
CaFet34p
Cu+
Fe3+
Fe2+
Cu+
Cu+
Cu+
Cu2+
Cu+Fe2+
Fe3+
Sfu1p
Sef1p ?
CaMAC1 Regulates C. albicans Copper Homeostasis Genes
• CaMAC1p was found to activate CaCTR1, CaFRE7 and CaMAC1 itself through promoter binding, in response to low copper conditions.
(Woodacre et al., 2008)
.
Disruption of CuRE Sites Affects CaMAC1 Copper Regulation
(Woodacre et al., 2008)
ScMAC1 ScMAC1
CuRE1 CuRE2 Target gene
Forms homo-dimers
Optimal transcriptional activity via ScMAC1p
Molecular functioning of ScMAC1p
β-galactosidase assays demonstrated that only a single functional CuRE (Copper response element) is required for optimal copper responsive regulation of CaFRE7and CaMAC1 by CaMAC1p in Candida albicans (Woodacre et al., 2008).
• Analyse whether or not CaMac1p can form dimers
• Is dimerisation essential for its functioning in vivo?
• Is the regulation of SEF1 dependent on iron or copper levels?
• How are the regulators linked? (Sef1p/Sfu1p/Mac1p)
• What is the sef1 regulon? (FREs/CTR1/FET3/FTR1)
Phase I
Phase II
C1 C2Cu fist NLS D
DNA binding domain
1 41 155 -177 264-279 322-337 388-406
417
RepI RepII D-helix
CxCxxxxCxCxxCxxH
C1 C2Cu fist
1 41 209-223 287-297
431
C1 C2
Zn finger
S. cerevisiae Mac1p
C. albicans Mac1p
C1 C2Cu fist
1 41 209-223 287-297
431
C1 C2
351 CDel 431NDel
• CaMAC1 shares 35% sequence similarity with ScMAC1
• 73.9% probability of being a nuclear protein
• ScMac1p functions as a homodimer
• Dimerisation may not be essential for activation by CaMac1p?
• Yeast two hybrid assay to test protien:protein interaction
Mac1 protein
Y2H Filter paper assay- Results• Yeast two hybrid (Y2H)
transformants were replica plated on filter paper
• Blue colouration indicates protein : protein interaction
1. wt CaMAC1 : AD2. wt CaMAC1 : wt CaMAC13. wt CaMAC1 : NDel*CaMAC14. wt CaMAC1 : CDel*CaMAC1
Quantitative ONPG assay to measure β-Gal activity in Y2H- Results
β- Gal
activity
0
1
2
3
4
5
6
7
wt CaMac1p : wtCaMac1p
wt CaMac1p :NDel*CaMac1p
wt CaMac1p :CDel*CaMac1p
Average β-Gal activity
(3 biological replicates)
Discussion• CaMac1p is capable of self-interaction and can
form CaMac1p : CaMac1p dimers
• C-terminus is involved in protein : protein interactions
• N-terminus domain of CaMac1p is likely helping to increase binding activity of C-terminus domain
• In S. cerevisiae – deletion of N-terminus domain showed increased interaction via C-terminus which is different from our observation in C.albicans
• CaMac1p folds in a different manner than ScMac1p?
Previous evidence…
Limiting Iron
Aft1/ Aft2
Sfu1
Sef1
Limiting Copper
Mac1
Iron acquisition & uptake regulon (FREs/CTR1/FET3/FTR1)
S. cerevisiae C. albicans Both species
Phase II- Role of Sef1p in iron uptake
S. cerevisiae
C. albicans
Mac1pCu+
Cu+
Ctr1p Cu+
Ctr3p Cu+
Cu2+
Fe2+
Fe3+
Aft2p
Aft1p
Cu+
F Ftr1
p
Fet3p
Cu+
Fe3+
Fe2+
Cu+
Cu+
Cu+
CaMAC1p
Cu2+
Cu2+
CaCTR1p Cu+
CaFtr1
p
CaFet34p
Cu+
Fe3+
Fe2+
Cu+
Cu+
Cu+
Cu2+
Cu+Fe2+
Fe3+
Sfu1p
Sef1p ?
• Analyse whether or not CaMac1p can form dimers
• Is dimerisation essential for its functioning in vivo
1. Is the regulation of SEF1 dependent on iron or copper levels?
2. What is the sef1 regulon? (FREs/CTR1/FET3/FTR1)
3. How are the regulators linked? (Sef1p/Sfu1p/Mac1p)
Phase I
Phase II
Phenotypic plate observations -Results
CONDITION Wild type SC5314
Mutant sef1∆∆
YPA (pH 6.4) XXXX XXXX
Very high Copper (CuCl2 5mM) XX
Very high Iron (FeCl3 5mM) XX XX
BPS (iron chelator 100 µM) XXX X
BCS (copper chelator 100 µM) XXXX XXXX
BPS and BCS XXX X
• sef1ΔΔ shows loss of ability to form hyphae in response to serum at 37°
(Dr. Jonathan Baker)
1. Regulation of SEF1 – Is it Fe/Cu regulated?
(Iron chelator)
Level of SEF1 transcript in high and low Fe & Cu conditions- Results
10
11
12
13
14
15
16
17
18
WT ↑Fe↑Cu WT ↑Fe↓Cu WT ↓Fe↑Cu
WT ↑Fe↑Cu
WT ↑Fe↓Cu
WT ↓Fe↑Cu
2. What is the sef1 regulon? (FREs/CTR1/FET3/FTR1)
• Surface reductaseconverts Fe3+ Cu2+ to soluble Fe2+ Cu1+ which can be imported by transporter proteins.
• At very high copper levels in media, Internal copper levels rise leading to toxicity and growth defect.
Sef1p
Cu+
Fe2+
Fe3+
Cu+
Fe2+
Cu+
Fe2+
Wild-type cell
CaFtr1
p
CaFet34p
Cu+
Fe3+
Fe2+
Cu+
Cu+
Cu+
CaCTR1pCu+
Cu+Cu+
Cu2+
Very high copper levels
• If a functional Sef1p is required for the expression of surface reductases or copper transporter….
• ….then the mutant will lack these surface proteins and toxicity would be reduced as little/no copper (Cu2+) can enter the cell.
Sef1p
Fe3+
Cu2+ Fe3+
Cu2+Fe3+
Cu2+
sef1∆∆ mutant cell
Hypothesis
CaFtr1
p
CaFet34p
Cu+
Fe3+
Fe2+
Cu+
Cu+
Cu+
CaCTR1p
3.Regulation of SEF1 – Is it regulated by transcription factors SFU1 or CaMAC1?
Level of SEF1 transcript in high and low Fe conditions in wt, sfu1∆∆ and mac1∆∆ - Results
wt sfu1∆∆ mac1∆∆
10
12
14
16
18
20
22
↓Fe
Summary
Limiting Iron
Aft1/ Aft2
Sfu1
Sef1
Limiting Copper
Mac1
Iron and Copper acquisition & uptake regulon (FREs/CTR1/FET3/FTR1)
S. cerevisiae C. albicans Both species
FREs/ Ctr1 ?
Current and future work
Repeat RT-PCR using a more controlled media
Perform RT-PCR using sef1∆∆ to identify targets of sef1p (FREs, Ctr1)
Study interaction between SEF1,MAC1 & SFU1
• Create sef1∆∆sfu1∆∆ double mutant
A big thank you to…………• Annette
• Jon Baker
• Alex Woodacre
• Everyone in lab 121
Thank you all for listening!!
All suggestions are welcome!
Yeast two hybrid
Reporter gene
Binding domain
Activating domain
Polymerase
CaMac1p
Activation sequence
Reporter gene
Binding domain
Activating domain
Polymerase
CaMac1p
Activation sequence