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