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Similar to Transcription Factors Regulating Iron and Copper Homeostasis in Candida albicans
Similar to Transcription Factors Regulating Iron and Copper Homeostasis in Candida albicans (20)
Transcription Factors Regulating Iron and Copper Homeostasis in Candida albicans
- 1. Transcription factors involved in iron and copper homeostasis in Candida albicans Gunjan Mukesh Wig Supervisor: Professor Annette Cashmore
- 2. 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
- 3. Hyphal growth obtained in serum at 37˚ Budding yeast and pseudo-hyphal growth
- 4. 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
- 5. Sacharomyces cerevsiae Mac1pCu+ Cu+ Ctr1p Cu+ Ctr3p Cu+ Cu2+ Fe2+ Fe3+ Aft2p Aft1p Cu+ Ftr1pFet3p Cu+ Fe3+ Fe2+ Cu+ Cu+ Cu+
- 6. Candida albicans • CaMac1p – functional homologue of ScMac1p • Major Cu-responsive transcription factor • Iron uptake related genes regulator – SFU1, SEF1? CaMAC1p Cu2+ Cu2+ CaCTR1p Cu+ CaFtr1p CaFet34p Cu+ Fe3+ Fe2+ Cu+ Cu+ Cu+ Cu2+ Cu+ Fe2+ Fe3+ Sfu1p Sef1p ?
- 7. 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)
- 8. . Disruption of CuRE Sites Affects CaMAC1 Copper Regulation (Woodacre et al., 2008)
- 9. ScMAC1 ScMAC1 CuRE1 CuRE2 Target gene Forms homo-dimers Optimal transcriptional activity via ScMAC1p Molecular functioning of ScMAC1p
- 10. β-galactosidase assays demonstrated that only a single functional CuRE (Copper response element) is required for optimal copper responsive regulation of CaFRE7 and CaMAC1 by CaMAC1p in Candida albicans (Woodacre et al., 2008).
- 11. • 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
- 12. 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
- 13. 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 : AD 2. wt CaMAC1 : wt CaMAC1 3. wt CaMAC1 : NDel*CaMAC1 4. wt CaMAC1 : CDel*CaMAC1
- 14. Quantitative ONPG assay to measure β-Gal activity in Y2H- Results β- Gal activity 0 1 2 3 4 5 6 7 wt CaMac1p : wt CaMac1p wt CaMac1p : NDel*CaMac1p wt CaMac1p : CDel*CaMac1p Average β-Gal activity (3 biological replicates)
- 15. 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?
- 16. 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
- 17. Phase II- Role of Sef1p in iron uptake S. cerevisiae C. albicans Mac1pCu+ Cu+ Ctr1p Cu+ Ctr3p Cu+ Cu2+ Fe2+ Fe3+ Aft2p Aft1p Cu+ FFtr1p Fet3p Cu+ Fe3+ Fe2+ Cu+ Cu+ Cu+ CaMAC1p Cu2+ Cu2+ CaCTR1p Cu+ CaFtr1p CaFet34p Cu+ Fe3+ Fe2+ Cu+ Cu+ Cu+ Cu2+ Cu+ Fe2+ Fe3+ Sfu1p Sef1p ?
- 18. • 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
- 19. 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)
- 20. 1. Regulation of SEF1 – Is it Fe/Cu regulated? (Iron chelator)
- 21. 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
- 22. 2. What is the sef1 regulon? (FREs/CTR1/FET3/FTR1)
- 23. • Surface reductase converts 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 CaFtr1p CaFet34p Cu+ Fe3+ Fe2+ Cu+ Cu+ Cu+ CaCTR1p Cu+ Cu+ Cu+ Cu2+ Very high copper levels
- 24. • 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 CaFtr1p CaFet34p Cu+ Fe3+ Fe2+ Cu+ Cu+ Cu+ CaCTR1p
- 25. 3.Regulation of SEF1 – Is it regulated by transcription factors SFU1 or CaMAC1?
- 26. 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
- 27. 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 ?
- 28. 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
- 29. A big thank you to………… • Annette • Jon Baker • Alex Woodacre • Everyone in lab 121 Thank you all for listening!! All suggestions are welcome!
- 30. Yeast two hybrid Reporter gene Binding domain Activating domain Polymerase CaMac1p Activation sequence Reporter gene Binding domain Activating domain Polymerase CaMac1p Activation sequence
Editor's Notes
- Good afternoon everyone! I am Gunjan Wig and this talk will give you a brief overview about my PhD project. I am trying to study the role of … in the opportunistic pathogen C. albicans
- Obligate commensal eukaryotic fungus. It is part of the normal human flora and present in most people on the surface of the gastro intestinal tract and mouth. However it is the most common opportunistic pathogen and it primarily causes a superficial skin & mucosal membrane infection known as Candidiasis which is accompanied by redness, discomfort, itching & localised inflammation. It is commonly also called as oral or vaginal thrush. However in immunocompromised people such as cancer patients or AIDS patients or people who have had organ transplants- it can affect the eosophagus and cause a life-threatening systemic bloodstream infection termed Candidaemia. In US Candida is the 4th most-common causative agent of bloodstream infections & the mortality rate is as high as 49% Ca is intrinsically resistant to most anti-fungal drugs & additionally over a period of time it has also been observed to acquire resistance to the drugs that are being used for its treatment and hence the high mortality rate. It has great clinical relevance & hence its virulence is vastly studied.
- One of the most imp virulence factors in Ca is its pleiomorphic property. It can exist in 3 different morphological forms: unicellular budding yeast, multicellular hyphae & an intermediate peudo-hyphae form. It can switch between these forms during infection which may be an adaption mechanism to help survival & proliferation of the organism in the mammalian host. This demonstrates that the regulation of two virulence determinants, the reductive iron uptake system and the morphological form of C. albicans, are linked (Jeeves et. al., 2011).
- Sc diverged from Ca 140 million years ago. Ca lacks a complete sexual cycle and hence both conventional and molecular-genetic analysis have proved difficult. Today reverse-genetic approaches (genes are first identified by their sequence and then both genomic copies are are sequentially deleted or mutated) is used and therefore a well established model organism such as Sc is used. Sc is a budding yeast used in the baking & brewing industry and is a non-pathogenic fungi. The mechanism of Fe & Cu uptake has been well studied in this organism & Ca shows considerable homology to Sc, we use it as a model organism. The uptake mechanism is similar however the regulation differs considerably, presumably because Sc is a non-pathogen while Ca is an opportunistic pathogen. I will briefly explain the Fe & Cu homeostasis in Sc & then draw comparisons with the info that is currently available on Ca. This is a simplified view of a Sc cell. Fe & Cu are usually available in the host environment in their insoluble Ferric & Cupric forms and have to converted to their more soluble Ferrous & Cuprous forms. This is brought about by cell surface ferric reductase proteins. Cu is transported within the cell by Cu transporters and Fe is transported across the cell membrane via a multi-copper oxidase-permease complex where Fet3p is not an iron transporter but is a oxidase that is requires Cu for its functioning and it is also required for localisation of the iron transporter at the plasma membrane. Hence the Fe & Cu uptake is intrinsically linked and Cu starvation conditions within the cell lead to secondary Fe starvation conditions. Mac1p regulates most Cu related and some Fe related genes. Aft1p & Aft2p are the major iron-related gene regulators in Sc.
- Now lets look at Ca. It has a similar Fe & Cu uptake system, however some of the proteins are still unidentified. By previous work done in our lab we have identified a functional homologue of ScMac1p- CaMac1p. Its transcription is regulated in response to Cu levels. Ca lacks functional homologues of Aft1p & Aft2p and it is not clear as to what controls the Fe-related genes in Ca. There are two likely candidates. I will be focussing my attention on CaMac1p & Sef1p for the rest of the talk.
- A C. albicans MAC1∆/MAC1∆ mutant was constructed and through using β-galactosidase reporters, the C. albicans MAC1 (CaMAC1) protein was found to be required for the induction of key copper homeostasis genes during copper starvation, similar to the homologous MAC1 of S. cerevisiae. (Fig. 2). However while ScMAC1 is constitutively expressed, CaMAC1 is itself copper responsive. This may be a copper homeostasis adaptation by C. albicans, in response to this yeast occupying a niche as a human pathogen. Figure 2: β-Galactosidase Activity of Wild-type Promoter Constructs. Assays for β-galactosidase activity were performed on strains growing at exponential phase in copper-restricted MD containing 100 mM CuCl2 (black bars) or 0 mM CuCl2 (white bars). Results are shown for C. albicans BWP17 (wild-type) and MEM-m2 (MAC1∆/MAC1∆), each transformed with either plac-poly, CaCTR1 promoter–lacZ fusion, CaFRE7 promoter–lacZ fusion or CaMAC1 promoter–lacZ fusion. The means of three independent experiments are shown; error bars show 1SD (Woodacre et al., 2008).
- Disruption of CuRE Sites Affects CaMAC1 Copper Regulation Using site directed mutagenesis, CuRE mutants were created from the previously described reporter constructs (Fig. 2). Subsequent β-galactosidase assays demonstrated that only a single functional CuRE is required for the copper responsive regulation of CaFRE7 and CaMAC1 (Fig. 4). Figure 4: β-galactosidase Activity of Mutant CaFRE7 and CaMAC1 Promoter–lacZ Fusions. The CaFRE7 and CaMAC1 promoters were mutated by site directed mutagenesis, with either one or two CuREs mutated. A schematic representation to the left of the graph of the CaFRE7 and CaMAC1 promoters shows wild-type CuRE sequences as white boxes and mutated CuRE sequences as grey boxes. All mutant reporter plasmids were transformed into BWP17 (wild-type). Assays for β-galactosidase activity were performed on strains growing at exponential phase in copper-restricted MD containing 100 mM CuCl2 (black bars) or 0 mM CuCl2 (white bars). The means of three independent experiments are shown; error bars show 1SD (Woodacre et al., 2008). Figure: β-galactosidase Activity of Mutant CaFRE7 and CaMAC1 Promoter–lacZ Fusions.
- Optimal transcription activity by ScMac1p requires presence of two CuREs in the target promoters which could be because ScMac1p functions as a homodimer and each monomer of ScMac1p binds to one CuRE sequence in the DNA.
- However it was found that promoters of CaFRE7 & CaMAC1 containing only one CuRE were able to mediate wt Cu-responsive transcription indicating that only one CuRE was required for wt activation by CaMac1p- suggesting that dimerisation may not be essential for activation by CaMac1p in vitro in Ca and that it may not be able to form homodimers.
- I wanted to focus on the mechanism of action of this regulatory protein and how it undergoes multiple prt-prt & prt-DNA interactions that are modulated by Cu.
- The transcription factor has a conserved N-terminal DNA binding domain which is composed of approximately 40 aa, termed as the copper-fist domain & has been found to be essential for DNA binding activity of similar transcription factors in other fungi such as Ace1p and Amt1p in S. cerevisiae and C. glabrata respectively. The copper-fist domain shows presence of conserved histidine & cysteine residues which can be a possible Zn-finger motif that maybe capable of directly binding a single Zn ion Nuclear localisation signal sequence KKXRX15KKXK It shows presence of two cysteine-rich domains in the C-terminus termed as C1 and C2 or the Rep motifs which are also conserved in CaMac1p. The C1 & C2 motifs are separated by 42 residues in ScMac1p whereas it is separated by 61 residues in CaMac1p. They are involved in Cu responsive activation activity of the prt and are directly capable of binding four Cu ions In the C-terminus a D-helix has been identified which takes part in prt : prt interactions in Sc This theory will be tested in our lab using yeast two hybrid studies. CaMAC1 was cloned into both these vectors to test CaMAC1 : CaMAC1 protein interaction. In addition, two deletion mutants were created; one with 38 amino acids deleted from the N-terminus and another with 81 amino acids deleted from the C-terminus.
- A positive result was observed using the wild type sequence, demonstrating that CaMAC1 does indeed interact with itself. I decided to perform a quantitative assay to measure the prt : prt interaction
- We saw that when the N terminus was deleted the prt : prt interaction decreased by apporx 51% When C terminus was deleted the prt : prt interaction decreased by approx 87% Standard deviation was calculated. 0.267706, 0.432049, 0.163299 P value was calculated using a Student’s T test was found to be 0.002543 and was found to be statistically significant.
- When a similar experiment was performed in Sc they observed different results. They predicted that this could be because during folding of the prt the N terminal domain was probably undergoing an intramolecular interaction with the C terminal domain and therefore masking it binding ability. So our results indicate that its likely that…
- To recap your memory, I mentioned that Aft1p & Aft2p are the major iron related gene regulators in Sc however Ca lacks functional homologues of these so there are two likely candidates in Candida and I will be focussing my attention on Sef1p which is a putative transcription activator
- I wanted to focus on the mechanism of action of this regulatory protein and how it undergoes multiple prt-prt & prt-DNA interactions that are modulated by Cu.
- The loss of ability to form hyphae is interesting as I have already mentioned before that Rose Jeeves has demonstrated that the regulation the reductive iron uptake system and the morphological form of C. albicans, are linked (Jeeves et. al., 2011).
- Quantify SEF1 transcript in SC5314 (wt) using qPCR in different conditions. Fe + Cu (high Fe + Cu). Fe + BCS (high Fe – low Cu) Cu + BPS (high copper – low iron)
- The cell will still be able to get iron from other sources.
- Quantify level of SEF1 transcript in sfu1∆∆, mac1∆∆ and wild-type in low iron conditions.
- All this is very initial data and I am still in the process of trying to figure out how these regulators are inter-linked however I would like to share what I think is possibly happening based on the observations I have made so far
- Thereby identify position of SEF1 in the regulatory network.
- pGBT9 contains Gal4 or Gal1 DNA binding domain? Because the strains contain the reporter genes fused to Gal1 promoters containing Gal1 activation sequence.