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Department of Genetics, University of Leicester, Leicester LE1 7RH, UK
Correspondence
Annette M. Cashmore
amc19{at}le.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Currently, the most common species causing candidaemia is Candida albicans (Pfaller & Diekema, 2002; Tortorano et al., 2004). The effect of infection is significant, and the mortality rate of Candida bloodstream infections was recently estimated to be 49 % (Gudlaugsson et al., 2003).
C. albicans infection is associated with risk factors that decrease the competence of the patient's immune system, e.g. surgery, intravascular catheterization and antibiotic treatment (Tortorano et al., 2004). Approximately 30–50 % of healthy people are colonized by Candida species, and several properties of C. albicans are associated with the change from commensal organism to pathogen, including the ability to grow in yeast and hyphal forms, adherence to surfaces via adhesins and resistance to anti-fungal drugs (Navarro-Garcia et al., 2001). The ability of the human body to restrict the amount of free iron available to invading micro-organisms is an important defence against infection, and the ability of pathogenic organisms to acquire iron from the human host is a well-studied virulence factor in many bacterial and fungal pathogens (Schaible & Kaufmann, 2004). In C. albicans the iron transporter gene CaFTR1 has been shown to be essential for virulence in the mouse systemic model of candidiasis (Ramanan & Wang, 2000).
Iron and copper are essential nutrients for almost all living organisms and are important for a wide variety of cellular processes (reviewed by Crichton & Pierre, 2001). The redox potential of these transition metals makes them ideal as components of molecules involved in electron transfer such as cytochrome c oxidase, which requires copper, haem, magnesium and zinc molecules to function (Tsukihara et al., 1995). Excess copper and iron in the cell can also increase production of toxic hydroxyl radicals that can damage DNA and proteins (Urbanski & Beresewicz, 2000). Metal toxicity in C. albicans is prevented by ATPase transporters, which remove metals from the cell (Riggle & Kumamoto, 2000; Weissman et al., 2000), and Cu/Zn-containing superoxide dismutases, which detoxify superoxides produced during Fenton chemistry (Hwang et al., 2002). Copper and iron homeostasis must therefore be tightly regulated to maintain these metals at the correct physiological concentrations without causing any toxic effects to the cell.
Copper homeostasis and iron homeostasis in C. albicans are intrinsically linked due to the requirement of copper for effective iron uptake (Knight et al., 2002). Copper and iron are most readily available in the ferric and cupric forms but must first be reduced by ferric reductases in order to be taken up by the high-affinity copper and iron transporters CaCTR1 and CaFTR1 (Knight et al., 2002). Previous work in our laboratory has shown that when copper levels are lowered by deletion of the copper transporter gene CaCTR1, high-affinity iron uptake decreases by 96 % (Marvin et al., 2004). It has been demonstrated that copper-dependent oxidase activity is required for high-affinity iron uptake in C. albicans, suggesting the presence of a permease/oxidase complex similar to the Ftr1p/Fet3p complex of S. cerevisiae (Knight et al., 2002; Stearman et al., 1996). There are five homologues of the FET3 gene, including CaFET3, which is required for growth in low-iron media, but the effects of CaFET3 on high-affinity iron uptake have not been determined, and one or more of the other multicopper oxidases may therefore be involved in the acquisition of iron (Braun et al., 2005; Eck et al., 1999). In addition to the role of copper in iron homeostasis and Cu/Zn superoxide dismutases, a number of recent transcriptional profiling experiments have indicated roles for copper transport and metabolism genes in responses to alkaline pH (Bensen et al., 2004), prostaglandin (Levitin & Whiteway, 2007), phagocytosis by macrophages (Lorenz et al., 2004) and fluconazole resistance (Xu et al., 2006). The copper transporter gene CaCTR1 and the transcriptional activator gene CaMAC1 have also been shown, by our laboratory and others, to affect filamentous growth (Huang et al., 2006; Marvin et al., 2003). Copper metabolism therefore has an important part to play in the response of C. albicans to the host environment, and is involved in many of the processes associated with infection.
Previous work in our laboratory identified the high-affinity copper transporter gene CaCTR1 and demonstrated that CaCTR1 transcription increases under copper-starvation conditions (Marvin et al., 2003). We have previously shown that three sequences identical to the S. cerevisiae copper response elements (CuREs) are present in the promoter of CaCTR1 and that heterologous expression of CaCTR1 in S. cerevisiae requires the transcriptional activator gene ScMAC1 (Marvin et al., 2003). We have also identified the CaMAC1 gene as a homologue of ScMAC1, and we have shown that transcription of CaCTR1 requires CaMAC1 (Marvin et al., 2004).
ScMac1p regulates genes involved in the copper and iron uptake systems in response to copper levels (De Freitas et al., 2004; Gross et al., 2000). ScMac1p regulates its target genes by direct binding to consensus binding sites in the promoter, known as CuREs (Martins et al., 1998; Yamaguchi-Iwai et al., 1997). Two CuREs are required for copper-responsive transcription of ScCTR1, and mutation of the CuREs in the promoter of ScFRE1 also abolishes copper-responsive transcription (Jensen et al., 1998; Labbé et al., 1997). One CuRE in the ScFRE7 promoter was sufficient for copper-responsive transcription, but this level of transcription was significantly reduced compared with a wild-type ScFRE7 promoter containing three CuREs (Martins et al., 1998). A truncated ScCTR3 promoter of 214 bp containing just one CuRE was able to activate lacZ transcription twofold under low-copper conditions, but transcription was again significantly reduced compared with the eightfold activation of lacZ by a full-length ScCTR3 promoter of 1116 bp (Labbé et al., 1997). At least two CuREs are therefore required to produce wild-type expression levels of ScCTR1, ScCTR3, ScFRE1 and ScFRE7.
The aim of this study was to identify additional genes in the CaMac1p regulon and to determine whether or not, in C. albicans, the CuRE sequences play a functional role in regulation by copper and CaMac1p. We show here that at least one CuRE is required for the copper-dependent regulation of CaCTR1 and the ferric reductase gene CaFRE7 by CaMac1p. The CaMAC1 gene itself is also transcriptionally autoregulated in response to copper concentrations, and this involves the single CuRE in the CaMAC1 promoter. These findings are in contrast to observations in the model organism S. cerevisiae, in which the ScMAC1 gene is constitutively transcribed and at least two CuREs are required for wild-type levels of copper-dependent transcription of ScCTR1, ScCTR3 and ScFRE7 (Jensen et al., 1998; Labbé et al., 1997; Martins et al., 1998; Zhu et al., 1998). The differences in copper regulation in S. cerevisiae and C. albicans reflect the contrasting natural environments of these two organisms. Copper regulation in C. albicans may be important for adaptation to the human host and in the subsequent disease process.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and were routinely cultured at 30 °C in yeast peptone media containing 2 % glucose (YPD). Synthetic defined medium (SD) was used for selection and contained 2 % (w/v) Bactoagar, 0.67 % (w/v) yeast nitrogen base without amino acids (Bio 101) and 2 % (v/v) glucose, and was supplemented with the appropriate amino acids. Arginine and histidine were added to a final concentration of 20 µg ml–1, and uridine was added to a final concentration of 50 µg ml–1. SD supplemented with 1 mg ml–1 5-fluoroorotic acid (5-FOA) and 50 µg uridine ml–1 was used to counterselect for CaURA3. Minimal defined medium (MD) was used to grow cultures for RNA extraction and for β-galactosidase assays, and was based on Wickerham's medium (Wickerham, 1946) with the addition of 20 mM tri-sodium citrate (pH 4.2) and the omission of niacin, riboflavin and para-aminobenzoic acid. Copper-restricted MD also contained 50 µM bathocuproinedisulfonic acid (BCS) and 100 µM FeCl3. Iron-restricted MD contained 50 µM bathophenanthrolinedisulfonic acid (BPS) and 100 µM CuCl2. The Escherichia coli strain XL10 Gold (Stratagene) was used for all molecular cloning and was grown in Luria–Bertani (LB) medium at 37 °C. Ampicillin was used as a selective agent at a final concentration of 100 µg ml–1. All liquid cultures underwent shaking at 200 r.p.m.
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). RNA was electrophoresed on a 1.5 % denaturing formaldehyde gel and transferred to a nylon filter by Northern blotting overnight. The filters were hybridized overnight at 65 °C in Church Gilbert's buffer (0.5 M Na2HPO4, 0.5 M NaH2PO4, 7 % SDS, 1 mM EDTA) with probes labelled with [
-32P]dCTP using the Klenow fragment of DNA polymerase I and random hexamers as primers. Probes were constructed by PCR using the primers listed in Table 2. Filters were washed with 3x saline sodium citrate (SSC)/0.1 % (w/v) SDS and exposed to X-ray film at –80 °C. The blots were then stripped of probe by washing the filters with 2–3 changes of boiling 0.1 % SDS and rinsing with 2x SSC. The blots were then hybridized with [-32P]dCTP-labelled CaACT1 probes as a loading control. The intensity of the bands on the autoradiographs was estimated using Image J densitometry analysis software (Abramoff et al., 2004).
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. The PCR products and the reporter plasmid plac-poly (A. J. Brown, personal communication) were digested with either SalI and XmaI (CaCTR1) or XhoI and XmaI (CaFRE7 and CaMAC1). The promoter fragments were ligated to the digested plasmid and transformed into XL10 Gold E. coli using the calcium chloride method (Mandel & Higa, 1970). Recombinant plasmid DNA was isolated using the Qiagen Miniprep extraction kit and each plasmid was transformed into BWP17 and FOA-treated MEM-m2 (mac1
/mac1) using the lithium acetate method, as described elsewhere (Gietz & Woods, 2002). The plac-poly plasmid integrates into the genome at CaRPS10 in the same way as CIp10 (Murad et al., 2000), and integration of one copy of all the plasmids was confirmed by Southern blotting using a fragment of the plac-poly bla gene as a probe (data not shown).
Site-directed mutagenesis.
The existing reporter plasmids containing the promoters of CaCTR1 (AWC1), CaFRE7 (AWF1) and CaMAC1 (AWM1) were used as templates to generate reporter plasmids containing mutated CuRE sequences. The PCR-based mutagenesis strategy was modelled on the QuikChange XL site-directed mutagenesis kit from Stratagene. Mutagenesis primers (Table 2) were designed to incorporate mutations of two or three bases in one of the CuRE sequences to alter it from the consensus sequence TTTGC(T/G)C(A/G). Previous work has shown that any deviation from this consensus sequence abolishes binding of ScMac1p and results in the loss of copper-dependent regulation in β-galactosidase reporter assays (Jensen et al., 1998; Labbé et al., 1997; Martins et al., 1998; Yamaguchi-Iwai et al., 1997). The mutagenesis primers all had a melting temperature (Tm) of at least 68 °C, and each pair of primers (forward and reverse) was complementary, and in opposite orientations. Pairs of primers were used to amplify both strands of the template reporter plasmid (AWC1, AWF1 or AWM1) using BIO-X-ACT Long DNA polymerase (BioLine), and the high Tm of the primers ensured that the primers containing the mutations remained annealed to the template throughout the extension step. Following amplification, the methylated template DNA was digested into small fragments with DpnI and the newly synthesized DNA was transformed into XL10 Gold E. coli. The mutations were confirmed by sequencing and the mutant plasmid was transformed into BWP17 (wild-type). Integration of one copy of the reporter plasmid was confirmed by Southern blotting (data not shown).
In the CaCTR1 promoter, CuRE 1 (located 397 bp upstream of the CaCTR1 ORF) was mutated to give strain AWC2, CuRE 2 (located at –275 bp) was mutated to give strain AWC3 and CuRE 3 (at –237 bp) was mutated to give strain AWC4. The single mutants were then used as templates to generate strains AWC 5–8 with the mutations in the CuREs indicated in Table 1. Table 1 also indicates the mutations made in the promoters of CaFRE7 and CaMAC1 to generate strains AWF2–4 and AWM2.
Assay for β-galactosidase activity.
Cultures of reporter strains were grown to exponential phase in 5 ml MD with different concentrations of copper, and were assayed for β-galactosidase activity by the permeabilized cell method, as described elsewhere (Rupp, 2002).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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3' direction or the 3'5' direction. The results of both searches were collated and 20 ORFs were identified that contained two or more putative binding sites in their promoters (Table 3). The genes identified in the search have a wide variety of functions, and 25 % are involved in copper and iron metabolism. They include the copper transporter gene CaCTR1, the iron permease gene CaFTR2, two ferric reductase-like genes (CaFRE3 and CaFRE7) and the iron-responsive transcriptional repressor gene SFU1. We have demonstrated previously that CaCTR1 (orf19.3646) is regulated by copper and CaMac1p (Marvin et al., 2004). It is interesting to note that the upstream motif search revealed that the CaMAC1 promoter itself contains a CuRE sequence 167 bp upstream of the ORF.
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). The putative CuREs in the promoters of CaFRE3 and CaFRE7 are also located in this region of the promoter, but the CuRE sequences in the SFU1 promoter are outside the normal locations for transcription factor binding sites. Orf19.7078 was also analysed because it is divergently transcribed from CaFRE7 and the two genes share their promoter and the two CuREs within it, although the CuRE sequences are approximately 900 bp upstream of orf19.7078.
Previous analysis of the putative C. albicans ferric reductases in our laboratory has placed CaFRE3 in a group of ferric reductases that are most similar to ScFRE1–6, and has also shown that CaFRE7 is the C. albicans ferric reductase gene that is most similar to ScFRE7 (unpublished data). SFU1 encodes a GATA-type transcriptional repressor that represses several genes with functions in iron uptake, including the ferric reductases FRE1 and FRE2, iron permeases and multicopper oxidases (Lan et al., 2004). orf19.6238 has no known function, but AOX2 encodes an alternative oxidase involved in cyanide-resistant respiration, and accepts electrons from the electron transfer chain (Huh & Kang, 2001).
Northern blot analysis of genes containing two CuRE sequences in their promoters
Total RNA was extracted from cultures of BWP17 (wild-type) and mac1/mac1 strains grown in copper-restricted MD media with and without additional 100 µM CuCl2 or in iron-restricted MD media with and without additional 100 µM FeCl3. Northern blots were hybridized with [-32P]-labelled fragments of the ORFs of CaFRE7, CaMAC1, SFU1, AOX2, CaFRE3, orf19.6238 and orf19.7078. The blots were also hybridized with CaACT1 as a loading control (see Methods and Table 2 for probe sequences).
Transcripts for CaFRE7 were only detected at low copper levels in the wild-type (BWP17), and no transcript was visible in the absence of CaMAC1 (Fig. 1). This indicates that CaFRE7 transcription is activated by CaMac1p in response to copper starvation, and this evidence suggests that CaFRE7 may be the functional homologue of ScFRE7 (Martins et al., 1998). We also demonstrated that CaMAC1 transcription in the wild-type is increased approximately fivefold in response to low copper levels and confirmed that no transcript is detected in the mac1/mac1 mutant (Fig. 1). There was no difference in the levels of AOX2 transcript under different copper conditions in the wild-type, and no transcript could be detected in the mac1/mac1 strain (Fig. 1).
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). SFU1 transcripts in the mac1/mac1 mutant were more abundant under all conditions tested, but regulation of SFU1 transcription by copper and iron levels was maintained. Transcription of SFU1 in the mac1/mac1 mutant was repressed threefold under low-copper conditions and sixfold under low-iron conditions (Fig. 2). A transcript for CaFRE3 could not be detected, and no change in the expression levels of orf19.6238 or orf19.7078 was observed in response to copper, iron or CaMAC1 (data not shown).
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β-Galactosidase reporter constructs containing the promoters of CaCTR1, CaFRE7 and CaMAC1 were transformed into BWP17 (wild-type) and mac1/mac1. Integration of one copy of the plasmid into the genome at CaRPS10 was confirmed by PCR and Southern blotting (data not shown). The reporter constructs with CaCTR1, CaFRE7 and CaMAC1 promoters showed an increase in β-galactosidase activity of 3.1-fold, 4.4-fold and 2.8-fold, respectively, under low-copper conditions in BWP17 (Fig. 3). In the mac1/mac1 strain, all constructs showed lower levels of activity than in the wild-type, and copper-dependent regulation of activity was abolished (Fig. 3). This data shows that transcription of CaCTR1, CaFRE7 and CaMAC1 is activated by CaMac1p in response to copper starvation.
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). The reporter strains were grown in copper-restricted MD media containing high and low levels of copper, and β-galactosidase activity was measured as described in Methods.
When only CuRE 1 was functioning (AWC7), there was a higher level of expression compared with the wild-type CaCTR1 promoter (Fig. 4). The amount of β-galactosidase activity increased under high-copper conditions from 1.68 units µl–1 for the wild-type promoter to 2.35 units µl–1 for AWC7, and from 5.27 to 10.93 units µl–1 under low-copper conditions. When only CuRE 2 was functioning (AWC6), there was a decrease in β-galactosidase activity compared with the wild-type promoter (Fig. 4). Activity decreased by 23 % under high-copper and by 60 % under low-copper conditions. When only CuRE 3 was functioning (AWC5), β-galactosidase activity decreased by 65–85 % compared with the wild-type and there was no difference in activity in response to copper levels (Fig. 4). In a mutant without any functioning CuRE sequences (AWC 8), β-galactosidase activity was also reduced by 65–85 % compared with the wild-type and was not copper-responsive (Fig. 4). This level of activity was similar to the activity of the CaCTR1 promoter–lacZ reporter in a mac1/mac1 strain (Fig. 3).
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). When CuREs 1 and 3 were functioning (AWC3), β-galactosidase activity was not significantly affected by high copper levels, but activity did show an increase of 40 % at low copper concentrations compared with the wild-type promoter (Fig. 4). When CuREs 2 and 3 were functioning (AWC2), there was a decrease of 
70 % in β-galactosidase activity under both high- and low-copper conditions (Fig. 4). Although the overall level of expression was lower in this construct, β-galactosidase activity was still copper-regulated and was higher under low-copper conditions. This evidence demonstrates that one copy of CuRE 1 or 2 in the CaCTR1 promoter is sufficient but necessary for copper-responsive activation of the lacZ gene. A reporter construct containing only CuRE 1 in the promoter demonstrated levels of copper-responsive expression that were at least equal to those of the wild-type promoter. The CuRE sequences do not contribute equally to the activation of lacZ; CuRE 1 had the most effect on transcription and CuRE 3 the least effect. This indicates that a complex interaction is taking place between the CaMac1 protein and the CuRE sequences that is influenced by different combinations of CuREs.
One CuRE is necessary and sufficient for activation of CaFRE7 and CaMAC1 under low-copper conditions
The CaFRE7 promoter–lacZ reporter construct was used as a template for site-directed mutagenesis of the CuRE sequences to construct strains AWF2–4, as described in Methods. Reporter strains were grown under high- and low-copper conditions, and β-galactosidase activity was measured. Mutation of CuRE 1 in the CaFRE7 promoter (AWF2) caused a 1.8-fold increase in β-galactosidase activity at low copper levels compared with the wild-type promoter (Fig. 5). This was accompanied by a decrease in activity of 37 % at high copper levels, and resembled the alteration in β-galactosidase activity when CuRE 2 in the CaCTR1 promoter was mutated (Fig. 4). In contrast, mutation of CuRE 2 in the CaFRE7 promoter (AWF3) resulted in a decrease in β-galactosidase activity of 96 % at high copper levels, but did not significantly affect activity under low-copper conditions (Fig. 5). Mutation of both CuREs (AWF4) reduced β-galactosidase activity to a level similar to that of the CaFRE7 construct in a mac1/mac1 background and also abolished copper regulation. One of the two CuREs in the CaFRE7 promoter is therefore necessary and sufficient for copper-responsive β-galactosidase activity equal to that of the wild-type promoter.
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). There was no significant difference in β-galactosidase activity under high- or low-copper conditions (Fig. 5), indicating that the CuRE is involved in copper-responsive regulation of CaMAC1 and demonstrating for the first time, to our knowledge, that CaMAC1 is transcriptionally autoregulated. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The finding that one CuRE is sufficient and necessary for activation by CaMac1p shows a key difference between C. albicans and S. cerevisiae, because ScMac1p has been shown to require two CuREs to activate target genes (Martins et al., 1998). Transcriptional autoregulation of the CaMAC1 gene in response to copper levels is in contrast to the regulation of ScMAC1, which is constitutively transcribed (Zhu et al., 1998). The copper-responsive transcription factors Ace1p and Amt1p from S. cerevisiae and Candida glabrata both show variation in the number of binding sites required for activation activity, depending on the target gene (Liu & Thiele, 1997). This may also be the case for CaMac1p, and analysis of the promoters of additional genes regulated by CaMac1p will determine whether or not this is the case.
The involvement of CaMac1p in the regulation of AOX2 and SFU1 suggests that gene activation by CaMac1p may also affect other aspects of metabolism in addition to copper uptake. The regulation of AOX2 is different from the regulation of alternative oxidase genes from other fungi, where the transcription of alternative oxidases increases under low-copper conditions because a shortage of copper impairs the activity of copper-containing respiratory enzymes such as cytochrome c oxidase (Borghouts et al., 2001; Downie & Garland, 1973).
Transcription of the iron-responsive transcriptional repressor gene SFU1 is repressed under iron-starvation conditions in both the wild-type and the mac1/mac1 mutant, and this results in derepression of iron uptake genes regulated by Sfu1p, such as the siderophore transporter CaARN1 and the ferric reductases CFL1 and CFL2 (Lan et al., 2004). SFU1 transcription shows a small decrease under low-copper conditions in the wild-type and a more significant decrease under low-copper conditions in the mac1/mac1 mutant. Copper is essential for iron uptake in C. albicans, and a copper-limiting environment may lead to a decrease in the ability of the cell to take up iron and therefore over time this environment may also become iron-limiting and cause repression of SFU1 transcription. The mac1/mac1 mutant is able to take up less copper than the wild-type strain due to the decreased level of CaCTR1 transcription and is likely to be more severely starved of copper and iron than the wild-type. Repression of SFU1 transcription in low-copper medium is therefore more pronounced in the mutant. The higher overall level of SFU1 transcription in the mac1/mac1 mutant does not, however, appear to be a response to copper or iron levels, as copper or iron starvation would likely result in decreased transcription of SFU1.
CaMac1p is unlikely to be directly activating transcription of SFU1 and AOX2 in a manner similar to the copper-responsive regulation of CaCTR1, CaFRE7 and CaMAC1. CaMac1p could therefore be activating an intermediate regulator or acting as a co-activator or co-repressor to cause an effect on transcription levels that is not copper-dependent. Irrespective of the mechanism by which CaMac1p is affecting transcription, the influence of copper-responsive CaMac1p on the expression of the iron-responsive transcription factor gene SFU1 is an important link between the regulation of the high-affinity iron and copper uptake systems, which both show major differences in regulation from their homologous S. cerevisiae systems.
A number of the copper metabolism genes investigated in this study have also been implicated in the response of C. albicans to host factors, including engulfment by macrophages and the secretion of prostaglandin. Transcript profiling studies have recently shown that expression of CaFRE7, CaMAC1, CaCTR1 and the ferric reductase-like gene orf19.6139 is repressed in response to prostaglandin (Levitin & Whiteway, 2007). Prostaglandin is produced by endothelial cells and is stimulated by contact with C. albicans (Filler et al., 1994). During the preparation of this manuscript, further analysis of CaFRE7 demonstrated that prostaglandin-mediated repression only occurs in the yeast growth phase and that a ScMac1p binding site sequence between 120 and134 bp upstream of the ORF, designated CuRE 2 in the present study, is involved in the regulation of CaFRE7 expression in YPD medium (Levitin & Whiteway, 2007). However, the effect of copper levels and the role of the second putative CaMac1p binding site at position –177 bp were not investigated in the study of Levitin & Whiteway. Three copper metabolism genes (CaFRE7, CaCTR1 and orf19.6139) are also upregulated during phagocytosis by macrophages (Lorenz et al., 2004). The change in expression of copper metabolism genes in response to the host immune system indicates that copper metabolism may have a more significant role when C. albicans is attacked by macrophages than when the organism comes into contact with prostaglandin-producing endothelial cells.
This study demonstrates that CaMac1p is autoregulated and can also regulate copper metabolism genes via one CuRE sequence in their promoters. This evidence shows that copper-dependent regulation by CaMac1p in the human pathogen C. albicans is different from copper-dependent regulation by ScMac1p in the model yeast S. cerevisiae. The transcriptional autoregulation of the CaMAC1 gene may be an adaptation to the host environment, and there are several possible advantages to this system of regulation. C. albicans has a high tolerance to copper, suggesting that some niches colonized by the organism may have very high copper concentrations (Riggle & Kumamoto, 2000; Weissman et al., 2000). As CaMAC1 is only expressed under conditions of copper starvation, the basal level of transcriptional activation of CaCTR1, CaFRE7 and other genes in the CaMac1p regulon would be very low in high-copper environments. This would prevent unnecessary transcription and translation of genes when they are not required, and would conserve energy and nutrients, as well as preventing uptake of potentially toxic levels of copper. In niches where the availability of copper is low, transcription of the CaMAC1 gene would be induced, and this would increase the number of CaMac1p molecules available to activate target genes such as CaCTR1 and help to alleviate copper starvation. Copper-dependent regulation of CaMAC1 transcription may therefore be an adaptation to help C. albicans to survive in both high- and low-copper environments, and this could be a competitive advantage in the human host.
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ACKNOWLEDGEMENTS |
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Edited by: J. F. Ernst
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Received 20 September 2007;
revised 7 February 2008;
accepted 12 February 2008.
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