Department of Genetics, University of Leicester, Leicester LE1 7RH, UK
Correspondence
Annette M. Cashmore
amc19{at}le.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Iron and copper uptake in Saccharomyces cerevisiae has been well characterized and many of the component genes have been identified (reviewed by Eide, 1998). High-affinity iron and copper uptake is initiated by the ScFre1p/ScFre2p cell-surface-associated ferric/cupric reductase. Reduction of Fe3+ and Cu2+ to Fe2+ and Cu+ separates both metals from their environmental ligands, to allow uptake by specific cell membrane transporters. High-affinity iron uptake is achieved by a transporter complex that consists of a ferrous permease (ScFtr1p) and a multi-copper oxidase (ScFet3p). High-affinity copper uptake is achieved by two functionally redundant but distinct copper transporters, ScCtr1p and ScCtr3p. On delivery to the cytosol, Cu+ ions are taken to the intracellular copper transporter ScCcc2p by the copper chaperone ScAtx1p. ScCcc2p is then responsible for the translocation of copper into the lumen of the Golgi, where four Cu+ ions are incorporated into ScFet3p to confer biological activity. Due to the fact that ScFet3p function has an absolute requirement for copper, a defect in the process which leads to its incorporation into this protein causes severely defective high-affinity iron uptake. S. cerevisiae also has a low-affinity iron and copper uptake system that is supplied by the activity of Fet4p (Dix et al., 1994
; Hassett et al., 2000
). A second mechanism for iron uptake has also evolved in S. cerevisiae that makes use of low-molecular-mass compounds called siderophores that have a high affinity for the ion. This iron uptake system is not dependent upon copper and consists of four transporter proteins, ScArn14p (Heymann et al., 1999
; Lesuisse et al., 1998
; Yun et al., 2000b
). Each transporter displays varying specificity to a range of ferri-siderophores from both the hydroxamate and catechol classes (Yun et al., 2000a
). However, siderophore production has never been unequivocally proven in S. cerevisiae, so the organism may rely on those that are produced by other species.
In bacteria the acquisition of iron has long been known to be important for virulence (reviewed by Ratledge & Dover, 2000). It has now been identified that this is also the case for the fungal pathogen Candida albicans (Ramanan & Wang, 2000
). Iron acquisition presents a particular problem to pathogenic micro-organisms due to the hostile environment of the human host. The level of free iron in the bloodstream is limited, as it is bound to proteins with a high affinity for the metal such as haemoglobin and transferrin. During an infection, systemic free-iron levels are further reduced by the hypoferraemic response which is mediated by the reticulo-endothelial system (reviewed by Ward & Bullen, 1999
). This results in iron being transported from the cardiovascular system and into the cells, where it is tightly bound due to increased synthesis of the iron storage protein ferritin. Conversion of ferritin to its insoluble degradation product, haemosiderin, further reduces the iron available to the intracellular pool and to serum transferrin. Pathogens such as C. albicans must therefore possess mechanisms that overcome the host physical barriers and defence mechanisms, to enable it to acquire iron and establish an infection.
Haemolytic activity has been identified in C. albicans and the pathogen has been proposed to obtain iron by attaching to and lysing complement-coated erythrocytes (Manns et al., 1994; Moors et al., 1992
). In addition, the inhibitory growth effects of transferrin on C. albicans can be reversed by the addition of haem or haemoglobin (Moors et al., 1992
). Although the genes that encode the haemolysins have not yet been identified, the mechanisms involved in acquiring extracellular iron have begun to be elucidated. As in S. cerevisiae, a reductive iron uptake system containing several component genes has been identified that is dependent upon copper. Cell-surface-associated ferric/cupric reductase activity has been observed in C. albicans and is regulated in response to iron and copper availability in the growth medium (Morrissey et al., 1996
). Two component genes (CaCFL1 and CaCFL95) have been characterized that have the ability to rescue S. cerevisiae mutants defective in cell-surface-associated reductase activity (Hammacott et al., 2000
; Knight et al., 2002
). The predicted proteins of CaCFL1 and CaCFL95 share significant sequence identity with ScFre1p and ScFre2p respectively. Furthermore, CaCFL1 is transcriptionally regulated in response to both iron and copper availability (Hammacott et al., 2000
). The function of a further ten ORFs whose predicted proteins share significant identity to those encoded by the S. cerevisiae FRE genes remains unknown. Two C. albicans genes which encode a high-affinity iron transporter complex have also been identified. The CaFET3 gene encodes a multi-copper oxidase with the ability to rescue a S. cerevisiae fet3-null mutant (Eck et al., 1999
) and CaFTR1 encodes an iron permease that is functionally homologous to ScFtr1p (Ramanan & Wang, 2000
). C. albicans strains that carry deletions in CaFET3 or CaFTR1 display defects in reductive high-affinity iron uptake, and most notably a Caftr1-null mutant was unable to set up a systemic infection in mice (Eck et al., 1999
; Ramanan & Wang, 2000
). A siderophore uptake system has also been identified in C. albicans that is encoded by CaARN1 (Hu et al., 2002
). However, siderophore production in this organism has not been unequivocally proven.
Reductive iron uptake in C. albicans has been shown to be dependent upon copper (Knight et al., 2002). This is proposed to be due to the requirement of the multi-copper oxidase component of the high-affinity iron transporter complex, CaFet3p. There are also four additional ORFs that may encode multi-copper oxidases present on the C. albicans genomic database (Knight et al., 2002
). Evidence for the requirement of copper for reductive iron uptake in C. albicans is further strengthened by the characterization of a gene homologous to ScCCC2 (Weissman et al., 2002
). A homozygous deletion of CaCCC2 results in strains with defective high-affinity iron uptake, presumably as a result of incorrect delivery and incorporation of copper into CaFet3p (Weissman et al., 2002
). However, although reductive iron uptake has been shown to be reliant on copper in C. albicans, Cafet3 and Caccc2-null mutants were still able to set up a systemic infection in mice (Eck et al., 1999
). Furthermore, the virulence of a Caarn1-null mutant was also unaffected (Hu et al., 2002
). These findings are in contrast to those found with a Caftr1-null mutant and may suggest that additional mechanisms for iron and copper metabolism in C. albicans have yet to be elucidated.
We have previously described CaCTR1, a gene that is required for copper uptake in C. albicans. A Cactr1-null mutant displayed phenotypes that were similar to those of a S. cerevisiae mutant defective in high-affinity copper uptake. Furthermore, this mutant grew predominantly in the filamentous form and also displayed aberrant morphology in response to copper-depleted growth conditions (Marvin et al., 2003). We demonstrate here that invasive growth by C. albicans is induced by low-copper conditions and that this is augmented in a Cactr1-null strain. We also demonstrate that high-affinity iron uptake is not detectable in a Cactr1-null strain. Interestingly, the phenotypes displayed by a Cactr1-null mutant did not coincide with a drop in cell membrane p-phenylenediamine (PPD) oxidase activity. CaCTR1 is transcriptionally controlled by the copper-sensing transactivator ScMac1p when it is heterologously expressed in S. cerevisiae (Marvin et al., 2003
). Here, we show that a similar transactivator exists in C. albicans and is required for expression of CaCTR1 in response to low-copper conditions.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Induction of hyphae.
Overnight cultures were grown in YPD medium and transferred to fresh YPD medium at a cell density of 1x106 cells ml1. Growth was allowed to continue at 37 °C with agitation. Following addition of 5 % (w/v) bovine calf serum, cells were examined every 15 min for germ tube formation.
Genetic techniques.
Transformation of C. albicans was achieved by the lithium acetate method (Braun & Johnson, 1997). Plasmid DNA was isolated and purified from E. coli by alkaline lysis (Ish-Horowicz & Burke, 1981
) and transformation of E. coli was performed using the calcium chloride method (Mandel & Higa, 1970
).
RNA isolation and Northern blot analysis.
Total RNA was extracted from mid-exponential-phase cultures of C. albicans (1x107 cells ml1) by the SDS-hot phenol method (Schmitt et al., 1990). Purified RNA was separated by electrophoresis on a 1·7 % denaturing agarose gel and then transferred to a nylon filter (Hybond-N) and hybridized overnight at 65 °C in 0·5 M sodium phosphate pH 7·4/7 % (w/v) SDS/1 mM EDTA (Church & Gilbert, 1984
) with [
-32P]CTP-labelled probes. The CTR1 probe was a 508 bp PCR product representing an internal fragment of the C. albicans CTR1 gene, generated using primers CaCTR1+60 and CaCTR1+568 (Table 2
). To ensure equal loading of samples, total RNA was stained with ethidium bromide and analysed and quantified on a Kodak Image Station 1000. Stringent washes were carried out at 65 °C using 3x SSC/0·1 % (w/v) SDS. The filter was then exposed to Fuji Medical X-ray film at 80 °C to visualize hybridizing bands.
Radioactive iron uptake assay.
Cultures were grown up in YPD to a cell density of 1x107 cells ml1 and harvested by centrifugation at 2770 g. The cells were then washed three times with sterilized distilled water and resuspended in assay buffer (10 mM trisodium citrate pH 6·5, 5 % glucose) containing 1 µM 55Fe (FeCl3; 370 MBq ml1; 712 MBq mg1) at a cell density of 1x107 cells ml1. The cells were then incubated at either 0 °C or 30 °C with continuous agitation. At intervals of 30 min a 1 ml aliquot of the culture was taken and added to 5ml ice-cold 0·25 M EDTA, pH 6·5, and mixed to quench any free extracellular radioactive iron. The cells were then harvested by vacuum filtration through a glass-fibre filter (Whatman GF/C; 25 mm) and washed three times with 5 ml ice-cold 0·25 M EDTA, pH 6·5, and twice with ice-cold sterilized distilled water. The filters were finally dried and 5 ml emulsifier-safe scintillant fluid was added (Canberra-Packard). All samples were counted in the tritium channel of a Minaxi Tri-Carb 400 series scintillation counter (Canberra-Packard).
Preparation of plasma membrane extracts.
Plasma membrane extracts were obtained from mid-exponential-phase cells (1x107 cells ml1) using a method based on that described by Yuan et al. (1995). Cells, reagents and materials were kept on ice at all times. The strain to be assayed was grown in MD medium containing BPS and BCS, with iron chloride or copper chloride added or omitted as appropriate. The cells were then harvested by centrifugation at 4000 r.p.m. at 4 °C and the supernatant was discarded. The cells were washed in sterilized distilled water, harvested and the supernatant discarded once more. They were then resuspended in breakage buffer (150 mM NaCl, 25 mM Tris/HCl pH 7·5, 5 % glycerol, 1 mM DTT, 1 mM PMSF) containing 10 µl of a yeast protease inhibitor cocktail (Sigma-Aldrich). Finally, 1 g acid-washed glass beads (425600 µm) was added to the breakage mix. The cells were then broken by agitation for 3 min in a multi-vortexer at 4 °C. This was repeated five times, with the cells placed on ice between agitations to prevent overheating and denaturation of the protein. The homogenate was then centrifuged at 55 000 rpm (100 000 g) for 40 min to harvest the cell membrane fraction. The supernatant was discarded and the pellet was washed with 1 ml breakage buffer. The membrane fraction was then solubilized in breakage buffer containing 1 % Triton X-100 and centrifuged for 30 min at 13 000 r.p.m. (16 060 g) to yield a clarified extract. Protein content was determined by Bradford assay.
p-Phenylenediamine (PPD) oxidase assays.
The method used was modified from that described by Spizzo et al. (1997). Liquid PPD oxidase assays were carried out using 25 µg of the cell membrane protein fraction. This was resuspended in 600 µl assay buffer (100 mM sodium acetate pH 5·7, 0·06 % Triton X-100, 0·05 % PPD) and incubated at 30 °C for 1 h. Colour development was followed in a spectrophotometer set at 530 nm at 30 min intervals for a total of 3 h. Oxidation rates were calculated after subtracting the rate of a blank sample that contained assay buffer with no added protein.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Iron uptake is defective in a C. albicans ctr1-null mutant
Iron uptake has been shown to be important for virulence in C. albicans and it has been demonstrated that a homozygous Caftr1-null mutant is unable to establish a systemic infection in a mouse model (Ramanan & Wang, 2000). In S. cerevisiae, it has been observed that deletions in genes that facilitate high-affinity copper uptake or delivery of copper ions to the multicopper oxidase ScFet3p result in strains that also display defective high-affinity iron uptake (Knight et al., 1996
; Lin et al., 1997
; Yuan et al., 1995
). Furthermore, growing evidence suggests that reductive iron uptake in C. albicans may also be dependent on copper uptake (Eck et al., 1999
; Knight et al., 2002
; Weissman et al., 2002
).
To ascertain whether a Cactr1-null mutant displayed defective iron uptake, 55Fe uptake assays were carried out on strain BWP17ctr1/HIS1 and the wild-type strain, DAY185. Assays were performed at both 30 °C and 0 °C as a negative control, and 55Fe uptake was shown to be linear for at least 3 h. The data generated demonstrated that double deletion of CaCTR1 results in defective 55Fe uptake in the resulting mutant strain. Iron uptake in strain BWP17ctr1
/HIS1 was reduced by an average of 96 % using the conditions described (Fig. 2
). However, a low rate of uptake was still detectable in the Cactr1-null mutant when the assays were performed at 30 °C. To ascertain whether the cells that had been incubated in assay buffer were still viable, serial dilutions of each strain were made after each experiment and plated out onto YPD. Following incubation at 30 °C for 3 days, counts of the subsequent colonies typically gave a mean value of around 71 % viability. The 55Fe incorporation assays were performed on four occasions and were quantitatively similar between experiments.
|
A sequence is present (orf6.8485) on the C. albicans genome database (http://www-sequence.stanford.edu/group/candida/search.html) which displays significant identity to ScMac1p. Direct comparison using the Needleman & Wunsch algorithm alignment (GAP) from the GCG package showed that the putative Mac1p sequence from C. albicans shared 35·0 % similarity and 26·2 % identity with ScMac1p. In addition, further analysis showed that the putative CaMac1 protein sequence contained specific motifs that were indicative of a ScMac1p homologue (Fig. 3). Analysis using the predicted CaMac1p sequence as a query against the Conserved Domain Database (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) revealed the presence of a copper fist motif at the amino terminus (Fig. 3a
; Altschul et al., 1997
). This motif is found in several copper-responsive transcription factors, including Mac1p and Ace1p of S. cerevisiae (Jungmann et al., 1993
; Szczypka & Thiele, 1989
), Amt1p of Candida glabrata (Zhou & Thiele, 1991
), Cuf1p of Schizosaccharomyces pombe (Labbe et al., 1999
) and Crf1p of Yarrowia lipolytica (Garcia et al., 2002
). In each corresponding organism, the motif facilitates the binding of protein to DNA in the presence of copper or silver (Furst & Hamer, 1989
; Jensen et al., 1998
; Jensen & Winge, 1998
).
|
Phenotypic analysis of a Camac1-null mutant
The features found during sequence analysis of the predicted protein product of orf6·8485 made this ORF a likely candidate to encode a protein functionally homologous to Mac1p of S. cerevisiae, and the gene is referred to as CaMAC1 from here onwards in the text. To investigate whether CaMAC1 was functional in C. albicans we constructed a Camac1-null mutant by disrupting the two genomic copies in strain BWP17 using PCR-directed mutagenesis (Wilson et al., 1999). We then reintegrated CaHIS1 into strain BWP17mac1
: : URA3/mac1
: : ARG4 and CaHIS1 along with a wild-type allele of CaMAC1 to construct strains BWP17mac1
/HIS1 and BWP17mac1
/HIS1 : : MAC1, respectively (see Methods).
It has been previously observed that a S. cerevisiae mac1-null mutant displays phenotypes similar to those of a strain which lacks high-affinity copper uptake. These phenotypes include respiratory deficiency and slow growth on low-copper and low-iron medium (Dancis et al., 1994a, b
; Knight et al., 1996
). The Camac1-null mutant (BWP17mac1
/HIS1) was therefore tested to see if it possessed similar mutant phenotypes (Fig. 4
). The results revealed that the Camac1-null mutant displayed similar phenotypes to a S. cerevisiae strain defective in Mac1p activity, and these phenotypes were also comparable to those previously observed in a Cactr1-null mutant (Marvin et al., 2003
). Like a Cactr1-null strain, the Camac1-null mutant displayed slow growth on low-copper medium (although this was not as marked as in a Cactr1-null strain), slow growth on low-iron medium, and was unable to grow on YP medium containing glycerol or ethanol as the sole carbon source. All these phenotypes could be rescued by the addition of 100 µM cupric chloride. Reintegration of a wild-type allele of CaMAC1 at the his1 : : hisG locus was sufficient to rescue all the mutant phenotypes. The Camac1-null mutant was also tested for the ability to form hyphae in response to the addition of bovine calf serum at 37 °C. The results indicated that the production of true hyphae was unaffected by a double disruption of CaMAC1 (data not shown). We also tested the ability of a Camac1-null strain to take up iron using the 55Fe uptake assay. Interestingly, no significant difference was observed between the uptake rate of strain BWP17mac1
/HIS1 and the wild-type strain, DAY185 (data not shown). However, we did observe a lag period in strain BWP17mac1
/HIS1 lasting 60 min, during which iron uptake was induced at some point between 30 and 60 min (Fig. 2
).
|
|
Cells were grown in unsupplemented MD-BCS/BPS medium, or MD-BCS/BPS medium that had been supplemented with 100 µM cupric chloride, 100 µM ferric chloride, or both. Total cell membrane fractions were obtained from these cells and the rate of oxidase activity was determined (Fig. 6). The results obtained were corrected for background oxidation by subtracting the rate obtained from a blank sample containing no protein extract. To check that the colour change observed in these assays was due to protein activity, a boiled membrane extract was also assayed as a negative control and found to contain activity identical to a blank sample (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The remaining 3·78 % of iron uptake activity may be attributed to a low-affinity iron uptake system or low-affinity copper uptake system. There are sequences present on the Institut Pasteur C. albicans genome database with significant identity to the S. cerevisiae low-affinity iron/copper transporter Fet4p and also the vacuolar copper transporter Ctr2p (Hassett et al., 2000; Kampfenkel et al., 1995
). These putative homologous genes may provide an alternative source of copper and iron to Cactr1p, and may account for the residual 55Fe uptake observed. In addition, a previous study in S. cerevisiae has proposed that ScCcc2p may have the ability to shuttle copper between intracellular compartments and the cell membrane (Pufahl et al., 1997
). The other alternative source of iron uptake may be a ferri-siderophore uptake system. However, although CaARN1 encodes a protein that facilitates the uptake of ferri-ferrichrome in C. albicans, the biosynthesis of siderophores in this organism has not been unequivocally proven (Hu et al., 2002
). Future studies using iron uptake assays should be carried out over longer time-periods and under different growth conditions to see if the ferri-siderophore uptake system is differentially regulated. The regulation of genes that facilitate iron uptake in C. albicans has been reported to be under the control of the CaTup1p transcriptional repressor (Knight et al., 2002
). Interestingly, cDNA micro-arrays have shown that transcriptional repression of ScFRE2, ScFET3, ScFTR1, ScCCC2 and the ScARN genes in response to high-iron conditions in S. cerevisiae is under the control of the protein kinase ScTpk2p (Robertson et al., 2000
). The ScTPK2 gene and its functional homologue in C. albicans are also involved in the regulation of morphogenesis via the cAMP-activated pathway (reviewed by Gancedo, 2001
; Sonneborn et al., 2000
). This raises the possibility of copper- or iron-induced filamentation in C. albicans being under the regulation of the cAMP pathway, which may involve both CaTpk2p and CaEfg1p activity. Alkaline conditions are also known to induce filamentation in C. albicans via the Rim101p-mediated pathway (Davis et al., 2000a
). Therefore, CaRim101p is another candidate for activating filamentation in response to low-copper and low-iron conditions, as defective CaCtr1p activity may lead to changes in intracellular pH. Future studies on filamentation in Catpk2-, Catup1- and Carim101-null mutants in response to low copper or iron may reveal if they are involved in this process.
Identification of CuREs in the promoter of CaCTR1 raised the possibility of a similar copper-responsive transactivator to ScMac1p operating in C. albicans (Marvin et al., 2003). We have previously shown that CaCTR1 is transcriptionally regulated by ScMac1p when heterologously expressed in S. cerevisiae. Here we have shown that a copper-sensing transactivator similar to ScMac1p operates in C. albicans and is essential for transcription of CaCTR1 in response to low-copper conditions. The predicted CaMac1p possessed an N-terminal copper fist motif (Altschul et al., 1997
) and C-terminal C1 (REP I) and C2 (REP II) motifs that facilitate copper-mediated control of expression in the ScMac1p transactivator (Graden & Winge, 1997
; Jensen et al., 1998
; Jensen & Winge, 1998
; Keller et al., 2000
; Labbe et al., 1997
; Yamaguchi-Iwai et al., 1997
; Zhu et al., 1998
). Future analysis of the CuRE sites in the promoter of CaCTR1 will reveal whether they facilitate transcriptional control by CaMac1p. Similar studies on the copper fist and the C1 and C2 motifs of CaMac1p will determine the importance of these motifs for metal-sensing and transactivation.
A Camac1-null mutant displays phenotypes that are directly comparable with those of a Cactr1-null mutant and are consistent with defective high-affinity copper uptake (Dancis et al., 1994a, b
; Knight et al., 1996
). Interestingly, the Camac1-null mutant displayed a similar rate of iron uptake to a wild-type strain, but had a lag period of approximately 1 h, with uptake being induced somewhere between 30 and 60 min. Although a Camac1-null mutant displayed slow growth on low-iron medium, this phenotype was not manifested so strongly as in a Cactr1-null mutant (Marvin et al., 2003
). These observations may simply reflect the effect of CaMAC1 deletion on the growth rate of C. albicans. It should be noted that CaMac1p appears to be a copper-sensing transactivator. Therefore, a basal level of expression of CaCTR1 may still be present in a Camac1-null mutant even though no transcript was detected following Northern blot analysis (Fig. 5
). This may explain the anomalies in the phenotypes observed between the Cactr1-null and Camac1-null strains. It should also be noted that in S. cerevisiae, the ScMAC1 regulon includes six genes, ScCTR1, ScCTR3, two metal-reductases, ScFRE1 and ScFRE7, and two unidentified ORFs, YFR055w and YJL277w (Gross et al., 2000
). This raises the possibility that the phenotypes observed in a Camac1-null mutant may result from reduced expression of additional genes in C. albicans, such as ferric reductases, that are involved in iron or copper metabolism and not necessarily be due only to reduced expression of CaCTR1. The observed anomalies between the two mutant strains also raise the possibility of alternative mechanisms in C. albicans for activating copper and iron transport into the cell in response to metal ion starvation.
It has recently been shown that in a wild-type C. albicans strain, iron acquisition is dependent upon the availability of copper; with uptake being severely inhibited in copper-depleted conditions (Knight et al., 2002). In the same report, PPD oxidase activity was also shown to be dependent upon copper and this dependency was proposed to be due to the requirement of CaFet3p. However, although a Caftr1-null mutant was unable to establish an infection in a systemic mouse model, virulence of a Cafet3-null mutant and a Caccc2-null mutant has been shown to be essentially the same as that of a wild-type strain (Eck et al., 1999
; Ramanan & Wang, 2000
; Weissman et al., 2002
). Therefore, it appears that there is a paradox with the requirement of multi-copper oxidase activity for reductive iron uptake in C. albicans, as deletions in CaFET3 and CaCCC2 would be expected to result in reduced virulence. However, CaCCC2 was isolated and characterized by functional rescue of a corresponding S. cerevisiae mutant and its complete biochemical properties have not been fully elucidated (Weissman et al., 2002
). There are also four additional ORFs that are present on the C. albicans genome database with predicted protein products that display significant sequence identity to CaFet3p. These may encode additional multi-copper oxidases that work alongside CaFTR1 in a high-affinity iron transport complex (Eck et al., 1999
; Knight et al., 2002
). Interestingly, we have now demonstrated that in the absence of CaCtr1p, PPD oxidase activity is no longer dependent on copper added to the growth medium. This activity might be attributed to any of the additional ORFs that encode putative multi-copper oxidases that may mobilize internal iron stores in response to copper starvation.
This paper has described important components of a high-affinity iron and copper uptake system that are necessary to enable C. albicans to acquire these essential metals from its various environmental niches. The phenotypes associated with a Cactr1-null mutant indicate an important role of copper uptake for high-affinity iron uptake in C. albicans. These studies have also shown that CaCTR1 is regulated by a copper-sensing transactivator in a manner that appears to be similar to the system described in S. cerevisiae. Future studies on Cactr1- and Camac1-null mutants in infection models will reveal the importance of copper uptake and its regulation for the virulence of C. albicans. These studies will ascertain whether CaCtr1p or CaMac1p are potential drug targets for future therapy of C. albicans infections. The studies described here, and future studies on virulence, will give a greater understanding of the relationship between copper and iron uptake in C. albicans and the role they may play in the survival and proliferation of this organism in the mammalian host.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Braun, B. R. & Johnson, A. D. (1997). Control of filament formation in Candida albicans by the transcriptional repressor TUP1. Science 277, 105109.
Church, G. M. & Gilbert, W. (1984). Genomic sequencing. Proc Natl Acad Sci U S A 81, 19911995.[Abstract]
Crichton, R. R. & Pierre, J. L. (2001). Old iron, young copper: from Mars to Venus. Biometals 14, 99112.[CrossRef][Medline]
Dancis, A., Haile, D., Yuan, D. S. & Klausner, R. D. (1994a). The Saccharomyces cerevisiae copper transport protein (Ctr1p). Biochemical characterization, regulation by copper, and physiologic role in copper uptake. J Biol Chem 269, 2566025667.
Dancis, A., Yuan, D. S., Haile, D., Askwith, C., Eide, D., Moehle, C., Kaplan, J. & Klausner, R. D. (1994b). Molecular characterization of a copper transport protein in S. cerevisiae: an unexpected role for copper in iron transport. Cell 76, 393402.[Medline]
Davis, D., Edwards, J. E., Jr, Mitchell, A. P. & Ibrahim, A. S. (2000a). Candida albicans RIM101 pH response pathway is required for host-pathogen interactions. Infect Immun 68, 59535959.
Davis, D., Wilson, R. B. & Mitchell, A. P. (2000b). RIM101-dependent and -independent pathways govern pH responses in Candida albicans. Mol Cell Biol 20, 971978.
Dix, D. R., Bridgham, J. T., Broderius, M. A., Byersdorfer, C. A. & Eide, D. J. (1994). The FET4 gene encodes the low affinity Fe(II) transport protein of Saccharomyces cerevisiae. J Biol Chem 269, 2609226099.
Eck, R., Hundt, S., Hartl, A., Roemer, E. & Kunkel, W. (1999). A multicopper oxidase gene from Candida albicans: cloning, characterization and disruption. Microbiology 145, 24152422.
Eide, D. J. (1998). The molecular biology of metal ion transport in Saccharomyces cerevisiae. Annu Rev Nutr 18, 441469.[CrossRef][Medline]
Eide, D., Davis-Kaplan, S., Jordan, I., Sipe, D. & Kaplan, J. (1992). Regulation of iron uptake in Saccharomyces cerevisiae. The ferrireductase and Fe(II) transporter are regulated independently. J Biol Chem 267, 2077420781.
Furst, P. & Hamer, D. (1989). Cooperative activation of a eukaryotic transcription factor: interaction between Cu(I) and yeast ACE1 protein. Proc Natl Acad Sci U S A 86, 52675271.[Abstract]
Gancedo, J. M. (2001). Control of pseudohyphae formation in Saccharomyces cerevisiae. FEMS Microbiol Rev 25, 107123.[CrossRef][Medline]
Garcia, S., Prado, M., Degano, R. & Dominguez, A. (2002). A copper-responsive transcription factor, CRF1, mediates copper and cadmium resistance in Yarrowia lipolytica. J Biol Chem 277, 3735937368.
Gillum, A. M., Tsay, E. Y. H. & Kirsch, D. R. (1984). Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae URA3 and E. coli pyrF mutations. Mol Gen Genet 198, 179182.[Medline]
Graden, J. A. & Winge, D. R. (1997). Copper-mediated repression of the activation domain in the yeast Mac1p transcription factor. Proc Natl Acad Sci U S A 94, 55505555.
Gross, C., Kelleher, M., Iyer, V. R., Brown, P. O. & Winge, D. R. (2000). Identification of the copper regulon in Saccharomyces cerevisiae by DNA microarrays. J Biol Chem 275, 3231032316.
Halliwell, B. & Gutteridge, J. M. C. (1999). Free Radicals in Biology and Medicine. Oxford: Oxford University Press.
Hammacott, J. E., Williams, P. H. & Cashmore, A. M. (2000). Candida albicans CFL1 encodes a functional ferric reductase activity that can rescue a Saccharomyces cerevisiae fre1 mutant. Microbiology 146, 869876.
Hassett, R., Dix, D. R., Eide, D. J. & Kosman, D. J. (2000). The Fe(II) permease Fet4p functions as a low affinity copper transporter and supports normal copper trafficking in Saccharomyces cerevisiae. Biochem J 351, 477484.[CrossRef][Medline]
Heymann, P., Ernst, J. F. & Winkelmann, G. (1999). Identification of a fungal triacetylfusarinine C siderophore transport gene (TAF1) in Saccharomyces cerevisiae as a member of the major facilitator superfamily. Biometals 12, 301306.[CrossRef][Medline]
Hu, C. J., Bai, C., Zheng, X. D., Wang, Y. M. & Wang, Y. (2002). Characterization and functional analysis of the siderophore-iron transporter CaArn1p in Candida albicans. J Biol Chem 277, 3059830605.
Ish-Horowicz, D. & Burke, J. F. (1981). Rapid and efficient cosmid cloning. Nucleic Acids Res 9, 29892998.[Abstract]
Jensen, L. T. & Winge, D. R. (1998). Identification of a copper-induced intramolecular interaction in the transcription factor Mac1 from Saccharomyces cerevisiae. Embo J 17, 54005408.
Jensen, L. T., Posewitz, M. C., Srinivasan, C. & Winge, D. R. (1998). Mapping of the DNA binding domain of the copper-responsive transcription factor Mac1 from Saccharomyces cerevisiae. J Biol Chem 273, 2380523811.
Jungmann, J., Reins, H. A., Lee, J., Romeo, A., Hassett, R., Kosman, D. & Jentsch, S. (1993). MAC1, a nuclear regulatory protein related to Cu-dependent transcription factors is involved in Cu/Fe utilization and stress resistance in yeast. Embo J 12, 50515056.[Abstract]
Kampfenkel, K., Kushnir, S., Babiychuk, E., Inze, D. & Van Montagu, M. (1995). Molecular characterization of a putative Arabidopsis thaliana copper transporter and its yeast homologue. J Biol Chem 270, 2847928486.
Keller, G., Gross, C., Kelleher, M. & Winge, D. R. (2000). Functional independence of the two cysteine-rich activation domains in the yeast Mac1 transcription factor. J Biol Chem 275, 2919329199.
Knight, S. A., Labbe, S., Kwon, L. F., Kosman, D. J. & Thiele, D. J. (1996). A widespread transposable element masks expression of a yeast copper transport gene. Genes Dev 10, 19171929.[Abstract]
Knight, S. A., Lesuisse, E., Stearman, R., Klausner, R. D. & Dancis, A. (2002). Reductive iron uptake by Candida albicans: role of copper, iron and the TUP1 regulator. Microbiology 148, 2940.
Labbe, S., Zhu, Z. & Thiele, D. J. (1997). Copper-specific transcriptional repression of yeast genes encoding critical components in the copper transport pathway. J Biol Chem 272, 1595115958.
Labbe, S., Pena, M. M., Fernandes, A. R. & Thiele, D. J. (1999). A copper-sensing transcription factor regulates iron uptake genes in Schizosaccharomyces pombe. J Biol Chem 274, 3625236260.
Lesuisse, E., Simon-Casteras, M. & Labbe, P. (1998). Siderophore-mediated iron uptake in Saccharomyces cerevisiae: the SIT1 gene encodes a ferrioxamine B permease that belongs to the major facilitator superfamily. Microbiology 144, 34553462.[Abstract]
Lin, S. J., Pufahl, R. A., Dancis, A., O'Halloran, T. V. & Culotta, V. C. (1997). A role for the Saccharomyces cerevisiae ATX1 gene in copper trafficking and iron transport. J Biol Chem 272, 92159220.
Linder, M. C. & Hazegh-Azam, M. (1996). Copper biochemistry and molecular biology. Am J Clin Nutr 63, 797S811S.[Abstract]
Mandel, M. & Higa, A. (1970). Calcium dependent bacteriophage DNA infection. J Mol Biol 53, 154.
Manns, J. M., Mosser, D. M. & Buckley, H. R. (1994). Production of a hemolytic factor by Candida albicans. Infect Immun 62, 51545156.[Abstract]
Marvin, M. E., Williams, P. H. & Cashmore, A. M. (2003). The Candida albicans CTR1 gene encodes a functional copper transporter. Microbiology 149, 14611474.
Moors, M. A., Stull, T. L., Blank, K. J., Buckley, H. R. & Mosser, D. M. (1992). A role for complement receptor-like molecules in iron acquisition by Candida albicans. J Exp Med 175, 16431651.[Abstract]
Morrissey, J. A., Williams, P. H. & Cashmore, A. M. (1996). Candida albicans has a cell-associated ferric-reductase activity which is regulated in response to levels of iron and copper. Microbiology 142, 485492.[Abstract]
Pufahl, R. A., Singer, C. P., Peariso, K. L., Lin, S. J., Schmidt, P. J., Fahrni, C. J., Culotta, V. C., Penner-Hahn, J. E. & O'Halloran, T. V. (1997). Metal ion chaperone function of the soluble Cu(I) receptor Atx1. Science 278, 853856.
Ramanan, N. & Wang, Y. (2000). A high-affinity iron permease essential for Candida albicans virulence. Science 288, 10621064.
Ratledge, C. & Dover, L. G. (2000). Iron metabolism in pathogenic bacteria. Annu Rev Microbiol 54, 881941.[CrossRef][Medline]
Robertson, L. S., Causton, H. C., Young, R. A. & Fink, G. R. (2000). The yeast A kinases differentially regulate iron uptake and respiratory function. Proc Natl Acad Sci U S A 97, 59845988.
Schmitt, M. E., Brown, T. A. & Trumpower, B. L. (1990). A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res 18, 30913092.[Medline]
Sherman, F., Fink, G. R. & Hicks, J. B. (1986). Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sonneborn, A., Bockmuhl, D. P., Gerads, M., Kurpanek, K., Sanglard, D. & Ernst, J. F. (2000). Protein kinase A encoded by TPK2 regulates dimorphism of Candida albicans. Mol Microbiol 35, 386396.[CrossRef][Medline]
Spizzo, T., Byersdorfer, C., Duesterhoeft, S. & Eide, D. (1997). The yeast FET5 gene encodes a FET3-related multicopper oxidase implicated in iron transport. Mol Gen Genet 256, 547556.[CrossRef][Medline]
Szczypka, M. S. & Thiele, D. J. (1989). A cysteine-rich nuclear protein activates yeast metallothionein gene transcription. Mol Cell Biol 9, 421429.[Medline]
Ward, C. G. & Bullen, J. J. (1999). Clinical and physiological aspects. In Iron and Infection: Molecular, Physiological and Clinical Aspects. Edited by D. J. Bullen & E. Griffiths: Wiley.
Weissman, Z., Shemer, R. & Kornitzer, D. (2002). Deletion of the copper transporter CaCCC2 reveals two distinct pathways for iron acquisition in Candida albicans. Mol Microbiol 44, 15511560.[CrossRef][Medline]
Wickerham, L. J. (1951). Taxonomy of yeast. US Dep Agric Tech Bull 1029, 1159.
Wilson, R. B., Davis, D. & Mitchell, A. P. (1999). Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J Bacteriol 181, 18681874.
Yamaguchi-Iwai, Y., Serpe, M., Haile, D., Yang, W., Kosman, D. J., Klausner, R. D. & Dancis, A. (1997). Homeostatic regulation of copper uptake in yeast via direct binding of MAC1 protein to upstream regulatory sequences of FRE1 and CTR1. J Biol Chem 272, 1771117718.
Yuan, D. S., Stearman, R., Dancis, A., Dunn, T., Beeler, T. & Klausner, R. D. (1995). The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake. Proc Natl Acad Sci U S A 92, 26322636.[Abstract]
Yun, C. W., Ferea, T., Rashford, J., Ardon, O., Brown, P. O., Botstein, D., Kaplan, J. & Philpott, C. C. (2000a). Desferrioxamine-mediated iron uptake in Saccharomyces cerevisiae. Evidence for two pathways of iron uptake. J Biol Chem 275, 1070910715.
Yun, C. W., Tiedeman, J. S., Moore, R. E. & Philpott, C. C. (2000b). Siderophore-iron uptake in Saccharomyces cerevisiae. Identification of ferrichrome and fusarinine transporters. J Biol Chem 275, 1635416359.
Zhou, P. B. & Thiele, D. J. (1991). Isolation of a metal-activated transcription factor gene from Candida glabrata by complementation in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 88, 61126116.[Abstract]
Zhu, Z., Labbe, S., Pena, M. M. & Thiele, D. J. (1998). Copper differentially regulates the activity and degradation of yeast Mac1 transcription factor. J Biol Chem 273, 12771280.
Received 23 December 2003;
revised 23 April 2004;
accepted 27 April 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |