Department of Genetics1 and Department of Microbiology and Immunology2, University of Leicester, Leicester LE1 7RH, UK
Author for correspondence: Annette M. Cashmore. Tel: +44 116 252 3439. Fax: +44 116 252 5101. e-mail: amc19{at}le.ac.uk
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ABSTRACT |
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Keywords: Candida albicans, CFL1, iron uptake, ferric reductase, Saccharomyces cerevisiae
Abbreviations: BCS, bathocuproine disulphonic acid; BPS, bathophenanthroline disulphonic acid; FNR, ferredoxin-NADP+ reductase
The EMBL accession number for the sequence reported in this paper is AJ387722.
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INTRODUCTION |
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It has been shown that C. albicans demonstrates reduced growth in human serum due to the presence of transferrin (Moors et al., 1992 ), and lactoferrin and ovotransferrin are also antifungal (Valenti et al., 1986
). C. albicans can acquire iron from haem, and can both bind and lyse erythrocytes (Manns et al., 1994
; Moors et al., 1992
). Moreover, siderophores of both phenolate and hydroxamate types have been reported (Ismail et al., 1985
; Sweet & Douglas 1991b
). However, it is not yet clear how iron is released from siderophores or haem for utilization by the cell. Previous work from our laboratory (Morrissey et al., 1996
) presented biochemical evidence that C. albicans possesses a cell-surface ferric reductase which is regulated in response to levels of iron and copper. This reductase is similar in its activity and regulation to the surface reductase encoded by the FRE1 and FRE2 genes of Saccharomyces cerevisiae, which play a pivotal role in iron acquisition in this organism.
S. cerevisiae has often been used as a tool and model in the study of C. albicans since genetic analysis of C. albicans is made difficult by its lack of a sexual cycle and perpetual diploid state. S. cerevisiae possesses a well-characterized reductive-iron-uptake system which is capable of obtaining iron from a wide range of sources. This system comprises a cell-surface reductase and a transporter complex. FRE1 and FRE2 encode components of the cell-surface ferric reductase (Dancis et al., 1990 ; Georgatsou & Alexandraki, 1994
) and the main transporter complex is encoded by genes FTR1 (Stearman et al., 1996
) and FET3 (Askwith et al., 1994
). Fet3p is a multicopper oxidase, which converts Fe2+ back to Fe3+ during the uptake process, and requires copper for activity. Thus iron and copper uptake are inextricably linked and, indeed, the cell-surface ferric reductase is capable of reducing copper as well as iron (Hassett & Kosman, 1995
). The genes encoding components of the iron-uptake system are transcriptionally regulated in response to iron concentrations in the growth media (Dancis et al., 1992
). In iron-replete conditions genes are expressed at basal levels, whilst in low-iron conditions transcription is up-regulated. In addition, FRE1 transcription is negatively regulated in response to copper (Hassett & Kosman, 1995
). In fact there are five other ferric reductase-like genes in S. cerevisiae, all of which are expressed and regulated in response to iron or copper, or both (Martins et al., 1998
). Their roles in iron uptake have not yet been established. They may be involved with the surface-associated complex or, alternatively, may have an intracellular role.
A C. albicans ferric reductase-like gene, CFL1, has been identified on the basis of sequence homology with S. cerevisiae FRE1 but no functional ferric reductase activity was observed to be associated with it (Yamada-Okabe et al., 1996 ). Moreover, a recent project to sequence the C. albicans genome (http://alces.med.umn.edu/Candida.html) has identified 12 different sequences showing some identity with ferric reductase genes, but it is not yet known whether any of these short sequences are indeed part of expressed genes.
In this paper we describe the use of functional complementation to isolate a gene from C. albicans capable of restoring reductase activity to S. cerevisiae mutants defective in FRE1. The gene we identified was, in fact, the same one, CFL1, that had previously been described by Yamada-Okabe et al. (1996) . However, our analysis shows that the sequence reported by these workers arose from a clone carrying non-contiguous pieces of C. albicans chromosomal DNA, which explains why no reductase activity was observed. Our work therefore represents the first demonstration of a functional ferric reductase gene from C. albicans.
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METHODS |
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Yeast cultures were grown at 30 °C in either YNB [0·67% yeast nitrogen base with ammonium sulphate (bio101)] or minimal defined medium (MD) based on Wickerhams nitrogen base recipe (Wickerham, 1951 ) with the addition of 20 mM sodium citrate (pH 4·2) (Eide et al., 1992
). Amino acids were added to YNB and MD media at the final concentrations used by Sherman et al. (1986)
and glucose was added at 2% to all media. For growth on solid medium, 2% agar was added to YNB or MD medium. MD medium was rendered iron restricted by the addition of 300 µM
,
'-dipyridyl (Sigma) (MD-dipyridyl), 50 µM BPS (bathophenanthroline disulphonic acid; Sigma), or 1 mM EDTA (MD-EDTA). Copper-depleted medium was made by the addition of 50 µM BCS (bathocuproine disulphonic acid; Sigma). E. coli cultures were grown at 37 °C in LuriaBertani medium [1% Bacto tryptone, 0·5% Bacto yeast extract, 0·5% NaCl, pH 7·2].
The C. albicans genomic library was obtained from P. Meacock, Dept. of Genetics, University of Leicester, UK. Plasmids were isolated from S. cerevisiae by the method of Holm et al. (1986) . The procedure for S. cerevisiae transformation was the lithium acetate method of Geitz et al. (1992)
. Plasmid DNA was extracted from E. coli by the alkaline lysis method of Ish-Horowicz & Burke (1981)
. E. coli transformations were performed using the cold calcium chloride method (Maniatis et al., 1982
). Transposon mutagenesis was performed using a derivative of the E. coli transposon Tn1000 containing the yeast HIS3 gene for selection (Sedgwick & Morgan, 1994
).
Qualitative solid-phase ferric reductase assay.
A modified version of a qualitative ferric reductase assay (Dancis et al., 1990 ) was used to identify extracellular ferric reductase activity. Cells were grown on YNB agar plates for 35 d and placed at 4 °C overnight. Colonies were transferred to nylon filters (Hybond-N) and incubated for 5 h on solid MD-dipyridyl media containing 300 µM FeCl3. The filters were removed from the plates, incubated for 5 min on Whatman 3MM paper soaked in assay buffer (50 mM sodium citrate, pH 6·5, 5% glucose), and then transferred to fresh 3MM paper soaked in assay buffer containing 1 mM FeCl3 and 1 mM BPS for a further 5 min. Colonies with ferric reductase activity stained the filter red due to the formation of BPS[Fe2+] complexes.
DNA manipulation and sequence analysis.
Restriction, cloning, agarose gel electrophoresis and Southern blot analysis were performed by standard methods (Maniatis et al., 1982 ). DNA sequencing was carried out using an ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) and DNA sequencer (ABI model 377XL). The protocol used was that provided by the manufacturers.
RNA extraction and Northern analysis.
C. albicans cells were grown in MD medium containing either 50 µM BCS or 50 µM BPS to restrict copper and iron availability, respectively. High-copper conditions were made by adding 100 µM CuCl2 to MD-BCS and high-iron conditions were made by adding 100 µM or 250 µM FeCl3 to MD-BPS. Total RNA was extracted from these cells using the SDS-hot phenol method of Schmidt et al. (1990) . RNA was electrophoresed through a 1·5% denaturing agarose gel, blotted onto nitrocellulose and hybridized overnight in ChurchGilbert buffer (0·5 M sodium phosphate, pH 7·4, 7% SDS, 1 mM EDTA; Church & Gilbert, 1984
) at 65 °C using 32P-labelled DNA as a probe. Stringent washes were carried out at 65 °C for 30 min in 3xSSC, 0·1% SDS followed by 1xSSC, 0·1% SDS and 0·5xSSC, 0·1% SDS. (20xSSC is 3 M sodium chloride, 0·3 M trisodium citrate). The filter was then exposed to Fuji Medical X-ray film at -80 °C for 2 weeks prior to developing.
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RESULTS |
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Localization and sequence analysis of the ferric reductase gene pJDF1.3
Restriction analysis of plasmid pJDF1.3 (Fig. 3) indicated that it carried an insert of 7·6 kb which was likely to contain more than one ORF, and so transposon mutagenesis was used to localize the region responsible for ferric reductase activity. Plasmid pJDF1.3 DNA was used to transform E. coli strain DH1/R388::Tn1000(HIS3), which was then used as the donor for conjugal matings with selection for the ampicillin-resistance marker of the vector. Plasmid DNA isolated from the transconjugants represented pJDF1.3 derivatives containing random transposon insertions; this DNA was used to transform fre1::Tn(HIS3), with selection for leucine prototrophy conferred by the LEU2 gene on the library plasmid, and the resulting colonies were assayed for reductase activity. Plasmid DNA was isolated from four colonies that showed no activity, retransformed into fre1::Tn(HIS3) and reassayed to confirm that the observed phenotype, failure to rescue ferric reductase deficiency, was indeed plasmid-associated. Transposon-insertion sites in these plasmids were located by restriction mapping; all four were in a 1·8 kb EcoRI fragment (Fig. 3
), indicating that the gene of interest lay in this region. One of these plasmids (designated pJDF1.3a) was picked for sequence analysis using primers to sequence outwards from the transposon (Fig. 4
). Searches of the C. albicans information pages (http://alces.med.umn.edu/Candida.html) revealed that the gene had previously been sequenced and named CFL1 (for Candida ferric reductase-like gene) (Yamada-Okabe et al., 1996
). However, these authors were unable to demonstrate rescue by CFL1 either of the ferric reductase deficiency of a S. cerevisiae fre1 mutant or of the characteristic poor growth of the fre1 mutant in low-iron conditions.
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We performed a comparative PCR analysis of pJDF1.3 plasmid and genomic DNA using primers designed to amplify the 5' terminus of CFL1 and the untranslated region upstream of CFL1 (Fig. 5). PCR products of identical sizes were obtained from both templates with each combination of primers used, including some that spanned the Sau3A1 site at which homology with the previously published sequence broke down. These data confirm that the DNA cloned in plasmid pJDF1.3 correctly represents contiguous sequences in the C. albicans strain S/01 genome. We have submitted the sequence to the EMBL database (accession number AJ387722).
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DISCUSSION |
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The predicted amino acid sequence of Cfl1p shows homology with ferric reductase genes from S. cerevisiae. In particular, it shows high conservation of several specific domains such as those implicated in FAD and NAD(P)H binding, which are also found to be conserved between the wider family of FNR proteins as well as other domains with no known function. The conservation of the positioning of four histidine residues, essential for haem binding in Fre1p, is also interesting and indicates that similarly Cfl1p may also be haem-dependent.
CFL1 transcription is negatively regulated by both iron and copper. This is not unexpected, since previous biochemical evidence showed that the C. albicans ferric reductase activity is regulated by both copper and iron and is also capable of reducing both metals (Morrissey et al., 1996 ). Similarly, the S. cerevisiae cell-surface ferric reductase is capable of reducing copper as well as iron and FRE1 is transcriptionally regulated by both metals (Hassett & Kosman, 1995
). This transcriptional regulation has been shown to involve the interaction of two transcription factors, Aft1p and Mac1p, with specific sequences in the promoter of FRE1 (Yamaguichi-Iwai et al., 1995
; Labbe et al., 1997
). There is, however, no evidence that genes encoding similar transcription factors exist in C. albicans and Aft1p-like and Mac1p-like consensus sequences are not present in the promoter of CFL1. Furthermore, expression of CFL1 in the S. cerevisiae fre1 mutant is not regulated in response to iron levels.
CFL1 also rescues the slow growth on iron demonstrated by the S. cerevisiae fre1 mutant. Slow growth on low iron can be seen as a reflection of intracellular iron concentrations of a cell. A cell that is unable to acquire iron by a high-affinity mechanism is unable to grow in low-iron conditions, explaining why the fre1 mutant is unable to grow under these conditions. That CFL1 is able to rescue this phenotype suggests that it must be restoring the high-affinity iron-uptake mechanism of the cell, suggesting that it may be playing a direct role in iron accumulation by the cell. This would imply that Cfl1p is indeed being directed to the cell surface as the presence of a hydrophobic signal sequence suggests.
Thus, to date there are two parallel lines of evidence indicating that C. albicans uses a ferric reductase in iron acquisition in a similar manner to S. cerevisiae. Previous biochemical evidence (Morrissey et al., 1996 ) showed that it possesses a cell-surface ferric reductase activity whilst genetic evidence presented here demonstrates that C. albicans CFL1 is capable of rescuing a S. cerevisiae ferric reductase mutant and shows homology to other known ferric reductase genes. Furthermore, the Candida genome sequencing project has revealed the presence of a FET3-like gene in C. albicans. FET3 is a S. cerevisiae gene encoding a ferrous oxidase that forms part of the iron-transporter complex that internalizes iron after it has been reduced by the reductase (Askwith et al., 1994
). This provides additional support for the hypothesis that it uses a similar reductive mechanism to S. cerevisiae in iron acquisition. All studies carried out to date have been performed in vitro and it is not clear how this may relate to the in vivo situation. It is known that C. albicans can acquire iron from siderophores and haem, both sources that are likely to be available in vivo. The mechanism by which it may acquire iron from such sources and the role of the reductase in this have yet to be assessed. However, with the isolation of genes implicated in iron acquisition, we are now in a position to be able to construct mutant strains with deletions of these genes and assay for the affects on growth and ability to acquire iron from a range of different sources.
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Received 14 July 1999;
revised 17 December 1999;
accepted 6 January 2000.