Iron Acquisition from Transferrin by Candida albicans Depends on the Reductive Pathway
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感染与免疫杂志 2005年第9期
University of Pennsylvania, Department of Medicine, Division of Hematology/Oncology, 731 BRB II/III, 421 Curie Blvd., Philadelphia, Pennsylvania 19104-6160
Laboratoire d'Ingenierie des Proteines et Contrle Metabolique, Institut Jacques Monod, Tour 43, CNRS, Universites Paris 6 and Paris 7, 2 Place Jussieu, 75251 Paris Cedex 05, France
ABSTRACT
Host-pathogen interactions that alter virulence are influenced by critical nutrients such as iron. In humans, free iron is unavailable, being present only in high-affinity iron binding proteins such as transferrin. The fungal pathogen Candida albicans grows as a saprophyte on mucosal surfaces. Occasionally it invades systemically, and in this circumstance it will encounter transferrin iron. Here we report that C. albicans is able to acquire iron from transferrin. Iron-loaded transferrin restored growth to cultures arrested by iron deprivation, whereas apotransferrin was unable to promote growth. By using congenic strains, we have been able to show that iron uptake by C. albicans from transferrin was mediated by the reductive pathway (via FTR1). The genetically separate siderophore and heme uptake systems were not involved. FRE10 was required for a surface reductase activity and for efficient transferrin iron uptake activity in unbuffered medium. Other reductase genes were apparently up-regulated in medium buffered at pH 6.3 to 6.4, and the fre10–/– mutant had no effect under these conditions. Experiments in which transferrin was sequestered in a dialysis bag demonstrated that cell contact with the substrate was required for iron reduction and release. The requirement of FTR1 for virulence in a systemic infection model and its role in transferrin iron uptake raise the possibility that transferrin is a source of iron during systemic C. albicans infections.
INTRODUCTION
Candida albicans is a common fungal saprophyte for humans, growing on mucosal surfaces, including mouth and gut, of up to 60% of normal individuals (23). Under special circumstances, this pleiomorphic fungus becomes invasive and disseminates via the bloodstream. Disseminated Candida infections, occurring most frequently in the setting of neutropenia, cause tremendous morbidity and mortality (2, 57). Iron is an essential nutrient for C. albicans, and iron uptake may play a special role in promoting virulent infections (54). However, the iron sources that support intravascular infection and the fungal iron uptake systems that mediate iron acquisition in the intravascular space have not been defined.
An adult human contains about 3.5 g of iron. The majority of this iron circulates as hemoglobin in erythrocytes, and a smaller amount is bound to transferrin. Transferrin is a monomeric protein, with an approximate molecular mass of 80 kDa. The protein consists of two structurally similar lobes, each of which binds a single ferric iron atom in the presence of carbonate anion. In vertebrates, transferrin functions as the primary carrier of iron in blood. At blood pH (7.4), transferrin binds one or two ferric iron atoms with high affinity (Kd = 10–20 M) (1). Iron bound in the interior of the folded protein is protected from hydration, and this is critical because hydrated ferric iron forms insoluble ferric hydroxides that precipitate as rust. Iron in transferrin is also protected from oxidation-reduction reactions, except under special circumstances, such as those involved in physiological iron delivery. In mammalian cells, the surface structure of transferrin facilitates binding to transferrin receptors, as shown by a cocrystal structure (9). This is followed by rapid internalization into an endocytic compartment (16, 35). Conformational change, acidification, and iron reduction mediate iron release from transferrin, although the endosomal reductase has not yet been identified (9, 17, 22). Finally, the membrane permease DMT1 transports reduced (ferrous) iron from endosomal lumen to cytoplasm (7).
To acquire iron, C. albicans has separate iron uptake systems for different iron-containing substrates. There are independent systems for acquiring iron from siderophores, from heme, and from varied ferric iron chelates. Siderophores are small-molecule chelators that are synthesized and secreted by microorganisms (69). These have different structures, but all can bind and solubilize iron with extremely high affinity, and in some cases (e.g., Aspergillus fumigatus) these have been able to steal iron from transferrin (29). Although C. albicans lacks the enzymatic machinery to synthesize and secrete such molecules, it is equipped with a transporter for recovery of ferrichrome-type siderophores (30, 38). Presumably, in settings where mixed flora or mixed infections exist, the ability to scavenge siderophores secreted by other organisms would provide an advantage. A genetic knockout of SIT1 (ARN1), the siderophore transporter, did not confer loss of virulence in a mouse model of systemic infection, although the knockout strain did show a decreased ability to invade keratinocyte layers in a model system (28).
The majority of iron in a healthy human exists as heme in hemoglobin, and uptake systems for heme iron exist. C. albicans has a hemolytic activity, and plasma membrane proteins capable of binding hemin and hemoglobin have been identified (42, 45, 52, 58, 65, 66). The uptake pathway for hemin iron depends on the intracellular enzyme heme oxygenase (Hmx1p) to release iron from the porphyrin chelate (51, 58). The role of this system during intravascular infection has not been tested.
Finally, a reductive system exists, located in the plasma membrane of C. albicans and consisting of three activities, each encoded by multiple genes. A surface ferric reductase in C. albicans is able to reduce extracellular ferric chelates and has an activity that is approximately 10-fold greater than the activity of Saccharomyces cerevisiae. Two genes FRE10 (RBT2, CFL95) and FRE1 (CFL1) have been identified by complementation of S. cerevisiae reductase-deficient mutants, and a total of 12 genes can be identified by sequence homology searching of the C. albicans genome (25, 32, 36). Reduced ferrous iron generated by the surface reductase is, in turn, captured by a protein complex consisting of a multicopper oxidase and a ferrous permease. C. albicans has five genes with similarity to multicopper oxidase genes, and a requirement of the multicopper oxidase activity for ferrous uptake has been shown, although which gene is responsible is unknown (18, 32, 36). The ferrous permease activity is encoded by two highly homologous genes, with opposite regulations: FTR1 is induced by iron deprivation, and FTR2 is induced by iron loading (36, 54). Only FTR1 has been implicated in cellular iron uptake in that the knockout lacks high-affinity ferrous transport, whereas the FTR2 knockout has no phenotype. Importantly, deletion of FTR1 established a clear connection of iron with C. albicans virulence, as strains with FTR1 deleted were unable to establish a systemic infection in mice following intravenous tail vein injection (54).
The multiplicity of iron uptake systems in C. albicans and the multiplicity of genes involved for each have made it challenging to correlate genes and functions. In addition, little is known about which genes/functions operate in different clinically relevant niches. Here we focus on the mechanism of iron acquisition from transferrin, an intravascular iron binding protein present at high concentrations in the blood. Transferrin iron has generally been considered to be unavailable as an iron source for most microorganisms due to its high affinity for iron. We find here that C. albicans can acquire iron from transferrin by using a reductive strategy.
MATERIALS AND METHODS
Strains. C. albicans SC5314 was used as the wild-type reference strain. Strain BWP17 (ura3::imm434/ura3::imm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG), a gift from A. Mitchell (68), was used as the starting strain for FTR1 and SIT1 homozygous disruption mutants. BCa15-2 harbors a homozygous deletion of RBT2 (CFL95, FRE10) (rbt2::hisG/rbt2::hisG-URA3-hisG) and was a gift from B. Braun and A. Johnson (5). (RBT2/CFL95 is referred to as FRE10 in this paper; this is the name suggested by the annotators of the Candida genome at the Canadian Bioinformatics Resource of the National Research Council and, as we demonstrate in this report, describes the function [ferric reductase] of the gene product.) The homozygous knockout hmx1::ARG4/hmx1::URA3 strain and reconstructed hmx1::ARG4/hmx1::URA3 HMX1-HIS1::his1::hisG/his1::hisG strain have been described previously (58). A homozygous hmx1 knockout strain auxotrophically matched to the hmx1 reconstructed strain was created by target integration of NruI-cut pGEM-HIS1 (68) (hmx1::ARG4/hmx1::URA3 HIS1::his1::hisG/his1::hisG).
To create strains with FTR1 and SIT1 disrupted, we used the technique of gene disruption with short homology regions described by Wilson et al. (68) with plasmids generously provided by Aaron Mitchell. The DNA construct to create the homozygous FTR1 deletion (strain CT87.4) was generated with primers 5'-CGTTCAAATTTTCTTCATCGTTTTCAGAGAATCTTTGGAAGCTATCATTGTTGTTTCAGTGCTTTTGGCGTGGAATTGTGAGCGGATA-3' and 5'-GTCTCTTGCCTTATTCTTTTAGTTGTTGAATAATAATTAACTAAGTTTATTTGTTTTCTTTGGATTCGTTTCCCAGTCACGACGTT-3'. These primers were used to amplify the ARG4 and URA3 cassettes from pGEM-URA3 and pRS-ARG4SpeI and contain flanking homology to FTR1 of 69 and 68 nucleotides. Homologous recombination using the PCR product resulted in 1,177 bp, removing the majority of the open reading frame (ORF) from base 85 (where base 1 is A of the initiator ATG) and 47 bp of 3' untranslated region. Successful integration was verified by PCR with primers 5'-ATTACTTTGACAGAAACACC-3' and 5'-AATGAAACCAATATTTTCCC-3' directed outside the region of recombination. To reintroduce FTR1 into CT87.4, primers 5'-CGTGGCATAAATAACTGAATC-3' and 5'-GTGTGGCGACTTTGTAAAC-3' were used to amplify the FTR1 ORF with 997 bp 5' and 337 bp 3' of flanking sequence from SC5314 genomic DNA. The PCR product was ligated into pCR2.1-TOPO to create p020403. Two independently generated PCR products were sequenced. Both contained a single nucleotide difference, at different locations, from the sequence of ORF 6.8119 of the C. albicans sequencing project (sequence data for C. albicans were obtained from the Stanford Genome Technology Center website at http://www-sequence.stanford.edu/group/candida). To eliminate these differences, both plasmids were cut with ScaI, which cuts p020403 twice, and the two fragments which did not contain errors were ligated together to create p022103, which was sequence verified. p022103 was cut with SacI and NsiI, and the 2.6-kb DNA fragment harboring FTR1 was ligated into the corresponding sites of pGEM-HIS1 to generate p031003. This plasmid and pGEM-HIS1 were linearized with NruI and transformed into the homozygous FTR1 knockout mutant, CT87.4, to create the reconstructed strain (CT123.2) and an auxotrophically matched homozygous knockout strain (CT128.1). Correct insertion at a his1::hisG locus was verified by PCR using oligonucleotides 5'-ATTACTTTGACAGAAACACC-3' and 5'-AATGAAACCAATATTTTCCC-3'.
The SIT1 homozygous knockout strain (CT27.1) was constructed with similar methodology using primers 5'-CCAGTCTTCCAATAATCATTCTTCAGAAGAAGATAAACACTTGTCCGGAGATGAAAAGACGTTTTCGTGGAATTGTGAGCGGATA-3' and 5'-GCTACTCTTTTCTTCTTGAAATTGCCGAAGAAATTGGCCAACGAGTCCTTCTCTTCTTGCTTTTCTTTTCCCAGTCACGACGTT-3'. Homologous recombination with the PCR product resulted in a 1,668-bp deletion of the majority of the ORF from base 78. To create a reconstructed strain, primers 5'-CGCCTGAACAGGGTTTTATCG-3' and 5'-GTATGTGTGAATAGTGAGCGG-3' were used to amplify the SIT1 ORF with 1,082 bp of 5' and 315 bp of 3' flanking regions from wild-type genomic DNA. As for FTR1, subcloning of independently created PCR products was required to obtain a SIT1 product with sequence identity to that of assembly 6 in the Candida database. The sequence-verified SIT1 was subcloned into pGEM-HIS1 at ApaI/SacII to create plasmid p101002. The homozygous SIT1 knockout strain (CT27.1, sit1::ARG4/sit1::URA3 his1::hisG/his1::hisG) was transformed with NruI-linearized pGEM-HIS1 and p101002 to create the auxotrophically matched knockout strain (CT94.1, sit1::ARG4/sit1::URA3 HIS1::his1::hisG/his1::hisG) and reconstructed strain (CT91.1, sit1::ARG4/sit1::URA3 SIT1-HIS1::his1::hisG/his1::hisG).
To reinsert FRE10 into BCa15-2, plasmid p1A2, selected from a C. albicans genomic DNA library (generously provided by G. Fink) by its ability to complement an S. cerevisiae fre1 fre2 mutant (36), was used as the DNA source. A 4.5-kb SacII-ApaI fragment from p1A2 was subcloned into the corresponding sites of pGEM-URA3 to create plasmid p062804. A Ura– derivative (CT187.2) of BCa15-2 was created by selection on 5-fluoroorotic acid-containing medium as described previously (20). Plasmid p062804 was linearized at the PshA1 site in the 5' noncoding region of FRE10 and transformed into strain CT187.2. Ura+ transformants were selected, screened for ferric reductase activity, and verified by Southern blotting (see Results). An auxotrophically matched control strain for the FRE10 reconstructed strain was also created. Plasmid p062804 was digested with HindIII and religated to create plasmid p082404. This process removed the majority of the FRE10 ORF and all the 3' noncoding region. Only the noncoding 5' region and 190 bp of the FRE10 ORF remained. Plasmid p082404 was digested with PshA1, and the linear DNA was used to transform CT187.2. Ura+ transformants were selected and correct integrants identified by Southern blotting (see Results).
Saccharomyces cerevisiae strains used were YPH499 (MATa ura3-52 lys2-801[amber] ade2-101[ochre] trp1-63 his3-200 leu2-1) (62) and the congenic ferric reductase knockout strain 49912 (MATa ura3-52 lys2-801[amber] ade2-101[ochre] trp1-63 fre1::LEU2 fre2::HIS3) (19).
Growth conditions and growth media. For all experiments, strains were streaked from –80°C glycerol stocks onto yeast extract-peptone-dextrose agar medium, grown for 2 days at 30°C, and then transferred to room temperature, where they were maintained for a maximum of 10 days before being discarded. All experiments were initiated from inoculation of a single colony to a single culture. For biological replicates, multiple single colonies were individually inoculated into multiple cultures. Defined iron-deficient medium was prepared from complete synthetic medium (CSM) (0.79 g/liter) (Q-BioGene); yeast nitrogen base without amino acids, Cu, and Fe (6.7 g/liter) (Q-BioGene); 2% D-glucose; and 1 μM copper sulfate. The pH of this unbuffered medium was 4.3 to 4.4. In some experiments, 50 mM MES (morpholinethanesulfonic acid) was added to this medium and the pH maintained at 5.8 to 5.9. In other experiments 50 mM HEPES was added to the medium and the pH maintained at 6.3 to 6.4. For selection of transformants, CSM was replaced with the appropriate amino acid/base dropout mixture, and Difco yeast nitrogen base was used (Becton Dickinson & Co.). For growth of Ura– strains CSM was supplemented with 100 mg uridine/liter. Milli-Q (Millipore) water was used for all growth media and chemical solutions. All iron-deficient media were prepared in plastic containers and filter sterilized, and cells were grown in polypropylene and polystyrene vessels.
Growth assays. C. albicans was starved for iron by growth at 30°C in yeast extract-peptone-dextrose medium containing uridine and 0.5 mM bathophenanthroline disulfonate (BPS) to chelate ferrous iron. After 20 h the cells were subinocluated into medium with the same composition and grown at 30°C for another 20 h. Cells were harvested by centrifugation, washed in Milli-Q water, and resuspended in Milli-Q water to an optical density at 600 nm (OD600) of 5.0 (Cary Olis spectrophotometer). These cells were the starting point for the cell growth experiments, which were performed in 96-well plates with 200 μl of medium per well. The cells suspended in water were inoculated into HEPES-buffered, low-iron CSM medium with an appropriate iron source to give an OD600 of 0.1 (Cary Olis spectrophotometer). This OD corresponds to an OD720 of 0.01 to 0.02 when measured in the 96-well spectrophotometer (Bio-Tek EL800) and represents the lower limit of detection for this instrument. Cells were grown at 30°C without shaking, and growth was determined by resuspending the cells and measuring cell density with the 96-well spectrophotometer. Greater than 99.9% of cells were in the yeast form throughout the growth period.
Preparation of 55Fe-transferrin and 55Fe-ferrichrome. Human holotransferrin and apotransferrin were purchased from Calbiochem. 55Fe-labeled transferrin was prepared by the addition of a threefold molar excess of 55FeCl3 to 100 μM apotransferrin in transferrin loading buffer (0.1 M HEPES, pH 7.5, 0.15 M sodium chloride, and 10 mM sodium bicarbonate). After incubation for 30 min at 22°C and 30 min on ice, the labeled transferrin was separated from unbound 55Fe by using a Sephadex G-25 spin column equilibrated with transferrin loading buffer. Iron-loaded transferrin was greater than 99% saturated and was efficiently separated from iron by the column.
A 10% molar excess of iron-free ferrichrome from Ustilago sphaerogena (Sigma) was incubated with 55FeCl3 in 1 M Tris, pH 7.4, for 5 min at 22°C to a give final concentration of 100 μM 55Fe-ferrichrome.
Ferric reductase and high-affinity iron uptake assays. The ferric reductase assays rely on the formation of a red BPS-Fe(II) complex, which is quantified by measuring absorbance at 515 nm (13). Sources of ferric iron were ammonium ferric sulfate and holotransferrin. For reductase assays, cells were washed three times with 50 mM citrate buffer (pH 6.6)-5% D-glucose and incubated in 50 mM citrate buffer (pH 6.6)-5% D-glucose containing BPS and a ferric substrate for up to 10 min at 30°C. Unless indicated, BPS and ferric substrate concentrations were 1 mM. Assay mixtures containing no cells were used to determine blank values, which were subtracted from values obtained in assays with cells. Reductase activity was normalized with cell number determined from optical measurements of culture turbidity. Greater than 99.9% of cells were in the yeast form.
High-affinity iron uptake was measured as described previously (15). Cells were washed three times in citrate-glucose buffer and resuspended in the same buffer. Iron sources of 1 μM ferrous (55Fe)-ascorbate, 55Fe-holotransferrin, and 55Fe-ferrichrome were added to the suspended cells and incubated at 30°C. After 1 h, cells were resuspended and optical densities of the cultures were measured. The cells were harvested and washed free of 55Fe label by using either a 24-well cell harvester (PHD Cambridge Technologies) or a 96-well harvester (Tomtec). Cell-associated 55Fe was measured by liquid scintillation counting (Beckman LS6500 or Perkin-Elmer 1450 MicroBeta). Assay mixtures containing no cells were used to calculated blank values for correction.
Requirement for cell contact. Wild-type C. albicans was grown in two sequential overnight cultures of HEPES-buffered, low-iron defined medium, subinoculated into medium of the same composition, and grown for an additional 2 h at 30°C. The cells were washed three times in Milli-Q water and resuspended in citrate-glucose buffer at an OD600 of 1. Dialysis tubing (Spectra/Por 2) was prepared by soaking in 0.1 M EDTA for 20 min followed by copious rinsing with Milli-Q water. Holotransferrin (0.1 ml, 2 mM) was placed in the dialysis bags, which were then incubated with 5 ml of cells in the presence of 0.1 mM BPS for 30 min at 30°C.
RESULTS
Holotransferrin promotes growth of iron-starved C. albicans. To determine if C. albicans can use human transferrin as an iron source, wild-type C. albicans strain SC5314 was starved for iron and inoculated into low-iron defined medium buffered with HEPES, pH 6.4. Iron was added back, in parallel cultures, as holotransferrin (0.2 μM), ferric ammonium sulfate (0.4 μM), or ferrichrome (0.4 μM), and growth was monitored by frequent turbidity measurements. Throughout the growth period the cells grew with yeast morphology. The concentrations of iron sources were chosen to ensure that iron uptake from these sources would be via a high-affinity iron uptake system. The concentration of holotransferrin was half that of the other iron sources, because each molecule of transferrin binds two atoms of ferric iron. Growth of cultures supplemented with iron salts, ferrichrome, or diferric transferrin was enhanced compared to that of cultures with apotransferrin or no addition (Table 1; Fig. 1A). Analysis of the growth curves showed that logarithmic growth of the cells supplemented with iron occurred at between 4 and 9 h postinoculation. In the absence of iron supplementation C. albicans cells exhibited a prolonged lag time, and logarithmic growth occurred between 8 to 15 h. Doubling times for the cultures were calculated during the time of logarithmic growth (Table 1). Wild-type cells grown in the presence of the three iron sources (holotransferrin, ferric ammonium sulfate, or ferrichrome) showed similar doubling times, which were at least 3.5-fold faster than those of cells grown in the absence of iron or in the presence of apotransferrin. During the first 24 h of incubation, the pH of the cultures decreased to pH 6.3. During the same time period, 4 nM iron dissociated from 55Fe-transferrin in control cultures without cells, as determined by liquid scintillation counting of the flowthrough following separation of the medium through a Centricon filtration device.
The FTR1 iron permease is required for optimal growth with a transferrin iron source. C. albicans has at least three iron uptake systems, raising the question of which system mediates iron acquisition from transferrin. The ftr1–/– mutant, lacking the ferrous iron permease, is impaired in the reductive iron uptake system. We evaluated the ftr1–/– mutant for iron-dependent growth with diferric transferrin as the iron source, and a pronounced effect was observed (Table 1; Fig. 1B). The lag time of the ftr1–/– mutant was 5 h, compared to 4 h for the wild-type and reconstructed ftr1–/– + FTR1 strains. The doubling time of the ftr1–/– mutant was, however, greatly increased to 10.5 h, compared to 1.8 h and 1.6 h for the wild-type and reconstructed strains, respectively. At later time points (after 13 h), the growth rate of the ftr1–/– mutant increased substantially, and there was increased variability between the replica cultures. A similar acceleration of growth rate was also observed for the ftr1–/– mutant grown with ferric ammonium sulfate 6 h following inoculation. The cause of this growth rate change is unknown; it might be due to genetic change, but this has not been ascertained. Without iron addition to the medium, the ftr1–/– mutant was growth arrested (Table 1). In contrast, the wild-type and ftr1–/– + FTR1 strains were able to grow under these iron-starved conditions, although their growth was impaired. It is likely that small amounts of contaminating iron salts are still present in the low-iron medium and that cells may have access to this iron via Ftr1p.
Ferrichrome iron uptake bypasses growth inhibition of the ftr1–/– mutant exposed to apotransferrin. C. albicans can acquire iron from ferrichrome through the permease Sit1p; thus, iron-related effects of apotransferrin could be bypassed by supplying iron to the ftr1–/– strain as ferrichrome. In the presence of apotransferrin alone, the ftr1–/– strain grew extremely slowly. Wild-type and ftr1–/– + FTR1 cultures grew with doubling times that were two to three times longer than those of holotransferrin supplemented cultures (Table 1; Fig. 2). When the iron-starved cells were incubated with apotransferrin plus ferrichrome, the doubling times of the ftr1–/– and ftr1–/– + FTR1 strains were shortened. The two strains had equivalent doubling times, indicating that FTR1 was not required for high-affinity uptake of iron from ferrichrome. Intriguingly, the doubling times were about 20 min longer than those with ferrichrome alone for both strains (Table 1). It is possible that apotransferrin may inhibit growth of C. albicans independent of effects on iron availability. Lactoferrin, a homologous iron binding protein, has been shown to have similar effects unrelated to iron chelation (43). Alternatively, apotransferrin may compete with ferrichrome for ferric iron, and the resulting monoferric transferrin may be a poorer iron source than diferric transferrin or ferrichrome.
High-affinity iron uptake from transferrin requires C. albicans FTR1. The growth advantage that C. albicans strains harboring C. albicans FTR1 have, when provided with a transferrin iron source, is likely due to Ftr1p-mediated uptake of iron from transferrin. Iron uptake from transferrin was measured directly using radiolabeled 55Fe-diferric transferrin. When ftr1–/– cells were grown under iron-depleted conditions (Fig. 3A), the uptake of Fe from transferrin was 17% of the wild-type level. When a single copy of FTR1 was reinserted (Fig. 3A), iron uptake in this auxotrophically matched reconstructed strain was increased threefold compared to that in the homozygous knockout. This corresponds to 50% of the wild-type level. Similar results were seen with ferrous ascorbate as the iron source (Fig. 3B). One possible explanation is that two copies of FTR1 are required for full high-affinity ferrous uptake under inducing (low-iron) conditions. Alternatively, the promoter region, 998 bp 5' of the FTR1 ORF, used in the reconstructed DNA FTR1 allele might be insufficient for maximal expression. This second explanation is supported by the observation that the heterozygous mutant (ftr1::ARG4/FTR1) did not show diminished iron uptake from ferrous ascorbate compared to the wild type (data not shown). The absolute value of iron uptake from 55Fe-transferrin was approximately double the uptake from ferrous ascorbate, perhaps because equivalent molar amounts (1 μM) of iron sources were used and the diferric transferrin molecule carries two atoms of iron per molecule. Following growth in iron-supplemented medium, uptake of iron from holotransferrin or ferrous ascorbate was down-regulated in the wild-type and ftr1–/– + FTR1 strains to levels comparable to those in the ftr1–/– mutant (data not shown). C. albicans mutants with mutations in the siderophore uptake pathway (sit1–/–) and heme iron utilization pathway (hmx1–/–) were not altered in their ability to acquire iron from either holotransferrin or ferrous ascorbate (Fig. 3A and B). We also observed that the ftr1–/– mutant was unaffected in 55Fe-ferrichrome uptake (Fig. 3C), as shown previously (28), indicating independence of the reductive and siderophore uptake systems.
Iron uptake from transferrin is copper dependent. Ferrous iron uptake in C. albicans is copper dependent (36, 46, 67), and optimal growth of C. albicans under low-iron conditions requires a multicopper oxidase, encoded by FET3 (18). Wild-type C. albicans cultures were grown in low-iron defined medium containing 1 or 0.1 mM of the copper chelator bathocuproinedisulfonic acid (BCS). These cells exhibited only 0.6 and 16%, respectively, of the level of iron uptake from transferrin that copper adequate cells displayed (Fig. 4). This requirement for copper strongly suggests the involvement of a multicopper oxidase, a partner protein required with the ferrous permease for high-affinity ferrous iron uptake. Overall these biochemical results, together with the analysis of the ftr1–/– strain, indicate that iron uptake from transferrin depends on the reductive uptake pathway.
FRE10 encodes a cell surface ferric reductase. Iron bound to transferrin is coordinated as a ferric chelate (+3 valence) with synergistically bound carbonate ions (1). The Ftr1p/Fetp iron uptake system mediates transport of ferrous iron (+2 valence), and thus a ferric reductase that is able to convert the iron to the required oxidation state for transport must exist. A gene, RBT2, with homology to the ferric reductases of S. cerevisiae was identified in a screen for Tup1-repressed target genes (5). This gene was also independently cloned by its ability to complement an S. cerevisiae mutant lacking cell surface ferric reductases (CFL95) (36). (From here on we will refer to this gene as FRE10, as suggested by the BRI annotation group [http://candida.bri.nrc.ca/candida/index.cfm] and listed in Table 2.) The homozygous diploid fre10 knockout strain (BCa15-2) was converted to Ura–, and a wild-type copy of FRE10 was reinserted at its native locus. The source of the wild-type FRE10 allele was the clone with complementary activity for the S. cerevisiae ferric reductase mutant (36). To generate an auxotrophically matched control strain, a truncated nonfunctional fre10 allele was inserted at the same locus. The genotypes of these strains were confirmed by Southern blotting after digestion of genomic DNA with EcoRI (Fig. 5A and B), and XbaI plus XhoI (not shown). These strains were then examined for cell surface reductase activity for ferric iron salts (ferric ammonium sulfate). The cells were grown in low-iron defined medium and assayed for ferric reductase activity when they reached logarithmic growth. Wild-type SC5314 was used as a reference strain. The original fre10 homozygous mutant (BCa15-2) exhibited only 2% of the activity of the wild-type strain (Fig. 5C, compare bars 1 and 2). The cell surface ferric reductase activity of the Ura– derivative of BCa15-2, strain CT182.2, was not different from that of BCa15-2 (compare bar 3 with bar 2). When a single copy of FRE10 was reinserted into CT182.2, creating strains CT218.2 and CT222.1, cell surface ferric reductase activity was restored to wild-type levels (bars 4a and 4b). In contrast, the strains (CT212.2 and CT216.2, bars 5a and 5b) in which a truncated copy of fre10 had been reinserted maintained the very low activity of the deleted fre10 strains. In a separate experiment (not shown), the rates of the ferric reductase reactions were measured in a time course assay and were 0.34 nmol Fe(II)/million cells/min for SC5314 and 0.03 nmol Fe(II)/million cells/min for BCa15-2. These reactions were linear over the 10-min assay time. Overall these results provide strong evidence that FRE10 encodes or positively regulates a major cell surface ferric reductase.
Iron uptake from transferrin is impaired in fre10 mutants. C. albicans cells were grown in low-iron defined medium and analyzed for iron uptake from 55Fe-labeled transferrin and 55Fe-ferric chloride. Under these conditions, cellular uptake of radioactive iron from transferrin by strain BCa15-2 was 25% of that of the wild type (Fig. 6). A similar magnitude of difference was observed between the auxotrophically matched reconstructed strains CT218.2, harboring a single copy of FRE10, and CT212.2, harboring a truncated allele of fre10. These experiments were repeated with an equimolar amount of iron in the form of 55Fe-ferric chloride as the iron source. Iron uptake from transferrin or ferric chloride was compromised to a similar degree in the reductase-deficient mutants, indicating an important role of FRE10 in iron uptake from transferrin under these conditions.
Induction of an alternative reductase(s) in the absence of FRE10. The results from the experiments described above indicate that C. albicans has the ability to reduce and to take up iron from human transferrin, using this iron to support growth. However, although FRE10 was needed for maximal rates of iron uptake from transferrin under some conditions (see above), under other conditions FRE10 was dispensable. Using the same growth conditions outlined for Fig. 1, C. albicans wild-type, reconstructed mutant (fre10–/– + FRE10), and fre10 mutant () strains were grown in low-iron defined medium containing 1 μM copper sulfate and buffered with 50 mM HEPES to give a final pH 6.3 to 6.4. Iron was added back to this medium as 0.4 μM ferric ammonium sulfate, 0.4 μM ferrichrome, or 0.2 μM human holotransferrin. Surprisingly, there were no large differences in doubling times among the strains when they were grown with transferrin (Fig. 7A) or with the other iron sources (not shown). The logarithmic-phase doubling time for all strains with holotransferrin as the iron source was 1.5 h. This compares to doubling times of 1.2 to 1.3 h when the strains were grown with ferric iron as the iron source and of 1.3 to 1.6 h when the strains were grown with ferrichrome. The unmanipulated wild type exhibited a shorter lag time than the other strains, perhaps related to URA3 location or copy number, but the effect was not iron dependent (Fig. 7A).
A possible explanation for the lack of effect of the fre10 mutants is that other cell surface reductases might be active under these growth conditions. Eight hours following inoculation into transferrin-supplemented low-iron medium, all strains, with or without the FRE10 gene, exhibited similar cell surface ferric reductase activities (Fig. 7B). This result indicates that in the HEPES-buffered medium (pH 6.3 to 6.4) at least one other surface ferric reductase was active that was repressed in the nonbuffered medium (pH 4.3 to 4.4) utilized for some experiments (Fig. 5 and 6). Table 2 lists open reading frames of C. albicans that have homology to FRE10 and might encode such an alternative cell surface ferric reductase. One of these, FRE1 (CFL1), has been shown to complement an S. cerevisiae mutant lacking cell surface reductase activity (25). We examined the steady-state level of FRE1 mRNA by using Northern blots on RNA prepared from cells grown on low-iron HEPES-buffered medium. FRE1 mRNA was expressed at equal levels in the wild type and in strains with FRE10 disrupted, and there did not appear to be any regulatory compensation of CFL1 expression for lack of FRE10 expression (data not shown).
Iron uptake from 55Fe-transferrin was tested in cells grown in buffered medium. Iron uptake from transferrin (Fig. 7C) and ferric ammonium sulfate (not shown) were uniformly higher than in the same strains grown in nonbuffered medium (compare Fig. 7B to Fig. 6). For the wild type this was a fivefold difference, and for the two strains lacking FRE10, the differences were 17-fold and 15-fold compared with cells grown in nonbuffered medium. Similar magnitudes of difference were observed when ferric chloride was used as the iron source. Overall these results suggest that the reductive iron acquisition system is modulated by pH as well as by iron. The small contribution of FRE10 to reductase activity and iron uptake from ferric iron sources when cells are grown in medium buffered at pH 6.3 to 6.4 with 50 mM HEPES or MOPS (morpholinepropanesulfonic acid) (data not shown), compared to the large contribution it makes when cells are grown at pH 4.4, agrees with the recent observation that FRE10 expression is down-regulated at alkaline pH (3).
Contact of transferrin with C. albicans promotes iron release. A cell surface reductase involved in iron acquisition might physically interact with its substrate on the cell surface, or, alternatively, it might act on soluble or secreted reducing intermediates. To distinguish these possibilities, the following experiment was performed. Transferrin was placed in a dialysis bag to physically separate it from a suspension of iron-starved wild-type cells that had been grown in HEPES-buffered defined medium. The cells were washed and resuspended in citrate buffer, pH 6.6, containing BPS to measure reduced/released iron. When cells where physically separated from transferrin, very little pink coloration was observed, indicating lack of formation of ferrous-BPS (Fig. 8). Furthermore, the holotransferrin within the dialysis bag retained its brown color, indicating a failure to release the bound iron. In contrast, when the dialysis bag was punctured, allowing transferrin to come directly in contact with the cells, the bright pink color of a ferrous-BPS complex quickly formed. In the absence of cells, there was no indication of formation of an iron-BPS complex when the dialysis bag was punctured.
Reductive iron uptake from transferrin is highly elevated in C. albicans compared to S. cerevisiae. The nonpathogenic yeast S. cerevisiae has a reductive iron uptake system with components homologous to that of C. albicans. Wild-type S. cerevisiae, a S. cerevisiae ferric reductase mutant, and wild-type C. albicans were grown in low-iron defined medium and in media of the same composition buffered to pH 6.3 with 50 mM HEPES or 50 mM MOPS. While in log phase the cells were analyzed for iron uptake from 55Fe-labeled transferrin. Under all three conditions wild-type S. cerevisiae was able to acquire some iron from transferrin, whereas the ferric reductase mutant had undetectable and diminished iron uptake, indicating that iron uptake from transferrin occurred through the reductive pathway (Fig. 9). The most striking finding, however, was that the rate of iron acquisition from transferrin was considerably greater in C. albicans than in S. cerevisiae.
DISCUSSION
C. albicans, which is generally a saprophyte on mucosal surfaces, in some settings disseminates within the circulation; in order to survive, it must find ways to access critical nutrients (such as iron) in the hostile host environment. Within the host circulation, iron is present as heme in hemoglobin or as transferrin. Here we show that C. albicans can access transferrin iron via the high-affinity reductive system. Interestingly, C. albicans components involved in transferrin iron acquisition mirror those of the host pathway, including components for iron reduction and ferrous iron transport.
Use of the reductive pathway. C. albicans was starved for iron until growth was retarded. Holotransferrin added to these iron-depleted cells was able to restore growth, indicating that it was being used as a source of iron. Growth promotion was quite good as assessed by effects on lag time and doubling time, although it was still inferior to that with ferrichrome or ferric salts. Thus, transferrin iron can be efficiently used as an iron source. Radionuclide iron uptake from transferrin was also measured and was efficient compared with that from iron salts. Uptake- and growth-promoting effects were dependent on components of the reductive iron uptake pathway, including ferric reductase, multicopper oxidase, and ferrous permease.
For iron uptake into mammalian cells, diferric or monoferric transferrin binds to cell surface transferrin receptors. The transferrin-transferrin receptor complex is endocytosed, and the pH of the endosome is lowered, allowing protonation of the carbonate and amino acid ligands for iron. Interaction of transferrin with the receptor induces a change in transferrin conformation, increasing the redox potential of the bound iron to a value compatible to reduction by an NADPH-dependent reductase (17). The reduction of ferric to ferrous iron would favor release, because the transferrin affinity for ferrous iron is 1014-fold lower than that for ferric iron (26). Finally, the DMT ferrous transporter moves the released ferrous iron into the cytoplasm. The C. albicans system for iron acquisition from transferrin is similar. Specific transferrin binding to the C. albicans cell surface can occur (S. A. B. Knight et al., unpublished observations), and a surface reductase facilitates iron reduction and release, although other factors such as transferrin conformational changes, anions, and protonation may also contribute to iron release. A requirement for C. albicans reductase activity was observed, in that fre10–/– strains lacking surface reductase activity were also deficient in iron uptake from transferrin. Following or coincident with ferrous iron release, a high-affinity transporter-multicopper oxidase transports the ferrous iron into the C. albicans cell. Dependence of this process on multicopper oxidase is supported by data showing that C. albicans starved of copper was unable to take up iron from transferrin. The specific multicopper oxidase gene(s) responsible has not been identified among multiple candidates. Finally, the growth-promoting effect and radionuclide uptake were entirely dependent on FTR1, being abrogated in the ftr1–/– mutant, which lacks the ferrous permease (analogous to DMT1 of the host). In summary, independent data showing requirements for reductase, oxidase, and permease components support a role for the reductive pathway in iron uptake from transferrin. The other iron uptake pathways, for heme and siderophores, were not involved in iron uptake from transferrin.
Which reductase S. cerevisiae Fre1p has been characterized as a prototype ferric reductase. It is an integral plasma membrane protein and requires cofactors of heme and flavin (19). The reductase has broad specificity and utilizes electrons donated from intracellular NADPH to reduce metals (Fe3+ and Cu2+) or dye substrates (14, 27, 61). C. albicans too has a cell surface reductase activity, and the C. albicans FRE10 open reading frame has been shown to complement S. cerevisiae mutants lacking ferric reductase (36). Complementing activity corrected slow growth of the reductase mutant in low-iron media, and surface reductase activity was also restored. In the C. albicans genome there are at least 12 ORFs with sequence similarity to C. albicans Fre10p. These are recognizable by the presence of signature motifs, including four conserved histidine residues, predicted to coordinate two hemes within the membrane, and a FAD isoalloxazine binding motif (HPFT). The 12 ORFs with highest homology to Fre10p are listed in Table 2.
In studies with the fre10–/– mutant, we found that ferric reductase activity was almost completely lacking (<5% residual activity) compared with a wild-type control when the strains were grown in low-iron defined medium. The implication is that FRE10 encodes the major ferric reductase under these conditions. Of note is that the pH of the growth medium declined to 3.2 to 3.3 during the course of the experiment with unbuffered medium. When the experiment was repeated with growth medium buffered by the addition of HEPES KOH (or MOPS [data not shown]) at pH 6.3, the fre10–/– mutant and wild type were now indistinguishable in terms of surface reductase activity. Furthermore, the activities were markedly increased (about twofold) compared to those of cells grown in unbuffered medium. The implication is that the alkaline pH buffering resulted in induction of another reductase gene(s).
The identification of the FRE genes responsible for this alternative reductase activity may be facilitated by recently published microarray studies. In those studies, FRE1, FRE2, FRE4, and FRE5 transcripts were up-regulated by iron limitation (37). FRE7 was induced by macrophage phagocytosis (41). FRE1, FRE2, FRE7, and FRE9 were up-regulated at pH 8 (3). Thus, in the iron-starved HEPES-buffered medium, these genes would be candidates for providing the backup activity. FRE1 expression in wild-type and fre10–/– strains grown in HEPES-buffered defined medium was measured by Northern blotting, and compensatory induction was not observed (not shown). However, FRE1 has been shown to have complementing activity for S. cerevisiae reductase-deficient mutants (25) and has not been ruled out as the gene responsible for the major reductase under less acidic conditions. In summary, FRE10 encodes the major cell surface ferric reductase in unbuffered medium, but it is not required for virulence in a systemic infection model (5) and is not a major contributor to the activity under conditions of buffered pH above 6.3.
Role of transferrin iron uptake in virulence. Host-pathogen interactions that determine virulence are influenced by critical nutrients such as iron (55, 59). During intravascular infections, heme and transferrin represent the likely major iron sources for pathogens. To protect these sources, the host mobilizes an iron-withholding defense. Microbial lipopolysaccharides trigger increased expression of the peptide mediator hepcidin, a component of the type II acute-phase response (47, 53). This in turn leads to inhibition of duodenal iron absorption and an increase of iron retention in macrophages (48). This can culminate in anemia of inflammation, lowering transferrin iron saturation and limiting the availability of iron to an invading pathogen (21, 33). In contrast, there are also clinical conditions in which serum iron and transferrin saturation increase, conditions that favor some pathogens. Patients with leukemia and individuals who have undergone bone marrow transplants have increased susceptibility to fungal infections, and sera from these patients have been shown to promote rather than inhibit Candida growth (8, 31, 44).
From the point of view of the pathogen, the problem is to circumvent this iron-withholding defense. For a number of pathogens (Neisseria gonorrhoeae [11, 12], Neisseria meningitidis [56], and Haemophilus influenzae [24]), iron uptake from transferrin requires specific outer membrane binding proteins, and without these proteins, these bacteria have substantially reduced virulence. For Vibrio vulnificus and Aeromonas hydrophila, secreted siderophores are able to capture transferrin iron and return it to the pathogen (39, 63). A similar mechanism operates in the fungal pathogen Aspergillus fumigatus (29), and siderophore biosynthesis is essential for the virulence of this fungus (60). Histoplasma capsulatum expresses a multicomponent reductive system that may capture iron from transferrin in the intravascular space (64). For fungi that do not synthesize siderophores, a highly active reductive system maybe critical for iron acquisition from transferrin and might distinguish pathogenic yeasts from nonpathogenic yeasts that have less active systems.
C. albicans lives primarily as a saprophyte on mucosal surfaces, but in the setting of neutropenia, mucosal integrity may be compromised and the organism may gain access to the systemic circulation. Within the blood, C. albicans partially transforms to hyphal growth and binds to endothelial cells, invading and proliferating into microabcesses. Target tissues frequently include liver, spleen, and heart valves. Transferrin iron could be critical during the transit time in the circulation or during the invasive period of growth. Transferrin iron could also sustain C. albicans viability during passage through an intravascular compartment such as the phagosome. Phagocytosed C. albicans yeast cells are able to evade the macrophage by physically puncturing the wall of the phagosome and the phagocytic cell (40). Transferrin receptor, and by extension transferrin, have been found with C. albicans in phagosomes of mouse macrophages (34), and iron availability within this compartment has been shown to be important for virulent infections (49). Other pathogens (Mycobacterium tuberculosis [10, 50], Leishmaina amazonensis [4], H. capsulatum [70], and Paracoccidioides brasiliensis [6]) have been shown to pirate iron from transferrin while in phagosome or phagosome-like compartments.
Ftr1p, the ferrous iron permease, is required for propagation of C. albicans infections in a mouse intravenous infection model (54). FTR1 encodes the ferrous iron permease of the reductive iron uptake system, raising the question of where the critical ferrous iron substrate for supporting these intravenous infections originates. A possibility supported by data presented here is that the iron pool needed to support virulent infections in this model system originates from transferrin iron.
ACKNOWLEDGMENTS
This work was supported by grants from the Center for AIDS Research University of Pennsylvania, the American Cancer Society, and the NIH (RO1 AI052384) to A.D. Sequencing of the C. albicans genome at Stanford was accomplished with the support of the NIDR and the Burroughs Wellcome Fund.
We are very grateful to Aaron Mitchell (Columbia University), Gerry Fink (Whitehead Institute), and Alexander Johnson (UCSF) for yeast strains and plasmids. We thank Joel Bennett and the Abramson Cancer Center for use of their gamma counter and 96-well liquid scintillation counter. We also thank the anonymous reviewers whose suggestions improved the manuscript.
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Laboratoire d'Ingenierie des Proteines et Contrle Metabolique, Institut Jacques Monod, Tour 43, CNRS, Universites Paris 6 and Paris 7, 2 Place Jussieu, 75251 Paris Cedex 05, France
ABSTRACT
Host-pathogen interactions that alter virulence are influenced by critical nutrients such as iron. In humans, free iron is unavailable, being present only in high-affinity iron binding proteins such as transferrin. The fungal pathogen Candida albicans grows as a saprophyte on mucosal surfaces. Occasionally it invades systemically, and in this circumstance it will encounter transferrin iron. Here we report that C. albicans is able to acquire iron from transferrin. Iron-loaded transferrin restored growth to cultures arrested by iron deprivation, whereas apotransferrin was unable to promote growth. By using congenic strains, we have been able to show that iron uptake by C. albicans from transferrin was mediated by the reductive pathway (via FTR1). The genetically separate siderophore and heme uptake systems were not involved. FRE10 was required for a surface reductase activity and for efficient transferrin iron uptake activity in unbuffered medium. Other reductase genes were apparently up-regulated in medium buffered at pH 6.3 to 6.4, and the fre10–/– mutant had no effect under these conditions. Experiments in which transferrin was sequestered in a dialysis bag demonstrated that cell contact with the substrate was required for iron reduction and release. The requirement of FTR1 for virulence in a systemic infection model and its role in transferrin iron uptake raise the possibility that transferrin is a source of iron during systemic C. albicans infections.
INTRODUCTION
Candida albicans is a common fungal saprophyte for humans, growing on mucosal surfaces, including mouth and gut, of up to 60% of normal individuals (23). Under special circumstances, this pleiomorphic fungus becomes invasive and disseminates via the bloodstream. Disseminated Candida infections, occurring most frequently in the setting of neutropenia, cause tremendous morbidity and mortality (2, 57). Iron is an essential nutrient for C. albicans, and iron uptake may play a special role in promoting virulent infections (54). However, the iron sources that support intravascular infection and the fungal iron uptake systems that mediate iron acquisition in the intravascular space have not been defined.
An adult human contains about 3.5 g of iron. The majority of this iron circulates as hemoglobin in erythrocytes, and a smaller amount is bound to transferrin. Transferrin is a monomeric protein, with an approximate molecular mass of 80 kDa. The protein consists of two structurally similar lobes, each of which binds a single ferric iron atom in the presence of carbonate anion. In vertebrates, transferrin functions as the primary carrier of iron in blood. At blood pH (7.4), transferrin binds one or two ferric iron atoms with high affinity (Kd = 10–20 M) (1). Iron bound in the interior of the folded protein is protected from hydration, and this is critical because hydrated ferric iron forms insoluble ferric hydroxides that precipitate as rust. Iron in transferrin is also protected from oxidation-reduction reactions, except under special circumstances, such as those involved in physiological iron delivery. In mammalian cells, the surface structure of transferrin facilitates binding to transferrin receptors, as shown by a cocrystal structure (9). This is followed by rapid internalization into an endocytic compartment (16, 35). Conformational change, acidification, and iron reduction mediate iron release from transferrin, although the endosomal reductase has not yet been identified (9, 17, 22). Finally, the membrane permease DMT1 transports reduced (ferrous) iron from endosomal lumen to cytoplasm (7).
To acquire iron, C. albicans has separate iron uptake systems for different iron-containing substrates. There are independent systems for acquiring iron from siderophores, from heme, and from varied ferric iron chelates. Siderophores are small-molecule chelators that are synthesized and secreted by microorganisms (69). These have different structures, but all can bind and solubilize iron with extremely high affinity, and in some cases (e.g., Aspergillus fumigatus) these have been able to steal iron from transferrin (29). Although C. albicans lacks the enzymatic machinery to synthesize and secrete such molecules, it is equipped with a transporter for recovery of ferrichrome-type siderophores (30, 38). Presumably, in settings where mixed flora or mixed infections exist, the ability to scavenge siderophores secreted by other organisms would provide an advantage. A genetic knockout of SIT1 (ARN1), the siderophore transporter, did not confer loss of virulence in a mouse model of systemic infection, although the knockout strain did show a decreased ability to invade keratinocyte layers in a model system (28).
The majority of iron in a healthy human exists as heme in hemoglobin, and uptake systems for heme iron exist. C. albicans has a hemolytic activity, and plasma membrane proteins capable of binding hemin and hemoglobin have been identified (42, 45, 52, 58, 65, 66). The uptake pathway for hemin iron depends on the intracellular enzyme heme oxygenase (Hmx1p) to release iron from the porphyrin chelate (51, 58). The role of this system during intravascular infection has not been tested.
Finally, a reductive system exists, located in the plasma membrane of C. albicans and consisting of three activities, each encoded by multiple genes. A surface ferric reductase in C. albicans is able to reduce extracellular ferric chelates and has an activity that is approximately 10-fold greater than the activity of Saccharomyces cerevisiae. Two genes FRE10 (RBT2, CFL95) and FRE1 (CFL1) have been identified by complementation of S. cerevisiae reductase-deficient mutants, and a total of 12 genes can be identified by sequence homology searching of the C. albicans genome (25, 32, 36). Reduced ferrous iron generated by the surface reductase is, in turn, captured by a protein complex consisting of a multicopper oxidase and a ferrous permease. C. albicans has five genes with similarity to multicopper oxidase genes, and a requirement of the multicopper oxidase activity for ferrous uptake has been shown, although which gene is responsible is unknown (18, 32, 36). The ferrous permease activity is encoded by two highly homologous genes, with opposite regulations: FTR1 is induced by iron deprivation, and FTR2 is induced by iron loading (36, 54). Only FTR1 has been implicated in cellular iron uptake in that the knockout lacks high-affinity ferrous transport, whereas the FTR2 knockout has no phenotype. Importantly, deletion of FTR1 established a clear connection of iron with C. albicans virulence, as strains with FTR1 deleted were unable to establish a systemic infection in mice following intravenous tail vein injection (54).
The multiplicity of iron uptake systems in C. albicans and the multiplicity of genes involved for each have made it challenging to correlate genes and functions. In addition, little is known about which genes/functions operate in different clinically relevant niches. Here we focus on the mechanism of iron acquisition from transferrin, an intravascular iron binding protein present at high concentrations in the blood. Transferrin iron has generally been considered to be unavailable as an iron source for most microorganisms due to its high affinity for iron. We find here that C. albicans can acquire iron from transferrin by using a reductive strategy.
MATERIALS AND METHODS
Strains. C. albicans SC5314 was used as the wild-type reference strain. Strain BWP17 (ura3::imm434/ura3::imm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG), a gift from A. Mitchell (68), was used as the starting strain for FTR1 and SIT1 homozygous disruption mutants. BCa15-2 harbors a homozygous deletion of RBT2 (CFL95, FRE10) (rbt2::hisG/rbt2::hisG-URA3-hisG) and was a gift from B. Braun and A. Johnson (5). (RBT2/CFL95 is referred to as FRE10 in this paper; this is the name suggested by the annotators of the Candida genome at the Canadian Bioinformatics Resource of the National Research Council and, as we demonstrate in this report, describes the function [ferric reductase] of the gene product.) The homozygous knockout hmx1::ARG4/hmx1::URA3 strain and reconstructed hmx1::ARG4/hmx1::URA3 HMX1-HIS1::his1::hisG/his1::hisG strain have been described previously (58). A homozygous hmx1 knockout strain auxotrophically matched to the hmx1 reconstructed strain was created by target integration of NruI-cut pGEM-HIS1 (68) (hmx1::ARG4/hmx1::URA3 HIS1::his1::hisG/his1::hisG).
To create strains with FTR1 and SIT1 disrupted, we used the technique of gene disruption with short homology regions described by Wilson et al. (68) with plasmids generously provided by Aaron Mitchell. The DNA construct to create the homozygous FTR1 deletion (strain CT87.4) was generated with primers 5'-CGTTCAAATTTTCTTCATCGTTTTCAGAGAATCTTTGGAAGCTATCATTGTTGTTTCAGTGCTTTTGGCGTGGAATTGTGAGCGGATA-3' and 5'-GTCTCTTGCCTTATTCTTTTAGTTGTTGAATAATAATTAACTAAGTTTATTTGTTTTCTTTGGATTCGTTTCCCAGTCACGACGTT-3'. These primers were used to amplify the ARG4 and URA3 cassettes from pGEM-URA3 and pRS-ARG4SpeI and contain flanking homology to FTR1 of 69 and 68 nucleotides. Homologous recombination using the PCR product resulted in 1,177 bp, removing the majority of the open reading frame (ORF) from base 85 (where base 1 is A of the initiator ATG) and 47 bp of 3' untranslated region. Successful integration was verified by PCR with primers 5'-ATTACTTTGACAGAAACACC-3' and 5'-AATGAAACCAATATTTTCCC-3' directed outside the region of recombination. To reintroduce FTR1 into CT87.4, primers 5'-CGTGGCATAAATAACTGAATC-3' and 5'-GTGTGGCGACTTTGTAAAC-3' were used to amplify the FTR1 ORF with 997 bp 5' and 337 bp 3' of flanking sequence from SC5314 genomic DNA. The PCR product was ligated into pCR2.1-TOPO to create p020403. Two independently generated PCR products were sequenced. Both contained a single nucleotide difference, at different locations, from the sequence of ORF 6.8119 of the C. albicans sequencing project (sequence data for C. albicans were obtained from the Stanford Genome Technology Center website at http://www-sequence.stanford.edu/group/candida). To eliminate these differences, both plasmids were cut with ScaI, which cuts p020403 twice, and the two fragments which did not contain errors were ligated together to create p022103, which was sequence verified. p022103 was cut with SacI and NsiI, and the 2.6-kb DNA fragment harboring FTR1 was ligated into the corresponding sites of pGEM-HIS1 to generate p031003. This plasmid and pGEM-HIS1 were linearized with NruI and transformed into the homozygous FTR1 knockout mutant, CT87.4, to create the reconstructed strain (CT123.2) and an auxotrophically matched homozygous knockout strain (CT128.1). Correct insertion at a his1::hisG locus was verified by PCR using oligonucleotides 5'-ATTACTTTGACAGAAACACC-3' and 5'-AATGAAACCAATATTTTCCC-3'.
The SIT1 homozygous knockout strain (CT27.1) was constructed with similar methodology using primers 5'-CCAGTCTTCCAATAATCATTCTTCAGAAGAAGATAAACACTTGTCCGGAGATGAAAAGACGTTTTCGTGGAATTGTGAGCGGATA-3' and 5'-GCTACTCTTTTCTTCTTGAAATTGCCGAAGAAATTGGCCAACGAGTCCTTCTCTTCTTGCTTTTCTTTTCCCAGTCACGACGTT-3'. Homologous recombination with the PCR product resulted in a 1,668-bp deletion of the majority of the ORF from base 78. To create a reconstructed strain, primers 5'-CGCCTGAACAGGGTTTTATCG-3' and 5'-GTATGTGTGAATAGTGAGCGG-3' were used to amplify the SIT1 ORF with 1,082 bp of 5' and 315 bp of 3' flanking regions from wild-type genomic DNA. As for FTR1, subcloning of independently created PCR products was required to obtain a SIT1 product with sequence identity to that of assembly 6 in the Candida database. The sequence-verified SIT1 was subcloned into pGEM-HIS1 at ApaI/SacII to create plasmid p101002. The homozygous SIT1 knockout strain (CT27.1, sit1::ARG4/sit1::URA3 his1::hisG/his1::hisG) was transformed with NruI-linearized pGEM-HIS1 and p101002 to create the auxotrophically matched knockout strain (CT94.1, sit1::ARG4/sit1::URA3 HIS1::his1::hisG/his1::hisG) and reconstructed strain (CT91.1, sit1::ARG4/sit1::URA3 SIT1-HIS1::his1::hisG/his1::hisG).
To reinsert FRE10 into BCa15-2, plasmid p1A2, selected from a C. albicans genomic DNA library (generously provided by G. Fink) by its ability to complement an S. cerevisiae fre1 fre2 mutant (36), was used as the DNA source. A 4.5-kb SacII-ApaI fragment from p1A2 was subcloned into the corresponding sites of pGEM-URA3 to create plasmid p062804. A Ura– derivative (CT187.2) of BCa15-2 was created by selection on 5-fluoroorotic acid-containing medium as described previously (20). Plasmid p062804 was linearized at the PshA1 site in the 5' noncoding region of FRE10 and transformed into strain CT187.2. Ura+ transformants were selected, screened for ferric reductase activity, and verified by Southern blotting (see Results). An auxotrophically matched control strain for the FRE10 reconstructed strain was also created. Plasmid p062804 was digested with HindIII and religated to create plasmid p082404. This process removed the majority of the FRE10 ORF and all the 3' noncoding region. Only the noncoding 5' region and 190 bp of the FRE10 ORF remained. Plasmid p082404 was digested with PshA1, and the linear DNA was used to transform CT187.2. Ura+ transformants were selected and correct integrants identified by Southern blotting (see Results).
Saccharomyces cerevisiae strains used were YPH499 (MATa ura3-52 lys2-801[amber] ade2-101[ochre] trp1-63 his3-200 leu2-1) (62) and the congenic ferric reductase knockout strain 49912 (MATa ura3-52 lys2-801[amber] ade2-101[ochre] trp1-63 fre1::LEU2 fre2::HIS3) (19).
Growth conditions and growth media. For all experiments, strains were streaked from –80°C glycerol stocks onto yeast extract-peptone-dextrose agar medium, grown for 2 days at 30°C, and then transferred to room temperature, where they were maintained for a maximum of 10 days before being discarded. All experiments were initiated from inoculation of a single colony to a single culture. For biological replicates, multiple single colonies were individually inoculated into multiple cultures. Defined iron-deficient medium was prepared from complete synthetic medium (CSM) (0.79 g/liter) (Q-BioGene); yeast nitrogen base without amino acids, Cu, and Fe (6.7 g/liter) (Q-BioGene); 2% D-glucose; and 1 μM copper sulfate. The pH of this unbuffered medium was 4.3 to 4.4. In some experiments, 50 mM MES (morpholinethanesulfonic acid) was added to this medium and the pH maintained at 5.8 to 5.9. In other experiments 50 mM HEPES was added to the medium and the pH maintained at 6.3 to 6.4. For selection of transformants, CSM was replaced with the appropriate amino acid/base dropout mixture, and Difco yeast nitrogen base was used (Becton Dickinson & Co.). For growth of Ura– strains CSM was supplemented with 100 mg uridine/liter. Milli-Q (Millipore) water was used for all growth media and chemical solutions. All iron-deficient media were prepared in plastic containers and filter sterilized, and cells were grown in polypropylene and polystyrene vessels.
Growth assays. C. albicans was starved for iron by growth at 30°C in yeast extract-peptone-dextrose medium containing uridine and 0.5 mM bathophenanthroline disulfonate (BPS) to chelate ferrous iron. After 20 h the cells were subinocluated into medium with the same composition and grown at 30°C for another 20 h. Cells were harvested by centrifugation, washed in Milli-Q water, and resuspended in Milli-Q water to an optical density at 600 nm (OD600) of 5.0 (Cary Olis spectrophotometer). These cells were the starting point for the cell growth experiments, which were performed in 96-well plates with 200 μl of medium per well. The cells suspended in water were inoculated into HEPES-buffered, low-iron CSM medium with an appropriate iron source to give an OD600 of 0.1 (Cary Olis spectrophotometer). This OD corresponds to an OD720 of 0.01 to 0.02 when measured in the 96-well spectrophotometer (Bio-Tek EL800) and represents the lower limit of detection for this instrument. Cells were grown at 30°C without shaking, and growth was determined by resuspending the cells and measuring cell density with the 96-well spectrophotometer. Greater than 99.9% of cells were in the yeast form throughout the growth period.
Preparation of 55Fe-transferrin and 55Fe-ferrichrome. Human holotransferrin and apotransferrin were purchased from Calbiochem. 55Fe-labeled transferrin was prepared by the addition of a threefold molar excess of 55FeCl3 to 100 μM apotransferrin in transferrin loading buffer (0.1 M HEPES, pH 7.5, 0.15 M sodium chloride, and 10 mM sodium bicarbonate). After incubation for 30 min at 22°C and 30 min on ice, the labeled transferrin was separated from unbound 55Fe by using a Sephadex G-25 spin column equilibrated with transferrin loading buffer. Iron-loaded transferrin was greater than 99% saturated and was efficiently separated from iron by the column.
A 10% molar excess of iron-free ferrichrome from Ustilago sphaerogena (Sigma) was incubated with 55FeCl3 in 1 M Tris, pH 7.4, for 5 min at 22°C to a give final concentration of 100 μM 55Fe-ferrichrome.
Ferric reductase and high-affinity iron uptake assays. The ferric reductase assays rely on the formation of a red BPS-Fe(II) complex, which is quantified by measuring absorbance at 515 nm (13). Sources of ferric iron were ammonium ferric sulfate and holotransferrin. For reductase assays, cells were washed three times with 50 mM citrate buffer (pH 6.6)-5% D-glucose and incubated in 50 mM citrate buffer (pH 6.6)-5% D-glucose containing BPS and a ferric substrate for up to 10 min at 30°C. Unless indicated, BPS and ferric substrate concentrations were 1 mM. Assay mixtures containing no cells were used to determine blank values, which were subtracted from values obtained in assays with cells. Reductase activity was normalized with cell number determined from optical measurements of culture turbidity. Greater than 99.9% of cells were in the yeast form.
High-affinity iron uptake was measured as described previously (15). Cells were washed three times in citrate-glucose buffer and resuspended in the same buffer. Iron sources of 1 μM ferrous (55Fe)-ascorbate, 55Fe-holotransferrin, and 55Fe-ferrichrome were added to the suspended cells and incubated at 30°C. After 1 h, cells were resuspended and optical densities of the cultures were measured. The cells were harvested and washed free of 55Fe label by using either a 24-well cell harvester (PHD Cambridge Technologies) or a 96-well harvester (Tomtec). Cell-associated 55Fe was measured by liquid scintillation counting (Beckman LS6500 or Perkin-Elmer 1450 MicroBeta). Assay mixtures containing no cells were used to calculated blank values for correction.
Requirement for cell contact. Wild-type C. albicans was grown in two sequential overnight cultures of HEPES-buffered, low-iron defined medium, subinoculated into medium of the same composition, and grown for an additional 2 h at 30°C. The cells were washed three times in Milli-Q water and resuspended in citrate-glucose buffer at an OD600 of 1. Dialysis tubing (Spectra/Por 2) was prepared by soaking in 0.1 M EDTA for 20 min followed by copious rinsing with Milli-Q water. Holotransferrin (0.1 ml, 2 mM) was placed in the dialysis bags, which were then incubated with 5 ml of cells in the presence of 0.1 mM BPS for 30 min at 30°C.
RESULTS
Holotransferrin promotes growth of iron-starved C. albicans. To determine if C. albicans can use human transferrin as an iron source, wild-type C. albicans strain SC5314 was starved for iron and inoculated into low-iron defined medium buffered with HEPES, pH 6.4. Iron was added back, in parallel cultures, as holotransferrin (0.2 μM), ferric ammonium sulfate (0.4 μM), or ferrichrome (0.4 μM), and growth was monitored by frequent turbidity measurements. Throughout the growth period the cells grew with yeast morphology. The concentrations of iron sources were chosen to ensure that iron uptake from these sources would be via a high-affinity iron uptake system. The concentration of holotransferrin was half that of the other iron sources, because each molecule of transferrin binds two atoms of ferric iron. Growth of cultures supplemented with iron salts, ferrichrome, or diferric transferrin was enhanced compared to that of cultures with apotransferrin or no addition (Table 1; Fig. 1A). Analysis of the growth curves showed that logarithmic growth of the cells supplemented with iron occurred at between 4 and 9 h postinoculation. In the absence of iron supplementation C. albicans cells exhibited a prolonged lag time, and logarithmic growth occurred between 8 to 15 h. Doubling times for the cultures were calculated during the time of logarithmic growth (Table 1). Wild-type cells grown in the presence of the three iron sources (holotransferrin, ferric ammonium sulfate, or ferrichrome) showed similar doubling times, which were at least 3.5-fold faster than those of cells grown in the absence of iron or in the presence of apotransferrin. During the first 24 h of incubation, the pH of the cultures decreased to pH 6.3. During the same time period, 4 nM iron dissociated from 55Fe-transferrin in control cultures without cells, as determined by liquid scintillation counting of the flowthrough following separation of the medium through a Centricon filtration device.
The FTR1 iron permease is required for optimal growth with a transferrin iron source. C. albicans has at least three iron uptake systems, raising the question of which system mediates iron acquisition from transferrin. The ftr1–/– mutant, lacking the ferrous iron permease, is impaired in the reductive iron uptake system. We evaluated the ftr1–/– mutant for iron-dependent growth with diferric transferrin as the iron source, and a pronounced effect was observed (Table 1; Fig. 1B). The lag time of the ftr1–/– mutant was 5 h, compared to 4 h for the wild-type and reconstructed ftr1–/– + FTR1 strains. The doubling time of the ftr1–/– mutant was, however, greatly increased to 10.5 h, compared to 1.8 h and 1.6 h for the wild-type and reconstructed strains, respectively. At later time points (after 13 h), the growth rate of the ftr1–/– mutant increased substantially, and there was increased variability between the replica cultures. A similar acceleration of growth rate was also observed for the ftr1–/– mutant grown with ferric ammonium sulfate 6 h following inoculation. The cause of this growth rate change is unknown; it might be due to genetic change, but this has not been ascertained. Without iron addition to the medium, the ftr1–/– mutant was growth arrested (Table 1). In contrast, the wild-type and ftr1–/– + FTR1 strains were able to grow under these iron-starved conditions, although their growth was impaired. It is likely that small amounts of contaminating iron salts are still present in the low-iron medium and that cells may have access to this iron via Ftr1p.
Ferrichrome iron uptake bypasses growth inhibition of the ftr1–/– mutant exposed to apotransferrin. C. albicans can acquire iron from ferrichrome through the permease Sit1p; thus, iron-related effects of apotransferrin could be bypassed by supplying iron to the ftr1–/– strain as ferrichrome. In the presence of apotransferrin alone, the ftr1–/– strain grew extremely slowly. Wild-type and ftr1–/– + FTR1 cultures grew with doubling times that were two to three times longer than those of holotransferrin supplemented cultures (Table 1; Fig. 2). When the iron-starved cells were incubated with apotransferrin plus ferrichrome, the doubling times of the ftr1–/– and ftr1–/– + FTR1 strains were shortened. The two strains had equivalent doubling times, indicating that FTR1 was not required for high-affinity uptake of iron from ferrichrome. Intriguingly, the doubling times were about 20 min longer than those with ferrichrome alone for both strains (Table 1). It is possible that apotransferrin may inhibit growth of C. albicans independent of effects on iron availability. Lactoferrin, a homologous iron binding protein, has been shown to have similar effects unrelated to iron chelation (43). Alternatively, apotransferrin may compete with ferrichrome for ferric iron, and the resulting monoferric transferrin may be a poorer iron source than diferric transferrin or ferrichrome.
High-affinity iron uptake from transferrin requires C. albicans FTR1. The growth advantage that C. albicans strains harboring C. albicans FTR1 have, when provided with a transferrin iron source, is likely due to Ftr1p-mediated uptake of iron from transferrin. Iron uptake from transferrin was measured directly using radiolabeled 55Fe-diferric transferrin. When ftr1–/– cells were grown under iron-depleted conditions (Fig. 3A), the uptake of Fe from transferrin was 17% of the wild-type level. When a single copy of FTR1 was reinserted (Fig. 3A), iron uptake in this auxotrophically matched reconstructed strain was increased threefold compared to that in the homozygous knockout. This corresponds to 50% of the wild-type level. Similar results were seen with ferrous ascorbate as the iron source (Fig. 3B). One possible explanation is that two copies of FTR1 are required for full high-affinity ferrous uptake under inducing (low-iron) conditions. Alternatively, the promoter region, 998 bp 5' of the FTR1 ORF, used in the reconstructed DNA FTR1 allele might be insufficient for maximal expression. This second explanation is supported by the observation that the heterozygous mutant (ftr1::ARG4/FTR1) did not show diminished iron uptake from ferrous ascorbate compared to the wild type (data not shown). The absolute value of iron uptake from 55Fe-transferrin was approximately double the uptake from ferrous ascorbate, perhaps because equivalent molar amounts (1 μM) of iron sources were used and the diferric transferrin molecule carries two atoms of iron per molecule. Following growth in iron-supplemented medium, uptake of iron from holotransferrin or ferrous ascorbate was down-regulated in the wild-type and ftr1–/– + FTR1 strains to levels comparable to those in the ftr1–/– mutant (data not shown). C. albicans mutants with mutations in the siderophore uptake pathway (sit1–/–) and heme iron utilization pathway (hmx1–/–) were not altered in their ability to acquire iron from either holotransferrin or ferrous ascorbate (Fig. 3A and B). We also observed that the ftr1–/– mutant was unaffected in 55Fe-ferrichrome uptake (Fig. 3C), as shown previously (28), indicating independence of the reductive and siderophore uptake systems.
Iron uptake from transferrin is copper dependent. Ferrous iron uptake in C. albicans is copper dependent (36, 46, 67), and optimal growth of C. albicans under low-iron conditions requires a multicopper oxidase, encoded by FET3 (18). Wild-type C. albicans cultures were grown in low-iron defined medium containing 1 or 0.1 mM of the copper chelator bathocuproinedisulfonic acid (BCS). These cells exhibited only 0.6 and 16%, respectively, of the level of iron uptake from transferrin that copper adequate cells displayed (Fig. 4). This requirement for copper strongly suggests the involvement of a multicopper oxidase, a partner protein required with the ferrous permease for high-affinity ferrous iron uptake. Overall these biochemical results, together with the analysis of the ftr1–/– strain, indicate that iron uptake from transferrin depends on the reductive uptake pathway.
FRE10 encodes a cell surface ferric reductase. Iron bound to transferrin is coordinated as a ferric chelate (+3 valence) with synergistically bound carbonate ions (1). The Ftr1p/Fetp iron uptake system mediates transport of ferrous iron (+2 valence), and thus a ferric reductase that is able to convert the iron to the required oxidation state for transport must exist. A gene, RBT2, with homology to the ferric reductases of S. cerevisiae was identified in a screen for Tup1-repressed target genes (5). This gene was also independently cloned by its ability to complement an S. cerevisiae mutant lacking cell surface ferric reductases (CFL95) (36). (From here on we will refer to this gene as FRE10, as suggested by the BRI annotation group [http://candida.bri.nrc.ca/candida/index.cfm] and listed in Table 2.) The homozygous diploid fre10 knockout strain (BCa15-2) was converted to Ura–, and a wild-type copy of FRE10 was reinserted at its native locus. The source of the wild-type FRE10 allele was the clone with complementary activity for the S. cerevisiae ferric reductase mutant (36). To generate an auxotrophically matched control strain, a truncated nonfunctional fre10 allele was inserted at the same locus. The genotypes of these strains were confirmed by Southern blotting after digestion of genomic DNA with EcoRI (Fig. 5A and B), and XbaI plus XhoI (not shown). These strains were then examined for cell surface reductase activity for ferric iron salts (ferric ammonium sulfate). The cells were grown in low-iron defined medium and assayed for ferric reductase activity when they reached logarithmic growth. Wild-type SC5314 was used as a reference strain. The original fre10 homozygous mutant (BCa15-2) exhibited only 2% of the activity of the wild-type strain (Fig. 5C, compare bars 1 and 2). The cell surface ferric reductase activity of the Ura– derivative of BCa15-2, strain CT182.2, was not different from that of BCa15-2 (compare bar 3 with bar 2). When a single copy of FRE10 was reinserted into CT182.2, creating strains CT218.2 and CT222.1, cell surface ferric reductase activity was restored to wild-type levels (bars 4a and 4b). In contrast, the strains (CT212.2 and CT216.2, bars 5a and 5b) in which a truncated copy of fre10 had been reinserted maintained the very low activity of the deleted fre10 strains. In a separate experiment (not shown), the rates of the ferric reductase reactions were measured in a time course assay and were 0.34 nmol Fe(II)/million cells/min for SC5314 and 0.03 nmol Fe(II)/million cells/min for BCa15-2. These reactions were linear over the 10-min assay time. Overall these results provide strong evidence that FRE10 encodes or positively regulates a major cell surface ferric reductase.
Iron uptake from transferrin is impaired in fre10 mutants. C. albicans cells were grown in low-iron defined medium and analyzed for iron uptake from 55Fe-labeled transferrin and 55Fe-ferric chloride. Under these conditions, cellular uptake of radioactive iron from transferrin by strain BCa15-2 was 25% of that of the wild type (Fig. 6). A similar magnitude of difference was observed between the auxotrophically matched reconstructed strains CT218.2, harboring a single copy of FRE10, and CT212.2, harboring a truncated allele of fre10. These experiments were repeated with an equimolar amount of iron in the form of 55Fe-ferric chloride as the iron source. Iron uptake from transferrin or ferric chloride was compromised to a similar degree in the reductase-deficient mutants, indicating an important role of FRE10 in iron uptake from transferrin under these conditions.
Induction of an alternative reductase(s) in the absence of FRE10. The results from the experiments described above indicate that C. albicans has the ability to reduce and to take up iron from human transferrin, using this iron to support growth. However, although FRE10 was needed for maximal rates of iron uptake from transferrin under some conditions (see above), under other conditions FRE10 was dispensable. Using the same growth conditions outlined for Fig. 1, C. albicans wild-type, reconstructed mutant (fre10–/– + FRE10), and fre10 mutant () strains were grown in low-iron defined medium containing 1 μM copper sulfate and buffered with 50 mM HEPES to give a final pH 6.3 to 6.4. Iron was added back to this medium as 0.4 μM ferric ammonium sulfate, 0.4 μM ferrichrome, or 0.2 μM human holotransferrin. Surprisingly, there were no large differences in doubling times among the strains when they were grown with transferrin (Fig. 7A) or with the other iron sources (not shown). The logarithmic-phase doubling time for all strains with holotransferrin as the iron source was 1.5 h. This compares to doubling times of 1.2 to 1.3 h when the strains were grown with ferric iron as the iron source and of 1.3 to 1.6 h when the strains were grown with ferrichrome. The unmanipulated wild type exhibited a shorter lag time than the other strains, perhaps related to URA3 location or copy number, but the effect was not iron dependent (Fig. 7A).
A possible explanation for the lack of effect of the fre10 mutants is that other cell surface reductases might be active under these growth conditions. Eight hours following inoculation into transferrin-supplemented low-iron medium, all strains, with or without the FRE10 gene, exhibited similar cell surface ferric reductase activities (Fig. 7B). This result indicates that in the HEPES-buffered medium (pH 6.3 to 6.4) at least one other surface ferric reductase was active that was repressed in the nonbuffered medium (pH 4.3 to 4.4) utilized for some experiments (Fig. 5 and 6). Table 2 lists open reading frames of C. albicans that have homology to FRE10 and might encode such an alternative cell surface ferric reductase. One of these, FRE1 (CFL1), has been shown to complement an S. cerevisiae mutant lacking cell surface reductase activity (25). We examined the steady-state level of FRE1 mRNA by using Northern blots on RNA prepared from cells grown on low-iron HEPES-buffered medium. FRE1 mRNA was expressed at equal levels in the wild type and in strains with FRE10 disrupted, and there did not appear to be any regulatory compensation of CFL1 expression for lack of FRE10 expression (data not shown).
Iron uptake from 55Fe-transferrin was tested in cells grown in buffered medium. Iron uptake from transferrin (Fig. 7C) and ferric ammonium sulfate (not shown) were uniformly higher than in the same strains grown in nonbuffered medium (compare Fig. 7B to Fig. 6). For the wild type this was a fivefold difference, and for the two strains lacking FRE10, the differences were 17-fold and 15-fold compared with cells grown in nonbuffered medium. Similar magnitudes of difference were observed when ferric chloride was used as the iron source. Overall these results suggest that the reductive iron acquisition system is modulated by pH as well as by iron. The small contribution of FRE10 to reductase activity and iron uptake from ferric iron sources when cells are grown in medium buffered at pH 6.3 to 6.4 with 50 mM HEPES or MOPS (morpholinepropanesulfonic acid) (data not shown), compared to the large contribution it makes when cells are grown at pH 4.4, agrees with the recent observation that FRE10 expression is down-regulated at alkaline pH (3).
Contact of transferrin with C. albicans promotes iron release. A cell surface reductase involved in iron acquisition might physically interact with its substrate on the cell surface, or, alternatively, it might act on soluble or secreted reducing intermediates. To distinguish these possibilities, the following experiment was performed. Transferrin was placed in a dialysis bag to physically separate it from a suspension of iron-starved wild-type cells that had been grown in HEPES-buffered defined medium. The cells were washed and resuspended in citrate buffer, pH 6.6, containing BPS to measure reduced/released iron. When cells where physically separated from transferrin, very little pink coloration was observed, indicating lack of formation of ferrous-BPS (Fig. 8). Furthermore, the holotransferrin within the dialysis bag retained its brown color, indicating a failure to release the bound iron. In contrast, when the dialysis bag was punctured, allowing transferrin to come directly in contact with the cells, the bright pink color of a ferrous-BPS complex quickly formed. In the absence of cells, there was no indication of formation of an iron-BPS complex when the dialysis bag was punctured.
Reductive iron uptake from transferrin is highly elevated in C. albicans compared to S. cerevisiae. The nonpathogenic yeast S. cerevisiae has a reductive iron uptake system with components homologous to that of C. albicans. Wild-type S. cerevisiae, a S. cerevisiae ferric reductase mutant, and wild-type C. albicans were grown in low-iron defined medium and in media of the same composition buffered to pH 6.3 with 50 mM HEPES or 50 mM MOPS. While in log phase the cells were analyzed for iron uptake from 55Fe-labeled transferrin. Under all three conditions wild-type S. cerevisiae was able to acquire some iron from transferrin, whereas the ferric reductase mutant had undetectable and diminished iron uptake, indicating that iron uptake from transferrin occurred through the reductive pathway (Fig. 9). The most striking finding, however, was that the rate of iron acquisition from transferrin was considerably greater in C. albicans than in S. cerevisiae.
DISCUSSION
C. albicans, which is generally a saprophyte on mucosal surfaces, in some settings disseminates within the circulation; in order to survive, it must find ways to access critical nutrients (such as iron) in the hostile host environment. Within the host circulation, iron is present as heme in hemoglobin or as transferrin. Here we show that C. albicans can access transferrin iron via the high-affinity reductive system. Interestingly, C. albicans components involved in transferrin iron acquisition mirror those of the host pathway, including components for iron reduction and ferrous iron transport.
Use of the reductive pathway. C. albicans was starved for iron until growth was retarded. Holotransferrin added to these iron-depleted cells was able to restore growth, indicating that it was being used as a source of iron. Growth promotion was quite good as assessed by effects on lag time and doubling time, although it was still inferior to that with ferrichrome or ferric salts. Thus, transferrin iron can be efficiently used as an iron source. Radionuclide iron uptake from transferrin was also measured and was efficient compared with that from iron salts. Uptake- and growth-promoting effects were dependent on components of the reductive iron uptake pathway, including ferric reductase, multicopper oxidase, and ferrous permease.
For iron uptake into mammalian cells, diferric or monoferric transferrin binds to cell surface transferrin receptors. The transferrin-transferrin receptor complex is endocytosed, and the pH of the endosome is lowered, allowing protonation of the carbonate and amino acid ligands for iron. Interaction of transferrin with the receptor induces a change in transferrin conformation, increasing the redox potential of the bound iron to a value compatible to reduction by an NADPH-dependent reductase (17). The reduction of ferric to ferrous iron would favor release, because the transferrin affinity for ferrous iron is 1014-fold lower than that for ferric iron (26). Finally, the DMT ferrous transporter moves the released ferrous iron into the cytoplasm. The C. albicans system for iron acquisition from transferrin is similar. Specific transferrin binding to the C. albicans cell surface can occur (S. A. B. Knight et al., unpublished observations), and a surface reductase facilitates iron reduction and release, although other factors such as transferrin conformational changes, anions, and protonation may also contribute to iron release. A requirement for C. albicans reductase activity was observed, in that fre10–/– strains lacking surface reductase activity were also deficient in iron uptake from transferrin. Following or coincident with ferrous iron release, a high-affinity transporter-multicopper oxidase transports the ferrous iron into the C. albicans cell. Dependence of this process on multicopper oxidase is supported by data showing that C. albicans starved of copper was unable to take up iron from transferrin. The specific multicopper oxidase gene(s) responsible has not been identified among multiple candidates. Finally, the growth-promoting effect and radionuclide uptake were entirely dependent on FTR1, being abrogated in the ftr1–/– mutant, which lacks the ferrous permease (analogous to DMT1 of the host). In summary, independent data showing requirements for reductase, oxidase, and permease components support a role for the reductive pathway in iron uptake from transferrin. The other iron uptake pathways, for heme and siderophores, were not involved in iron uptake from transferrin.
Which reductase S. cerevisiae Fre1p has been characterized as a prototype ferric reductase. It is an integral plasma membrane protein and requires cofactors of heme and flavin (19). The reductase has broad specificity and utilizes electrons donated from intracellular NADPH to reduce metals (Fe3+ and Cu2+) or dye substrates (14, 27, 61). C. albicans too has a cell surface reductase activity, and the C. albicans FRE10 open reading frame has been shown to complement S. cerevisiae mutants lacking ferric reductase (36). Complementing activity corrected slow growth of the reductase mutant in low-iron media, and surface reductase activity was also restored. In the C. albicans genome there are at least 12 ORFs with sequence similarity to C. albicans Fre10p. These are recognizable by the presence of signature motifs, including four conserved histidine residues, predicted to coordinate two hemes within the membrane, and a FAD isoalloxazine binding motif (HPFT). The 12 ORFs with highest homology to Fre10p are listed in Table 2.
In studies with the fre10–/– mutant, we found that ferric reductase activity was almost completely lacking (<5% residual activity) compared with a wild-type control when the strains were grown in low-iron defined medium. The implication is that FRE10 encodes the major ferric reductase under these conditions. Of note is that the pH of the growth medium declined to 3.2 to 3.3 during the course of the experiment with unbuffered medium. When the experiment was repeated with growth medium buffered by the addition of HEPES KOH (or MOPS [data not shown]) at pH 6.3, the fre10–/– mutant and wild type were now indistinguishable in terms of surface reductase activity. Furthermore, the activities were markedly increased (about twofold) compared to those of cells grown in unbuffered medium. The implication is that the alkaline pH buffering resulted in induction of another reductase gene(s).
The identification of the FRE genes responsible for this alternative reductase activity may be facilitated by recently published microarray studies. In those studies, FRE1, FRE2, FRE4, and FRE5 transcripts were up-regulated by iron limitation (37). FRE7 was induced by macrophage phagocytosis (41). FRE1, FRE2, FRE7, and FRE9 were up-regulated at pH 8 (3). Thus, in the iron-starved HEPES-buffered medium, these genes would be candidates for providing the backup activity. FRE1 expression in wild-type and fre10–/– strains grown in HEPES-buffered defined medium was measured by Northern blotting, and compensatory induction was not observed (not shown). However, FRE1 has been shown to have complementing activity for S. cerevisiae reductase-deficient mutants (25) and has not been ruled out as the gene responsible for the major reductase under less acidic conditions. In summary, FRE10 encodes the major cell surface ferric reductase in unbuffered medium, but it is not required for virulence in a systemic infection model (5) and is not a major contributor to the activity under conditions of buffered pH above 6.3.
Role of transferrin iron uptake in virulence. Host-pathogen interactions that determine virulence are influenced by critical nutrients such as iron (55, 59). During intravascular infections, heme and transferrin represent the likely major iron sources for pathogens. To protect these sources, the host mobilizes an iron-withholding defense. Microbial lipopolysaccharides trigger increased expression of the peptide mediator hepcidin, a component of the type II acute-phase response (47, 53). This in turn leads to inhibition of duodenal iron absorption and an increase of iron retention in macrophages (48). This can culminate in anemia of inflammation, lowering transferrin iron saturation and limiting the availability of iron to an invading pathogen (21, 33). In contrast, there are also clinical conditions in which serum iron and transferrin saturation increase, conditions that favor some pathogens. Patients with leukemia and individuals who have undergone bone marrow transplants have increased susceptibility to fungal infections, and sera from these patients have been shown to promote rather than inhibit Candida growth (8, 31, 44).
From the point of view of the pathogen, the problem is to circumvent this iron-withholding defense. For a number of pathogens (Neisseria gonorrhoeae [11, 12], Neisseria meningitidis [56], and Haemophilus influenzae [24]), iron uptake from transferrin requires specific outer membrane binding proteins, and without these proteins, these bacteria have substantially reduced virulence. For Vibrio vulnificus and Aeromonas hydrophila, secreted siderophores are able to capture transferrin iron and return it to the pathogen (39, 63). A similar mechanism operates in the fungal pathogen Aspergillus fumigatus (29), and siderophore biosynthesis is essential for the virulence of this fungus (60). Histoplasma capsulatum expresses a multicomponent reductive system that may capture iron from transferrin in the intravascular space (64). For fungi that do not synthesize siderophores, a highly active reductive system maybe critical for iron acquisition from transferrin and might distinguish pathogenic yeasts from nonpathogenic yeasts that have less active systems.
C. albicans lives primarily as a saprophyte on mucosal surfaces, but in the setting of neutropenia, mucosal integrity may be compromised and the organism may gain access to the systemic circulation. Within the blood, C. albicans partially transforms to hyphal growth and binds to endothelial cells, invading and proliferating into microabcesses. Target tissues frequently include liver, spleen, and heart valves. Transferrin iron could be critical during the transit time in the circulation or during the invasive period of growth. Transferrin iron could also sustain C. albicans viability during passage through an intravascular compartment such as the phagosome. Phagocytosed C. albicans yeast cells are able to evade the macrophage by physically puncturing the wall of the phagosome and the phagocytic cell (40). Transferrin receptor, and by extension transferrin, have been found with C. albicans in phagosomes of mouse macrophages (34), and iron availability within this compartment has been shown to be important for virulent infections (49). Other pathogens (Mycobacterium tuberculosis [10, 50], Leishmaina amazonensis [4], H. capsulatum [70], and Paracoccidioides brasiliensis [6]) have been shown to pirate iron from transferrin while in phagosome or phagosome-like compartments.
Ftr1p, the ferrous iron permease, is required for propagation of C. albicans infections in a mouse intravenous infection model (54). FTR1 encodes the ferrous iron permease of the reductive iron uptake system, raising the question of where the critical ferrous iron substrate for supporting these intravenous infections originates. A possibility supported by data presented here is that the iron pool needed to support virulent infections in this model system originates from transferrin iron.
ACKNOWLEDGMENTS
This work was supported by grants from the Center for AIDS Research University of Pennsylvania, the American Cancer Society, and the NIH (RO1 AI052384) to A.D. Sequencing of the C. albicans genome at Stanford was accomplished with the support of the NIDR and the Burroughs Wellcome Fund.
We are very grateful to Aaron Mitchell (Columbia University), Gerry Fink (Whitehead Institute), and Alexander Johnson (UCSF) for yeast strains and plasmids. We thank Joel Bennett and the Abramson Cancer Center for use of their gamma counter and 96-well liquid scintillation counter. We also thank the anonymous reviewers whose suggestions improved the manuscript.
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