Haemin uptake and use as an iron source by Candida albicans: role of CaHMX1-encoded haem oxygenase

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Haemin uptake and use as an iron source byCandida albicans: role of CaHMX1-encoded haemoxygenase

Renata Santos,1 Nicole Buisson,1 Simon Knight,2 Andrew Dancis,2

Jean-Michel Camadro1 and Emmanuel Lesuisse1

Correspondence

Emmanuel Lesuisse

[email protected]

1Laboratoire d’Ingenierie des Proteines et Controle Metabolique, Institut Jacques Monod,Tour 43, Universite Paris 6/Paris 7, 2 Place Jussieu, 75251 Paris cedex 05, France

2University of Pennsylvania, Department of Medicine, Division of Hematology/Oncology,BRBII/III Room 731, 431 Curie Blvd, PA 19104, USA

Received 7 November 2002

Revised 6 December 2002

Accepted 20 December 2002

Candida albicans, unlikeSaccharomyces cerevisiae, was able to use extracellular haemin as an iron

source. Haemin uptake kinetics by C. albicans cells showed two phases: a rapid phase of haemin

binding (with a Kd of about 0?2 mM) followed by a slower uptake phase. Both phases were strongly

induced in iron-deficient cells compared to iron-rich cells. Haemin uptake did not depend on the

previously characterized reductive iron uptake system and siderophore uptake system. CaHMX1,

encoding a putative haem oxygenase, was shown to be required for iron assimilation from haemin. A

double DCahmx1mutant was constructed. This mutant could not grow with haemin as the sole iron

source, although haemin uptake was not affected. The three different iron uptake systems

(reductive, siderophore and haemin) were regulated independently and in a complex manner.

CaHMX1 expression was induced by iron deprivation, by haemin and by a shift of temperature from

30 to 37˚C. CaHMX1 expression was strongly deregulated in a Defg1 mutant but not in a Dtup1

mutant. C. albicans colonies forming on agar plates with haemin as the sole iron source showed a

very unusual morphology. Colonies were made up of tubular structures that were organized into a

complex network. The effect of haemin on filamentation was increased in the double DCahmx1

mutant. This study provides the first experimental evidence that haem oxygenase is required for iron

assimilation from haem by a pathogenic fungus.

INTRODUCTION

Iron is required by most living systems. Iron availabilityplays a critical role in the host–pathogen relationship andin the virulence of bacteria and fungi [reviewed by Howard(1999) and Payne (1993)]. In the host, iron is predominantlyintracellular, occurring as haem, iron–sulphur proteins orferritin, with smaller quantities in other iron proteins. Thelimited amounts of extracellular iron are tightly held byproteins such as transferrin in the serum and lactoferrin onbody surfaces. The withholding of iron by the host has beenshown to block virulent infections by many pathogens, andhigh-affinity iron acquisition by pathogens often promotesvirulent infections (Ratledge & Dover, 2000). Candidaalbicans is a saprophytic organism for humans; it can befound inhabiting external surfaces of the body, includingthe skin and the mucosae of the gut and mouth in morethan half of the normal population. In some settingsC. albicans invades tissue and disseminates systemically.Prolonged neutropenia, following cancer chemotherapy or

bone marrow transplantation, is a major risk factor forinvasive fungal infections, and Candida species are themost frequently implicated organisms (Rex et al., 1995).Recurrent oropharyngeal and oesophageal candidiasis isencountered in most AIDS patients (Laine & Bonacini,1994). There has been a rapid rise in the frequency of drugresistance in clinical C. albicans infections (Rex et al., 1995);hence, there is a pressing need to identify new drug targets.

The first studies about iron uptake by C. albicansemphasized the possible role of siderophore productionby this organism to fulfil its iron requirement (Holzberg &Artis, 1983). Some authors described the excretion ofhydroxamates, phenolates or both kinds of siderophoresby C. albicans (Ismail et al., 1985; Sweet & Douglas, 1991).However, these studies were never reproduced, and sid-erophores secreted by C. albicans were never isolated andidentified. Although siderophore production by Saccharo-myces cerevisiae has never been shown and has not beenconfirmed for C. albicans, both S. cerevisiae and C. albicanshave the ability to take up some siderophores non-reductively, via specific siderophore transporters (ArdonAbbreviation: BPS, bathophenanthroline disulfonic acid.

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Microbiology (2003), 149, 579–588 DOI 10.1099/mic.0.26108-0


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et al., 2001; Heymann et al., 1999, 2000; Lesuisse et al., 1998,2002; Yun et al., 2000). The first eukaryotic siderophoretransporter, Sit1, was identified in S. cerevisiae (Lesuisseet al., 1998). A gene homologous to SIT1 (CaSIT1/CaARN1)was then identified and characterized in C. albicans and wasshown to encode a transporter for ferrichrome-type sid-erophores (Ardon et al., 2001; Heymann et al., 2002; Hu et al.,2002; Lesuisse et al., 2002). Siderophore-mediated ironuptake is not the only iron transport mechanism in fungi.Reductive iron uptake (i.e. iron removal from its ligandsby reduction outside the cell prior to transport) was shownto occur in Ustilago maydis (Emery, 1987) and S. cerevisiae(Lesuisse et al., 1987). Reductive iron uptake by S. cerevisiaewas extensively studied at the molecular level [reviewedby Eide (2000) and Van Ho et al. (2002)]. Two plasmamembrane reductases (Fre1 and Fre2) are involved inreleasing iron from its ligands by reduction; the ‘free’ iron isthen transported into the cell via a permease–oxidasecomplex (Fet3–Ftr1). Morrissey et al. (1996) showed that avery similar mechanism of reductive iron uptake was pre-sent in C. albicans. At the molecular level, reductive ironuptake by C. albicans involves proteins that are homo-logous to the components of the S. cerevisiae reductiveuptake system. Cfl95/CaFre1 is a plasma membrane ferrire-ductase, while CaFtr1 and CaFet3 are the components ofthe permease–oxidase complex (Hammacott et al., 2000;Knight et al., 2002; Ramanan & Wang, 2000). A knockoutstrain lacking CaFTR1 was shown to be avirulent in amouse model for systemic infection (Ramanan & Wang,2000), confirming the importance and non-redundancy ofreductive iron uptake.

Interestingly, the reductive and siderophore iron uptakesystems of C. albicans are regulated differently. Both systemsare induced when the cells are grown under iron-deficientconditions, but transfer of the cells from a synthetic medium(YNB) to a serum-based medium results in repressionof the reductive uptake system and in induction of thesiderophore uptake system (Lesuisse et al., 2002). Thisobservation was the first evidence that C. albicans can adaptits strategy for iron uptake to the physiological context. Inthe case of systemic infection, a possible iron source forC. albicans is haem, since this organism is known to secretehaemolytic factors (Manns et al., 1994; Moors et al., 1992).Indeed, recent preliminary studies have shown that haemincan be used by C. albicans as an iron source (Weissman et al.,2002). Up until now, haem uptake and use was studiedmainly in bacteria (reviewed by Genco & Dixon, 2001).Here, we investigate this third strategy of iron acquisitionby the pathogenic fungus C. albicans, and show that it isindependent from the reductive and siderophore mechan-isms of iron uptake.

METHODS

Strains, media and iron compounds. We used the followingstrains of C. albicans: SC5314 (wild-type); BWP17 (ura3D : : imm434/ura3D : : imm434 his1 : : hisG/his1 : : hisG arg4 : : hisG/arg4 : : hisG)

(Wilson et al., 1999); BCa02-10 (tup1D : : hisG/tup1D : : hisG : : p405-URA3, ura3D : : imm434/ura3D : : imm434) (Braun & Johnson, 1997);SS4 (efg1 : :ADE2/PCK1p : : efg1 ura3D : : imm434/ura3D : : imm434)(Stoldt et al., 1997). For control experiments, we used the followingwild-type strains of S. cerevisiae: S150-2B (MATa his3-D1 leu2-3,112trp1-289 ura3-52); YPH499 [MATa ura3-52 lys2-801 ade2-101(ochre)trp1-D63 his3-D200 leu2-D1].

Unless otherwise stated, cells were grown at 30 C in YPD medium(1 % yeast extract, 1 % peptone, 2 % glucose). For iron-deficient andiron-rich cultures, cells from an overnight pre-culture in YPD werediluted 10-fold in fresh YPD containing either 200 mM bathophenan-throline disulfonic acid (BPS) (iron-deficient culture) or 10 mM ferriccitrate (iron-rich culture) and grown for 5 h at 30˚C. Cells were thenharvested and washed with water before being resuspended in 50 mMcitrate (tri-Na+) buffer (pH 6?5) containing 5 % glucose. Cells werethen used for experiments. For experiments requiring RNA isolation,cells were grown in minimal YNB/glucose (without copper and iron)medium (Bio 101) plus the required amino acids. After overnight pre-culture, the cells were diluted 10-fold in the same fresh medium addedwith various supplements [10 mM ferric citrate, 10 mM ferrichrome,5 mM iron-saturated transferrin, 50 mM haemin, 50 % (final) fetalbovine serum or 200 mM BPS]. The cells were grown for 5 h at 30 C,before RNA isolation.

Radiolabelled iron compounds were prepared from 55FeCl3(50 mCi mg21; 1?85 GBq mg21). 55Fe-haemin was synthesized chemi-cally from protoporphyrin IX and 55FeCl3 in pyridine/acetic acid(1 : 50) under a nitrogen atmosphere, as described by Galbraith et al.(1985). 55Fe-haemin was taken into ether, washed extensively withwater and 2?7 M HCl to remove any remaining Fe and protoporphyrin.Ferric citrate was obtained by mixing FeCl3 in sodium citrate buffer(pH 6?5) to get a final Fe/citrate ratio of 1 : 20. Ferrichrome andtransferrin were purchased from Sigma.

Haemin uptake assays. Cells were suspended in 50 mM citrate(tri-Na+) buffer (pH 6?5) containing 5 % glucose and 0?05 %Tween 80, and pre-incubated for 15 min at 30 C under agitation.55Fe-haemin was added at a final concentration of 1 mM to the cellsuspension. Aliquots (100 ml) were withdrawn as a function of timeand added to 20 ml of 1 mM cold haemin kept on ice in the wells ofa microtitre plate. The cells were collected with a cell harvester(Brandel) and washed on the filter.

Strain construction. Molecular cloning techniques and gel electro-phoresis were performed as described (Sambrook et al., 1989). Todisrupt the two alleles of the CaHMX1 gene from strain BWP17, weused the primer-directed integration of Cahmx1D : :URA3 andCahmx1D : :ARG4 as described by Wilson et al. (1999). PCR primers59-CGTCAAGGTTTGCAAGCATTCTATCATGTATTTGCTAGTATT-GAAAAGGCCTTGTACAGACAGCTTGAAAAGTGGAATTGTGAGC-GGATA-39 and 59-GTCATGTTCGAAAATGTATTTTGATTCTTCA-ATGATTTCCAACTTTTGTTCTTCCGTCAAACCATTTCTTGTTTT-CCCAGTCACGACGTT-39 were used to amplify the URA3 andARG4 cassettes, from plasmids pGEM-URA3 and pRS-ARG4DSpeI,respectively, flanked by 71 nt of CaHMX1. After homologousrecombination, a 397 nt deletion (nucleotides 252–649, startingfrom ATG) was created. Successful integration was verified by PCRusing the external primers 59-ATGCAATACAAACTGAGTGGAGC-TACATCG-39 and 59-TTAGGTTAATTTATTGACAACTCTTCTTAA-39. For re-introduction of the CaHMX1 gene into the doubledisrupted strain, a 2 kb fragment containing the entire ORF wasamplified by PCR using the primers 59-GGCGGATCCGAGGGCA-ATGATACTGATTGGGCCATTATTTGG-39 and 59-GGAATCAACG-GCATGCGTTGATTGGCATTGTTGTGATATTTTCC-39, which carrya BamHI and a SphI restriction site (underlined), respectively. AfterBamHI/SphI digestion, the amplified fragment was inserted into theBamHI–SphI sites of the vector pGEM-HIS1 (Wilson et al., 1999).

580 Microbiology 149

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The resultant plasmid was linearized by using NruI, used for trans-formation of the double CaHMX1 knockout mutant and His+

clones were selected. Correct insertion at the HIS1 locus was verifiedby PCR using the primers 59-CTCGTGCCGTGTTGAATGTTTG-CTTC-39 and 59-CGAGTACCAATATATCGGTTGCACCAGC-39.The C. albicans CaHEM14, CaFTR1 and CaSIT1 knockouts wereobtained using the same method. The primers used to disruptthese genes were: CaHEM14, 59-GATTTGGATTCTCAAATAGAA-GTAATTAATGAAAAATGTAATGCCAATAAGAAATATATTCTTG-ATTCTTCGTGGAATTGTGAGCGGATA-39 and 59-TAATTTTGA-TGTCAATACATCTTTAACAATTTTCAAATTCACTGACGAAGGA-ATTGTCCAATTTGTATATTTCCCAGTCACGACGTT-39; CaFTR1,59-CGTTCAAATTTTCTTCATCGTTTTCAGAGAATCTTTGGAAG-CTATCATTGTTGTTTCAGTGCTTTTGGCGTGGAATTGTGAGCG-GATA-39 and 59-GTCTCTTGCCTTATTCTTTTAGTTGTTGAATA-ATAATTAACTAAGTTTATTTGTTTTCTTTGGATTCGTTTCCCAG-TCACGACGTT-39; CaSIT1, 59-CCAGTCTTCCAATAATCATTCT-TCAGAAGAAGATAAACACTTGTCCGGAGATGAAAAGACGTTTT-CGTGGAATTGTGAGCGGATA-39 and 59-GCTACTCTTTTCTTCT-TGAAATTGCCGAAGAAATTGGCCAACGAGTCCTTCTCTTCTTG-CTTTTCTTTTCCCAGTCACGACGTT-39. Genotypes of all strainswere confirmed by PCR and Southern blot hybridization (Sambrooket al., 1989).

RNA analysis. RNA was extracted as described by Kohrer &Domdey (1991). Northern blotting and hybridization, at 42 C in50 % (v/v) formamide, were done essentially as described (Knightet al., 2002; Lesuisse et al., 2002). The DNA fragments used asprobes for each gene were amplified by PCR using primers 59-ATGCAATACAAACTGAGTGGAGCTACATCG-39 and 59-TTAGG-TTAATTTATTGACAACTCTTCTTAA-39 for CaHMX1, and primers59-GATTCTTATGTTGGTGATGA-39 and 59-TCGTCGTATTCTTG-TTTTGA-39 for CaACT1. Probes for CFL95/CaFRE1, CaFTR1 andCaSIT1 have been described previously (Knight et al., 2002; Lesuisseet al., 2002). After Northern blotting, CaHMX1, CaFRE1, CaFTR1,CaSIT1 mRNA and 25S rRNA levels were quantified using theIMAGEQUANT software (version 1.2; Molecular Dynamics). Volumevalues for mRNA signals were normalized with volume values for25S rRNA and ACT1 mRNA.

RESULTS

Haemin is an iron source for C. albicans

Free haemin or haem bound to proteins as the prostheticgroup (cytochrome c, haemoglobin) could be used byC. albicans as the sole iron source. When a high con-centration (1 mM) of the iron chelator BPS was added toa complete growth medium (YPD), growth of C. albicanscells was completely inhibited. Addition of free haemin orhaem proteins restored growth. Similar growth arrest inBPS and growth restoration by haemin were seen forstrains disrupted for both copies of the high-affinityferrous iron permease CaFTR1 gene (reductive uptakesystem) or of the siderophore receptor CaSIT1/CaARN1(Fig. 1). We can rule out the possibility that DCasit1/DCasit1cells in Fig. 1 were able to take up iron from haemin viathe reductive uptake system, after extracellular removal ofiron from haemin. Indeed, in haemin iron is tightly boundto the porphyrin ring and cannot be removed by simplereduction (Buchler, 1975). Moreover, the presence ofBPS in the medium functionally inactivated the reductiveuptake system, as evidenced by the fact that addition of

10 mM ferric citrate to the BPS-containing plates did notrestore growth (not shown). Finally, we showed thathaemin uptake was copper-independent in a wild-type aswell as in a DCasit1/DCasit1 strain (see below). S. cerevisiaedid not behave in the same manner; haemin was unable torestore growth of cells in iron-deficient medium (Fig. 1).We conclude that in C. albicans, but not in S. cerevisiae,there is a third iron uptake system in addition to thereductive and siderophore uptake systems described pre-viously. This is consistent with the recent observation ofWeissman et al. (2002) that haemin and haemoglobin arepotential iron sources that can be used by C. albicans in aCaCcc2-independent manner.

Characteristics of haemin uptake kinetics

We determined the kinetic parameters of haemin uptakeby C. albicans using chemically synthesized 55Fe-haemin.Two phases were observed (Fig. 2), a rapid phase ofhaemin binding followed by a slower uptake phase. The

Fig. 1. C. albicans, unlike S. cerevisiae, can use haemin asthe sole iron source. Iron-deficient cells (pre-grown onYPD+200 mM BPS) were sequentially fivefold-diluted and thenplated onto YPD (+uridine) agar with (A) no addition, (B)1 mM BPS or (C) 1 mM BPS+50 mM haemin. Lanes: 1, S.cerevisiae (YPH499); 2, C. albicans (SC5314, wild-type); 3, C.

albicans (double DCaftr1 mutant); 4, C. albicans (doubleDCasit1 mutant).

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Haemin uptake by C. albicans


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uptake phase was more temperature dependent than thebinding phase, but both phases strongly depended on theiron status of the cells (Fig. 2). Binding and uptake ofhaemin were very low when the cells were previouslygrown in iron-rich medium (10 mM ferric citrate), andboth phases were induced when the cells were previouslygrown in iron-deficient medium (Fig. 2). Similar kineticsof haemin uptake were observed with a mutant deficientfor reductive uptake of ferric citrate (DCaftr1/DCaftr1),and with a mutant deficient for non-reductive uptakeof ferrichrome (DCasit1/DCasit1). When the siderophorepathway was inactivated by knockout of CaSIT1/CaARN1and the reductive pathway was inactivated by copper chela-tion (Knight et al., 2002) there was still no effect on haeminuptake (not shown). A Dtup1/Dtup1 mutant previouslyshown to misregulate both reductive and siderophore ironuptake also showed normal kinetics for haemin uptake(not shown). In contrast, no haemin uptake was observedwith S. cerevisiae cells grown either in iron-rich or in iron-deficient conditions (Fig. 2). Thus, C. albicans but notS. cerevisiae has a specific and inducible, iron-regulatedhaemin uptake system independent from the reductive andnon-reductive uptake systems. Protoplasts of C. albicansshowed the same kinetics of haemin uptake as intact cells,but treatment of protoplasts by proteinase K decreased theamount of haemin bound to the cells, while transportof ferrichrome by proteinase-treated protoplasts wasunaffected (not shown). The same effect of proteinase Ktreatment on cell-surface binding of haemin was recently

observed in the fungus Histoplasma capsulatum (Foster,2002). In that case, the authors concluded that haeminuptake by this organism involved a first stage of haeminbinding at the cell surface. The same is probably true forC. albicans: the two phases that are observed in the kinetics(Fig. 2) probably correspond to two steps of transport. Thefirst step is energy-independent and probably consists ofthe binding of haemin to a cell-surface receptor expressedunder low-iron conditions. The second step is energy-dependent and probably consists of the transport of cell-surface bound haemin into the cells.

We further studied the binding phase of haemin uptake.Haemin binding at the cell surface at 4 C was measured asa function of extracellular haemin concentration (Fig. 3).The contribution of non-specific binding of haemin wasdetermined by measuring 55Fe-haemin binding in thepresence of a 1000-fold excess of unlabelled haemin. Thevalues for non-specific binding were subtracted from thosefor total binding (binding in the absence of unlabelledhaemin) to give specific binding (Fig. 3). Experimentaldata for specific binding were fitted to the equationy=c[x]a/ba+[x]a, where b and c are the Kd and Bmax

values, respectively, and a is a constant. Resolution of thisequation gives the following values, Kd=195?4±6?1 nM,Bmax=1?21±0?017 pmol per 106 cells, a=2?02±0?08. TheBmax value allows one to calculate that a maximum ofabout 76105 molecules of haemin can be bound specifi-cally to one cell. Also, considering that 106 cells representabout 1 mg total protein and 0?01 mg plasma membraneprotein, a putative receptor protein of 30 kDa (arbitraryvalue) would represent about 1/5000 of total membraneprotein. The data on specific binding did not fit to stand-ard Scatchard curves. The curve representing haemin

Fig. 2. Kinetics of haemin uptake by C. albicans and S. cerevi-

siae. C. albicans cells (strain SC5314) grown under iron-defi-cient (#, n) or iron-rich (%) conditions were incubated in50 mM citrate buffer (pH 6?5) containing 5% glucose, 0?05%Tween 80 and 1 mM 55Fe-haemin at 30˚C (#, %) or 4˚C (n).S. cerevisiae cells (strain S150-2B) grown in iron-deficient (6)or iron-rich (+) conditions were incubated at 30˚C in assaybuffer as above. Haemin uptake was followed by withdrawingaliquots at the indicated intervals.

Fig. 3. Saturation of haemin binding at the cell surface. Iron-deficient C. albicans cells (strain SC5314) were incubated for5 min at 4˚C with increasing concentrations of 55Fe-haemin,with (m, non-specific binding) or without ($, total binding) a1000-fold excess of cold haemin. Specific binding of haemin atthe cell surface was calculated (&).




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dependence of specific haemin binding was sigmoidal,indicating that a process involving positive co-operativityoccurs at low haemin concentrations (Fig. 3).

CaHmx1 is required for iron assimilation fromhaemin

In haem, iron is tightly bound to the porphyrin ring.Haem-bound iron can be released by enzymic liganddestruction. An alternative possibility, enzymic ironremoval (reverse ferrochelatase), has not been described.In mammals, iron re-utilization from cellular haemopro-teins requires haem oxygenase activity (Poss & Tonegawa,1997). Haem oxygenase catabolizes haem to biliverdin,carbon monoxide and free iron. A gene encoding a putativehaem oxygenase is present in the C. albicans genome(orf6.7617) and has been named CaHMX1. This geneencodes a 291 residue protein with a theoretical Mr of33 973 and a pI value of 7?7. The CaHmx1 protein showed25 % identity with human haem oxygenase-1 (HO-1) and35 % identity with S. cerevisiae Hmx1p for the conservedcore of the enzyme. Crystal structures of the phylogeneti-cally distant human HO-1 and Neisseria meningitidishaem oxygenases (Schuller et al., 1999, 2001) show thatthe haem binding pocket is formed by two a-helices.Even though the structure of the active site is similar, thesequence of the distal helices of these enzymes is different(Schuller et al., 1999, 2001). Accordingly, sequence com-parison (not shown) revealed that only the proximalhelices residues Thr35, His39, Asp40, Ala42 and Asp43 areconserved in the CaHmx1 sequence.

We tested the role of CaHmx1 in iron utilization fromhaemin by deleting two copies of the CaHMX1 gene fromthe C. albicans genome. A wild-type allele of CaHMX1was then re-introduced into the Cahmx1D : :URA3/Cahmx1D : :ARG4 mutant (Fig. 4). Cells from the wild-type, from the single Cahmx1 and double Cahmx1

disruption strains and from the reconstituted Cahmx1/CaHMX1 strain were plated onto complete medium withhaemin as the sole iron source. The double Cahmx1 dele-tion strain grew normally on YPD medium (not shown)but was unable to grow in conditions where haemin wasthe sole iron source (Fig. 5, column 3). In the sameconditions, the reconstituted knockout grew normally(Fig. 5, column 4). This result shows that CaHmx1 isrequired for iron assimilation from haemin in C. albicans.Although CaHmx1 is involved in iron utilization fromhaemin, it is not involved in haemin uptake. Kineticsof haemin binding and transport were not significantlychanged in the double Cahmx1 deletion strain compared towild-type cells (not shown).

Regulation of CaHMX1 transcription

We tested various conditions for effects on CaHMX1expression by Northern blot (Fig. 6A). In standard condi-tions of growth (minimal medium at 30 C), the CaHMX1transcript was difficult to detect due to low expression, andthis level was further repressed when iron was added asferric citrate, ferrichrome or transferrin. Transcript levelswere strongly induced by iron deprivation and by haeminexposure. A shift of the temperature from 30 to 37 C wasalso a strong inducer. Incubation of the cells in fetal calfserum had no effect on the level of CaHMX1 transcripts(Fig. 6A). The observations that CaHMX1 expression wasupregulated by both iron deprivation and haemin suggestthat this gene has a complex pattern of regulation, sincehaemin is itself an iron source. To our knowledge, CaHMX1is the first C. albicans gene found to be regulated byhaem. While extracellular haemin induced transcriptionof CaHMX1, intracellular haem was not required forinduction. The Cahem14D : :URA3/Cahem14D : :ARG4haem-deficient mutant contained no trace of haem butresponded to iron-deficient conditions as the wild-typeby inducing CaHMX1 transcription (not shown).

Fig. 4. Confirmation of chromosomal disruption and re-introductionof gene CaHMX1 by PCR. The primers used are described inMethods. Amplified fragments were run on a 0?8% agarose geland stained using ethidium bromide. Lanes: 1, phage l

digested with HindIII; 2, BWP17 (wild-type); 3, BWP17Cahmx1D : :URA3/CaHMX1 (single knockout); 4, BWP17Cahmx1D : :URA3/Cahmx1D : :ARG4 (double knockout); 5,BWP17 Cahmx1D : :URA3/Cahmx1D : :ARG4/CaHMX1 (recon-structed strain).

Fig. 5. CaHMX1 knockout prevents growth of C. albicans withhaemin as the sole iron source. Iron-deficient cells (pre-grownon YPD+200 mM BPS) were sequentially fivefold-diluted andthen plated onto YPD (+uridine) agar containing either 1 mMBPS (A, iron-deficient condition) or 1 mM BPS+50 mM haemin(B, haemin as the sole iron source). Strains: 1, BWP17 (wild-type); 2, BWP17DCahmx1/CaHMX1; 3, BWP17DCahmx1/DCahmx1; 4, BWP17DCahmx1/DCahmx1/CaHMX1.




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We also investigated the pattern of CaHMX1 expression inmutants of the major transcription factors Tup1 and Efg1(Brown & Gow, 1999; Liu, 2001; Whiteway, 2000). Both thereductive and the siderophore pathways of iron uptakewere influenced by the Tup1 co-repressor (Knight et al.,2002; Lesuisse et al., 2002) and by the Efg1 activator(S. Knight & A. Dancis, unpublished data). These effectsmay be linked to the serum response, which is associated

with a shift from reductive to siderophore uptake pathways(Knight et al., 2002). In contrast, CaHMX1 expression wasalmost unchanged in a double Dtup1 mutant compared towild-type (Fig. 6B). This suggests that the haemin uptakepathway is regulated independently from the reductiveand non-reductive pathways of iron uptake. The effect ofEfg1 was also unique. CaHMX1 expression was deregulatedin the efg1 mutant (Fig. 6B), which fits well with the

Fig. 6. Regulation of CaHMX1 compared to genes of the reductive (CFL95, CaFTR1) and non-reductive (CaSIT1/CaARN1)uptake systems. Cells were grown in minimal medium (YNB without iron) with various additions. Northern blotting of theindicated genes was performed as described in Methods. (A) CaHMX1 expression in the wild-type (BWP17) grown undervarious conditions. Lanes: 1, no addition; 2, 200 mM BPS; 3, 10 mM ferric citrate; 4, 10 mM ferrichrome; 5, 5 mM iron-saturated transferrin; 6, 25 mM haemin; 7, 50% (v/v) fetal bovine serum; 8, no addition, 5 h shift at 37˚C. (B) CaHMX1

expression in various strains grown under different conditions. Lanes: 1, Wild-type (BWP17), 200 mM BPS; 2,BWP17DCahmx1/CaHMX1 (single knockout), 200 mM BPS; 3, BWP17DCahmx1/DCahmx1 (double knockout), 200 mMBPS; 4, Defg1/Defg1, no addition; 5, Defg1/Defg1, 200 mM BPS; 6, Defg1/Defg1, 10 mM ferric citrate; 7, Defg1/Defg1, 25 mMhaemin; 8, Defg1/Defg1, 50% (v/v) fetal bovine serum; 9, Dtup1/Dtup1, no addition; 10, Dtup1/Dtup1, 200 mM BPS; 11,Dtup1/Dtup1, 10 mM ferric citrate; 12, Dtup1/Dtup1, 25 mM haemin; 13, Dtup1/Dtup1, 50% (v/v) fetal bovine serum. (C)Expression of CFL95, CaFTR1 and CaSIT1/CaARN1 in wild-type (WT), Dtup1/Dtup1 and Defg1/Defg1 isogenic strains.Lanes: 1, no addition; 2, 200 mM BPS; 3, 10 mM ferric citrate. (D) Relative abundance of CaHMX1, CFL95, CaFTR1 andCaSIT1 transcripts in wild-type (WT), Dtup1 and Defg1 strains. 1, 200 mM BPS; 2, no addition; 3, 10 mM ferric citrate.




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observation that the promoter region of CaHMX1 con-tains four E-box sequences (Leng et al., 2001) (CATGTG,2684 to 2679; CACTTG, 2548 to 2543; CATCTG, 2409to 2404; CAATTG, 2385 to 2380). In unsupplementedmedia, the expression of the CaHMX1 gene was stronglyinduced in the efg1 mutant compared to the wild-type(Fig. 6B). Induction by BPS and haemin was almostidentical in both strains. However, addition of fetal serumstrongly induced the gene in the efg1 mutant compared tothe wild-type strain (Fig. 6B). These results suggest that thetranscriptional regulator Efg1 is a repressor of CaHMX1.Fig. 6(C) shows the expression patterns of CFL95/CaFRE1,CaFTR1 and CaSIT1/CaARN1 in wild-type, Dtup1 and efg1mutants. Expression of CFL95 was downregulated by ironexposure in the wild-type and derepressed in the Dtup1mutant, as observed previously (Knight et al., 2002). In theefg1 mutant CFL95 expression was unresponsive to ironrestriction and detectable at low levels under all conditions.Likewise, CaFTR1 expression did not respond to irondepletion in the efg1 mutant and was present at low, butnot fully repressed, levels similar to that in the Dtup1mutant. The expression of CaSIT1 was repressed underall conditions in the Dtup1 mutant and was barely detect-able in the efg1 mutant. The effect of EFG1 and TUP1deletions on the relative transcript abundance of CFL95/CaFRE1, CaFTR1, CaSIT1 and CaHMX1 is summarizedin Fig. 6(D). Thus, our results show that the three dif-ferent iron uptake systems are regulated independentlyand in a complex manner, although they were all inducedby iron deprivation.

Haemin effects on colony and cell morphology

C. albicans colonies forming on agar plates with haeminas the sole iron source showed a very unusual morphology.Colonies were made up of worm-like, tubular structuresorganized into a complex network (Fig. 7A). When observed

microscopically, some cells within the colonies were seento form a network of filaments enclosing other cells in theyeast form (Fig. 7B). When the colonies grew older, theproportion of filaments increased and the colonies took onthe consistency of a dried sponge (not shown). Thesecolonies with tubular structures appeared only whenhaemin was the sole iron source in the medium (haeminplus 1 mM BPS). Haemin added to complete mediumwithout the iron chelator BPS induced filamentation (notshown), but the morphological change of the colonies wasnot as striking (Fig. 7C). Others have previously reportedinduction of filamentation by haemin (Casanova et al.,1997). This effect of haemin on filamentation was specific,since it was not observed with any other iron source(ferric citrate, ferrichrome) or with protoporphyrin IX (notshown). Induction of filamentation by haemin increasedwith increasing extracellular concentrations of haemin, andit was always more pronounced in the double DCahmx1/DCahmx1 mutant than in the wild-type (Fig. 7D). Thedouble DCahmx1/DCahmx1 mutant showed unchangedhaemin uptake and decreased haemin degradation; thusmore haemin is expected to accumulate in these cells. Thissuggests that the inducer of filamentation may be intra-cellular haemin. The mechanisms by which intracellularhaemin promotes filamentation and morphological changeof the colonies, and the physiological significance of theseprocesses, remain to be investigated.

DISCUSSION

Several recent studies have greatly improved our knowledgeof the mechanisms of iron uptake by C. albicans (Ardonet al., 2001; Eck et al., 1999; Hammacott et al., 2000;Heymann et al., 2002; Hu et al., 2002; Knight et al., 2002;Lesuisse et al., 2002). These studies, together with thepresent one, show that there are at least three independent

Fig. 7. Haemin-induced morphological changeof C. albicans colonies. (A) Colony of a wild-type strain (BWP17) grown with haemin as thesole iron source (YPD+1 mM BPS+50 mMhaemin). (B) Light microscope view of cellsfrom the colony shown in (A). (C) Colony of awild-type strain (BWP17) grown in the presenceof haemin (YPD+50 mM haemin). (D) Coloniesof the wild-type (1) and of mutant strainsBWP17DCahmx1/CaHMX1 (2) and BWP17DCahmx1/DCahmx1 (3) grown on YPD+25 mMhaemin (upper view) or on YPD+75 mM haemin(lower view).




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iron uptake systems in this pathogenic fungus. The threesystems are induced under conditions of iron deprivation;however, fine regulation is probably complex, involvingseveral transcription factors, including Tup1 and Efg1. Theeffect of Efg1 on CaHMX1 transcription is especiallyinteresting. Efg1 is well known as a transcriptional activatorof many hyphal-specific genes. CaHMX1 seems to be thefirst gene shown to be directly under the negative controlof Efg1, though others have previously suggested that itmay function as both repressor and activator based on theeffects of shut-down and overexpression of EFG1 on hyphalgrowth (Stoldt et al., 1997). The complex regulatory patternsof the genes involved in the three iron uptake pathways ofC. albicans probably reflect the ability of C. albicans toreadily adapt to various ecological niches where iron maybe present in very different chemical forms. The reductiveuptake system is able to release iron from various ferricchelates at the surface of the cells, and the resulting ‘free’ferrous iron is taken up via a permease–oxidase complexof the plasma membrane homologous to that described inS. cerevisiae (Dancis et al., 1992; Stearman et al., 1996). Thesiderophore uptake system is able to mediate uptake ofsiderophores of the ferrichrome family, which enter the cellby a specific receptor, the CaSIT1/CaARN1 gene product.This uptake system is homologous to the siderophoreuptake system described in S. cerevisiae (Lesuisse et al., 1998;Yun et al., 2000). The haemin uptake system described inthis work mediates the use of extracellular haem as an ironsource, a process involving specific binding at the cellsurface, transport into the cell and degradation by haemoxygenase. This iron utilization system does not seem to bepresent in S. cerevisiae. In this yeast, there is a gene (HMX1)expected to encode a haem oxygenase, but its physiologicalrole remains unexplored. Haemin can enter S. cerevisiaecells, as shown by the ability of haemin added to theextracellular medium to restore growth of haem-deficientmutants. This effect may represent passive diffusion ofhaemin into the cells. Haem can diffuse passively acrossmodel lipid bilayers (Genco & Dixon, 2001), and we couldnot detect any transport of haemin into S. cerevisiae cells bykinetic studies. Moreover, S. cerevisiae was unable to growwith haemin as the sole iron source, unlike C. albicans. Thus,the role of haem oxygenase in S. cerevisiae, if any, couldbe restricted to re-utilization of iron from intracellularhaemoproteins, as in higher eukaryotes. The presence of ahaemin uptake system able to provide C. albicans cellswith iron could have important implications. It is wellknown that this pathogenic fungus can secrete haemolyticfactors, and thus haem proteins may provide an ironsource for cells during systemic infection (Manns et al.,1994; Moors et al., 1992). CaHmx1 is not directly involvedin haemin uptake, but it allows C. albicans to use haem asan iron source. The study of CaHMX1 regulation could beimportant, in this respect. CaHMX1 is involved in a crucialstep of iron assimilation from haemin and, as such, it isnot really surprising that it is induced under iron-deficientconditions. More interesting is the observation that haeminitself induces expression of CaHMX1. This observation will

form the basis of a strategy to find new components ofthe haemin uptake system, i.e. a search for haemin-inducedgenes. It would be surprising that CaHMX1 could be theonly gene positively regulated by haemin that is involved inhaemin iron utilization. The observation that incubationof cells with haemin increases haemin binding to the cellsurface is consistent with this hypothesis. Haemin mostlikely induces expression of several genes involved in thehaemin uptake/utilization pathway, and these genes maybe identified by DNA micro-array experiments. Identifica-tion of such new genes by this strategy is one of our prioritiesnow. Another important field to investigate based on theresults of the present study is the involvement of haem ironutilization in virulence. We are currently testing the effectof CaHMX1 double disruption on systemic infection inmice. CaHmx1 itself is an interesting protein to study, assuch. In a recent review, haem oxygenase-1 of mammalswas characterized as ‘the emerging molecule’ (Morse &Choi, 2002). This is because haem-oxygenase-relatedmechanisms play an important role in several aspects ofdifferent diseases, and increasing importance is given tohaem oxygenases in the defence of organisms againstoxidative stress. The mechanism by which haem oxygenaseconfers its protective effect is, as yet, poorly understood.In micro-organisms, the role of haem oxygenases in ironassimilation from haemin was often postulated, for evidentmechanistic reasons, but rarely shown experimentally. Toour knowledge, a direct role of haem oxygenase in ironassimilation has only been shown in Corynebacteriumdiphtheriae (Chu et al., 1999). Our study provides the firstexperimental evidence that haem oxygenase is required foriron assimilation from haem by a pathogenic fungus.

ACKNOWLEDGEMENTS

We thank R. Bryce Wilson and Aaron Mitchell (Columbia University,New York, USA) for the BWP17 strain and for the plasmids used forgene disruption, Burk Braun and Alexander Johnson (Universityof California, San Francisco, USA) for the Bca02-10 strain, andJoachim Ernst (Institut fur Mikrobiologie, Heinrich-Heine-Universitat,Dusseldorf, Germany) for the SS4 strain. Some of the sequence datafor C. albicans were obtained from the Stanford DNA Sequencingand Technology Center web-site (http://www-sequence.stanford.edu/group/candida), which is supported by funds from the NIDR and TheBurroughs Wellcome Fund. This work was supported by grants fromCNRS and the Ministere de la Recherche et de l’EnseignementSuperieur (Programme de Recherches Fondamentales en Micro-biologie et Maladies Infectieuses and Reseau Infections Fongiques),and by the American Cancer Society (Grant RPG-00-101-01-MBC).

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Haemin uptake and use as an iron source by Candida albicans: role of CaHMX1-encoded haem oxygenase