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EUKARYOTIC CELL, Feb. 2011, p. 207–225 Vol. 10, No. 21535-9778/11/$12.00 doi:10.1128/EC.00158-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Candida albicans Hap43 Is a Repressor Induced under Low-IronConditions and Is Essential for Iron-Responsive Transcriptional

Regulation and Virulence�

Po-Chen Hsu,1 Cheng-Yao Yang,2 and Chung-Yu Lan1,3*Institute of Molecular and Cellular Biology1 and Department of Life Science,3 National Tsing Hua University, No. 101,

Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan, Republic of China, and Division of Animal Medicine,Animal Technology Institute Taiwan, Chunan, Miaoli 35053, Taiwan, Republic of China2

Received 21 June 2010/Accepted 23 November 2010

Candida albicans is an opportunistic fungal pathogen that exists as normal flora in healthy human bodies butcauses life-threatening infections in immunocompromised patients. In addition to innate and adaptive immu-nities, hosts also resist microbial infections by developing a mechanism of “natural resistance” that maintainsa low level of free iron to restrict the growth of invading pathogens. C. albicans must overcome this iron-deprived environment to cause infections. There are three types of iron-responsive transcriptional regulatorsin fungi; Aft1/Aft2 activators in yeast, GATA-type repressors in many fungi, and HapX/Php4 in Schizosaccha-romyces pombe and Aspergillus species. In this study, we characterized the iron-responsive regulator Hap43,which is the C. albicans homolog of HapX/Php4 and is repressed by the GATA-type repressor Sfu1 underiron-sufficient conditions. We provide evidence that Hap43 is essential for the growth of C. albicans underlow-iron conditions and for C. albicans virulence in a mouse model of infection. Hap43 was not required for ironacquisition under low-iron conditions. Instead, it was responsible for repression of genes that encode iron-dependent proteins involved in mitochondrial respiration and iron-sulfur cluster assembly. We also demon-strated that Hap43 executes its function by becoming a transcriptional repressor and accumulating in thenucleus in response to iron deprivation. Finally, we found a connection between Hap43 and the globalcorepressor Tup1 in low-iron-induced flavinogenesis. Taken together, our data suggest a complex interplayamong Hap43, Sfu1, and Tup1 to coordinately regulate iron acquisition, iron utilization, and other iron-responsive metabolic activities.

Iron is the fourth most abundant element in the Earth’scrust, and the transition states of iron endow it with chemicalproperties essential to many biological processes. Iron hasbeen confirmed to be crucial for all organisms, with only twoexceptions (1, 66). The metal plays a structural or functionalrole in a variety of proteins responsible for DNA synthesis,respiration, electron transport, oxygen transport/storage, andmany central metabolic pathways (18). Excess iron content,however, does lead to deleterious oxidative damage as a resultof the Fenton reaction, in which free ferrous iron reacts withH2O2 or lipid peroxide to generate free radicals (28–30).Therefore, a precise regulation system for cellular iron ho-meostasis is necessary to maintain the intracellular level of ironin a balanced state between the minimal requirement andcytotoxicity.

Based on recent studies of fungal eukaryotes, a finely tunedsystem for the maintenance of iron homeostasis was identified(48). This system requires apparatuses for iron sensing, irontransportation, and storage. The iron-sensing mechanism canact as an iron-responsive regulator to modulate the activities orexpression of downstream effectors and proteins involved iniron uptake/utilization. In other words, a metabolic remodeling

event occurs in response to iron repletion or depletion duringthe process of iron homeostasis (45). When iron levels are low,the iron-responsive activator in some yeast species is activatedand induces the expression of genes encoding components foriron acquisition, whereas the expression of genes encodingiron-dependent proteins is repressed. In this way, the limitedamount of iron may be used more efficiently by the cells forpreventing cellular exhaustion, e.g., the iron can be used byvital enzymes that are involved in DNA synthesis, replication,repair, and transcription (18). In addition, the iron-responsiverepressor in most other fungi represses genes involved in ironuptake or iron transport under high-iron conditions. This reg-ulatory strategy avoids the detrimental consequences of ironoverload.

In Saccharomyces cerevisiae, iron deficiency leads to activa-tion and cytosol-to-nucleus translocation of two iron-regula-tory activators, Aft1 and Aft2 (3, 13, 65, 76, 90). Under iron-deprived conditions, Aft1 and Aft2 directly induce theexpression of iron uptake genes to increase the acquisition ofexternal iron sources and withdraw the deposited iron fromintracellular vacuoles. Furthermore, the withdrawal of storediron and the decrease in iron use are also regulated by the geneproduct of CTH2, which is also one of the Aft1/Aft2 targetgenes. Cth2 is a CCCH-type zinc finger mRNA-binding pro-tein and can couple with its iron-resistant paralog, Cth1, tobind coordinately to the AU-rich 3� untranslated region(UTR) of the target mRNA, which leads to its degradation (11,67, 68). Notably, these target mRNAs are mostly transcribed

* Corresponding author. Mailing address: Institute of Molecularand Cellular Biology, National Tsing Hua University, No. 101, Section2, Kuang-Fu Road, Hsinchu 30013, Taiwan, Republic of China. Phone:886-3-5742473. Fax: 886-3-5715934. E-mail: [email protected].

� Published ahead of print on 3 December 2010.

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from genes that encode iron-dependent proteins, and thereby,degradation of these mRNAs decreases the consumption andfacilitates the efficient usage of limited amounts of iron. Al-though Pfam prediction indicates that the Aft-type proteins(PF08731) exist in most hemiascomycota yeasts, only S. cerevi-siae and Kluyveromyces lactis Aft orthologs have so far beendemonstrated to possess iron-responsive transcriptional activ-ity via the PuCACCC binding motif in the promoter region ofthe iron regulon (12).

The second regulatory mechanism for the expression ofiron-responsive genes is mediated by GATA-type transcrip-tional repressors, which usually contain one or two Cys2/Cys2-type zinc finger domains separated by a conserved cysteine-richregion (26). The first fungal GATA-type repressor, Urbs1, wasidentified in the basidiomycete Ustilago maydis (86). Sincethen, orthologs of Urbs1 in ascomycetes with similar functionshave been described, such as Penicillium chrysogenum Srep(25), Neurospora crassa Sre (93), Schizosaccharomyces pombeFep1 (64), and Pichia pastoris Fep1 (58). Pathogenic fungi alsohave homologs of Urbs1, including Candida albicans Sfu1 (49),Aspergillus nidulans and Aspergillus fumigatus SreA (27, 60, 81),Histoplasma capsulatum Sre1 (10), and the basidiomycotaCryptococcus neoformans Cir1 (44). These repressors functionunder iron-replete conditions and negatively regulate the ex-pression of genes encoding iron assimilation proteins, espe-cially those involved in siderophore uptake and other reductiveiron uptake apparatuses. Furthermore, loss of the GATA fac-tor Cir1 attenuates the virulence of C. neoformans (44).

Recently, a third mode of transcriptional regulation in re-sponse to iron states was demonstrated as a negative modula-tion under iron-depleted conditions. In S. pombe, php4� en-codes a negative regulatory subunit of the heteromericCCAAT-binding complex (CBC) composed of Php2/Php3/Php5 (Php2/3/5) (54). The expression of php4� is partiallyrepressed by the GATA-type repressor Fep1 when iron issufficient, but it is upregulated in response to iron deficiency.Activated Php4 acts as a transcriptional repressor associatedwith the constitutively expressed Php2/3/5 complex to repressthe gene expression of iron-dependent proteins, especiallythose involved in the mitochondrial electron transport chain,tricarboxylic acid (TCA) cycle, iron-sulfur (Fe/S) cluster as-sembly, and heme biosynthesis (56, 57). Moreover, AspergillusHapX has been identified as a Php4 ortholog by sequencesimilarity (84). In A. nidulans, HapX is also controlled by SreA;the iron-dependent heme biosynthesis is repressed by HapX,together with the CBC factors HapB/HapC/HapE (HapB/C/E), and the siderophore biosynthesis pathway also requiresHapX (34). Moreover, HapX and HapB/C/E are essential forthe growth of A. nidulans under iron depletion. Deletion ofhapX also leads to significant attenuation of virulence in amouse model of invasive aspergillosis (79). In C. albicans,previous studies using DNA microarray analysis suggested thatthe expression of HAP43 (orf19.681), which encodes the or-tholog of Php4/HapX, is repressed by Sfu1 under high-ironconditions (49). Experimental evidence demonstrating theroles of C. albicans Hap2 and Hap3 is, however, still lacking.Moreover, C. albicans Hap5 was first revealed as a repressor ofmitochondrial electron transport components (43) and wasfurther demonstrated to govern the iron starvation-mediatedinduction of the gene encoding ferric reductase (Frp1). Hap43

may also partially participate in the Hap-mediated regulationof FRP1 (2). We do not, however, have a detailed understand-ing of the roles of C. albicans Hap43 in iron-responsive generegulation and virulence.

In this study, we characterized the function of C. albicansHap43. We first created a hap43� mutant and showed that lossof HAP43 leads to a defect in iron-dependent cell growth. Wefurther hypothesized that a pathogen unable to grow in aniron-deprived environment would lose its virulence to the hostbecause of the iron-withholding defenses within the host body(8) and demonstrated that deletion of HAP43 did indeed at-tenuate the virulence of C. albicans in a mouse model. More-over, the impeded growth of the hap43� strain under low-ironconditions did not result from its inability to acquire iron butwas possibly due to disruptions of iron-responsive transcrip-tional remodeling in genes encoding iron-dependent proteins.Interestingly, one-hybrid assays revealed that Hap43 becomesa repressor when the environmental iron cannot support theminimal requirement for the growth of C. albicans. This iron-dependent switch in the activity of Hap43 is possibly controlledby low-iron-induced nuclear accumulation of Hap43. Finally,Hap43 was essential for iron starvation-induced flavinogenesis.Taken together, our findings highlight the significance of C.albicans Hap43 in the virulence and transcriptional reprogram-ming of iron homeostasis, particularly under iron-depletedconditions.

MATERIALS AND METHODS

Yeast strains and growth conditions. All C. albicans strains used in this studyare listed in Tables 1 to 3. Cells were cultivated in YPD medium (1.0% yeastextract, 2.0% meat peptone, and 2.0% glucose), synthetic complete (SC) medium(0.67% yeast nitrogen base [YNB] with ammonium sulfate, 2.0% glucose, and0.079% complete supplement mixture), or synthetic minimal (SM) medium(0.67% yeast nitrogen base with ammonium sulfate and 2.0% glucose). Plateswere prepared with 1.5% agar for YPD-based medium and 2.0% agar for YNB-based medium. For the selection or growth of certain strains, SMU (SM plus 80�g/ml uridine) or YPDNou (YPD plus 200 �g/ml nourseothricin; Werner Bio-Agents, Jena, Germany) medium was used. For the induction of the SAP2promoter and the MAL2 promoter, yeast carbon base (YCB)-bovine serumalbumin (BSA) (23.4 g/liter yeast carbon base, 4 g/liter of BSA fraction V, pH4.0) medium and YPM (1.0% yeast extract, 2.0% meat peptone, and 2% mal-tose) medium were used, respectively (72). Cells were cultivated at 30°C withshaking at 180 rpm.

Iron-dependent growth analysis. Cells were grown in acidic noniron medium(NIM) or YPD-based medium. NIM was prepared as described previously(49). Briefly, NIM contains 0.17% YNB without Fe and Cu, 0.079% completesupplement mixture, 2% glucose, 0.5% ammonium sulfate, and 0.25 �MCuSO4 and is supplemented with 100 �M basophenanthrolinedisulfonatedisodium salt (BPS; Sigma), a ferrous iron chelator, to restrict the ironcontent of the medium. To support minimal cell growth, 10 �M ferrousammonium sulfate (FAS) was added to make the low-iron medium (LIM).Accordingly, high-iron medium (HIM) was prepared by adding 10-fold excessof FAS (100 �M). The second limited-iron medium was prepared by addingBPS to the YPD medium to restrain the free iron. YPD was defined as aniron-rich medium, whereas YPD plus 200 �M BPS was defined as an iron-poor medium. For pre-iron starvation, overnight cultures in YPD mediumwere diluted 100- to 1,000-fold into NIM or YPD plus 400 �M BPS and thengrown at 30°C with shaking for 20 to 24 h to achieve a steady state. For spotassays, iron-starved cells were harvested by centrifugation (Eppendorf 5810Rcentrifuge; A-4-62 rotor; 1,500 � g; 5 min; 25°C) and serially diluted to thedesired cell densities with sterile double-distilled water (ddH2O). Each dilu-tion was spotted onto agar plates (5 �l/spot) and incubated at 30°C for 1 dayor longer as indicated.

Gene manipulation. All deletion strains were generated from SC5314 using theSAT1 flipper method (72). The primers used are listed in Table 4. The HAP43deletion cassette was constructed as follows. An ApaI-XhoI DNA fragment com-

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posed of the 5� flanking region (nucleotides �816 to �64) of HAP43 was amplifiedfrom the SC5314 genome with the primer pair Orf19.681UP-1-ApaI andOrf19.681UP-2-XhoI. A SacII-SacI fragment composed of the 3� flanking region(nucleotides �1949 to �2228) of HAP43 was amplified from the SC5314 genomewith the primer pair Orf19.681DOWN-1-SacII and Orf19.681DOWN-2-SacI. TheseHAP43 5� and 3� flanking regions were cloned into the pSFS1A vector in order at theindicated restriction sites to generate pSFS1A-35fCaHap43. The plasmid pSFS2A-35fCaHap43 was constructed by replacing the XhoI-SacII fragment in pSFS1A-35fCaHap43 with the SAT1-PMAL2-FLIP cassette from pSFS2A. For the construc-tion of the DNA fragment used in the HAP43 reintegration, an ApaI-XhoI fragmentcomposed of the HAP43 promoter together with the full-length HAP43 codingsequence was amplified from the SC5314 genome with the primer pairOrf19.681UP-1-ApaI and CaHap43-ORF-2-XhoI. This fragment was then used to

replace the 5� flanking sequence of pSFS2A-35fCaHap43 to generate pSFS2A-35fCaHap43-ORF.

C. albicans strains were transformed by electroporation as described previ-ously (72) with some modifications. Cells from 5 ml of overnight culture in YPDmedium were collected by centrifugation (Eppendorf 5810R centrifuge; A-4-62rotor; 1,500 � g; 5 min; 25°C) and resuspended in sterile lithium buffer (8 ml ofddH2O, 1 ml of 10� Tris-EDTA [TE] [pH 8.0], and 1 ml of 1.0 M lithium acetate[pH 7.5]). After incubation at 30°C for 1 h with shaking, 250 �l of 1.0 Mdithiothreitol (DTT) was added to the cell suspension and incubated for 30 minat 30°C with shaking. Then, the cells were washed sequentially with 30 ml ofddH2O, 10 ml of ice-cold ddH2O, and 5 ml of ice-cold 1.0 M sorbitol; resus-pended in 500 �l 1.0 M sorbitol; and kept on ice until it was used. Deletion andreintegration cassettes from pSFS1A-35fCaHap43 (or pSFS2A-35fCaHap43)

TABLE 1. Strains used in gene deletion, iron-related assays, and RT-PCRa

Strain Genotype Background Source

SC5314 Wild type 23CaHap43A1 HAP43/hap43�::SAT1-SAP2p-FLIP SC5314 This studyCaHap43A2 HAP43/hap43�::SAT1-SAP2p-FLIP SC5314 This studyCaHap43B1-1 HAP43/hap43�::FRT CaHap43A1 This studyCaHap43B2-1 HAP43/hap43�::FRT CaHap43A2 This studyCaHap43C1-1-9 hap43�::SAT1-PMAL2-FLIP/hap43�::FRT CaHap43B1-1 This studyCaHap43C2-1-8 hap43�::SAT1-PSAP2-FLIP/hap43�::FRT CaHap43B2-1 This studyCaHap43D1-1-9-1 hap43�::FRT/hap43�::FRT CaHap43C1-1-9 This studyCaHap43D2-1-8-1 hap43�::FRT/hap43�::FRT CaHap43C2-1-8 This studyCaHap43R3 hap43�::FRT/hap43�::PHAP43-HAP43-SAT1-PMAL2-FLIP CaHap43D2-1-8-1 This studyCaHap43R3-1 hap43�::FRT/hap43�::PHAP43-HAP43-FRT CaHap43R3 This studyCaHap43R3-1-5 hap43�::PHAP43-HAP43-SAT1-PMAL2-FLIP/hap43�::PHAP43-HAP43-FRT CaHap43R3-1 This studyCaHap43R3-1-5-2 hap43�::PHAP43-HAP43-FRT/hap43�::PHAP43-HAP43-FRT CaHap43R3-1-5 This studyCaSfu1A1 SFU1/sfu1�::SAT1-SAP2p-FLIP SC4315 This studyCaSfu1B1-1 SFU1/sfu1�::FRT CaSfu1A1 This studyCaSfu1C1-1-3 sfu1�::SAT1-SAP2p-FLIP/sfu1�::FRT CaSfu1B1-1 This studyCaSfu1D1-1-3-1 sfu1�::FRT/sfu1�::FRT CaSfu1C1-1-3 This studyCaSfu1R2 sfu1�::FRT/sfu1�::PSFU1-SFU1-SAT1-PSAP2-FLIP CaSfu1D1-1-3-1 This studyCaSfu1R2-2 sfu1�::FRT/sfu1�::PSFU1-SFU1-FRT CaSfu1R2 This studyCaSfu1R2-2-2 sfu1�::PSFU1-SFU1-SAT1-PSAP2-FLIP/sfu1�::PSFU1-SFU1-FRT CaSfu1R2-2 This studyCaSfu1R2-2-2-1 sfu1�::PSFU1-SFU1-FRT/sfu1�::PSFU1-SFU1-FRT CaSfu1R2-2-2 This studyBca2-10 ura3�::�imm434/ura3�::�imm434 tup1�::hisG/tup1�::hisG::p405-URA3 Bca2-9 7

a RT, reverse transcription.

TABLE 2. Strains used in one-hybrid assays


CAI8 ura3�::�imm434/ura3�::�imm434 ade2::hisG/ade2::hisG CAI7 20COP1 ura3�::�imm434/ura3�::�imm434 ade2::hisG/ade2::hisG::�pCR-OPlacZ CAI8 This studyCCR1 ura3�::�imm434/ura3�::�imm434 ade2::hisG/ade2::hisG::�pCR-lacZ CAI8 This studyOC1 ura3�::�imm434/ura3�::�imm434 ade2::hisG/ade2::hisG::�pCR-OPlacZ

RPS1/RPS1::�CIplexA-F1COP1 This study

CC1 ura3�::�imm434/ura3�::�imm434 ade2::hisG/ade2::hisG::�pCR-lacZRPS1/RPS1::�CIplexA-F1

CCR1 This study

OG21 ura3�::�imm434/ura3�::�imm434 ade2::hisG/ade2::hisG::�pCR-OPlacZRPS1/RPS1::�CIplexA-F-CaGcn4-21

COP1 This study

CG21 ura3�::�imm434/ura3�::�imm434 ade2::hisG/ade2::hisG::�pCR-lacZRPS1/RPS1::�CIplexA-F-CaGcn4-21

CCR1 This study

ON18 ura3�::�imm434/ura3�::�imm434 ade2::hisG/ade2::hisG::�pCR-OPlacZRPS1/RPS1::�CIplexA-F-CaNrg1-18

COP1 This study

CN18 ura3�::�imm434/ura3�::�imm434 ade2::hisG/ade2::hisG::�pCR-lacZRPS1/RPS1::�CIplexA-F-CaNrg1-18

CCR1 This study

OH43-3 ura3�::�imm434/ura3�::�imm434 ade2::hisG/ade2::hisG::�pCR-OPlacZRPS1/RPS1::�CIplexA-F-CaHap43-3

COP1 This study

CH43-3 ura3�::�imm434/ura3�::�imm434 ade2::hisG/ade2::hisG::�pCR-lacZRPS1/RPS1::�CIplexA-F-CaHap43-3

CCR1 This study

COPGCRE-5 ura3�::�imm434/ura3�::�imm434 ade2::hisG/ade2::hisG::�pCR-OPGCRElacZ CAI8 This studyOGRH43-2 and

OGRH43-8ura3�::�imm434/ura3�::�imm434 ade2::hisG/ade2::hisG::�pCR-OPGCRElacZ

RPS1/RPS1::�CIplexA-F-CaHap43-3COPGCRE-5 This study

COGRE-2 andCOGRE-5

ura3�::�imm434/ura3�::�imm434 ade2::hisG/ade2::hisG::�pCR-OPGCRElacZRPS1/RPS1::�CIplexA-F1

COPGCRE-5 This study

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and pSFS2A-35fCaHap43-ORF, respectively, were excised by ApaI/SacI diges-tion and purified. Approximately 1 to 2 �g of linear DNA fragments was mixedwith 50 �l of electrocompetent cells, and the cells were transformed with theGene Pulser Xcell electroporator (Bio-Rad; 1,800 V; 200 ; 25 �F). Afterelectroporation, the cells were immediately washed with 1 ml of 1.0 M sorbitol,resuspended in 1 ml fresh YPD medium, and incubated for 3 to 4 h at 30°C withshaking. For the selection of cells with the genome-integrating cassette, thetransformants were spread onto YPDNou agar plates and grown at 30°C for 1 to2 days. Nou-resistant colonies were selected for verification by PCR. To pop outthe integrated SAT1-FLIP cassette from the HAP43 locus, the MAL2 promoteror SAP2 promoter was induced by YPM (22) or YCB-BSA (38) medium, re-spectively. Briefly, cells were inoculated into 5 ml YPM or YCB-BSA mediumand grown for 20 to 24 h at 30°C with shaking. Overnight-cultured cells were thenplated on YPM or YCB-BSA agar plates and incubated at 30°C for 2 days. Singlecolonies were then screened for Nou resistance by streaking them on both YPDand YPDNou plates, and the Nou-sensitive strains were picked for PCR diag-nostics. Two rounds of deletion cassette integration and excision were performedto manipulate two loci of HAP43 during the process of HAP43 deletion orHAP43 reconstitution. Homozygous hap43� strains were created from SC5314,whereas HAP43 reconstituted strains were created from one hap43� strain (Ta-ble 1).

Similar procedures were used for the deletion of SFU1. Briefly, an ApaI-XhoIfragment corresponding to the 5� flanking region (nucleotides �927 to �83) ofC. albicans SFU1 was amplified from the SC5314 genome with the primer pairCaSfu1up-1-ApaI and CaSfu1up-2-XhoI, whereas a SacII-SacI fragment corre-sponding to the 3� flanking region (nucleotides �1555 to �2042) was amplified

from the SC5314 genome with the primer pair CaSfu1down-1-SacII andCaSfu1down-2-SacI. The SFU1 5� and 3� flanking regions were cloned intopSFS1A to generate pSFS1A-35fCaSfu1. For the construction of the DNA frag-ment used in the SFU1 reintegration, an ApaI-XhoI fragment composed of theSFU1 promoter region together with the full-length SFU1 coding sequence wasamplified from the SC5314 genome with the primer pair CaSfu1up-1-ApaI andCaSfu1-ORF-2-XhoI to replace the 5� flanking sequence of pSFS1A-35fCaSfu1,generating pSFS2A-35fCaSfu1-ORF. These ApaI-SacI fragments were excisedfrom pSFS1A-35fCaSfu1 and pSFS2A-35fCaSfu1-ORF and used in yeast trans-formation to generate SFU1 homozygous deletion strains and reconstitutedstrains, respectively, as described above. All strains were verified by diagnosticPCR. For a successful deletion, the intra-open reading frame (ORF) PCRyielded no product, whereas a parental SC5314 yielded a matched product.

Southern blotting. Genomic DNA isolation for C. albicans was performed asdescribed previously (72) with modifications. Cells from 5 ml of overnight cul-tures in YPD medium were spun down, resuspended in 200 �l breaking buffer(10 mM Tris-Cl [pH 7.5], 100 mM NaCl, 1 mM EDTA, 2% Triton X-100, 1%SDS), and transferred to a microcentrifuge tube containing 0.3 g glass beads(Sigma) and 200 �l of PCIA (phenol-chloroform–isoamyl alcohol [25:24:1], 0.1%8-quinolinol, pH 7.0). The cells were lysed by vortexing them at maximum speedfor 5 min. The lysates were mixed with 200 �l TE buffer (pH 8.0) and centrifugedat maximum speed for 5 min (Eppendorf 5413D centrifuge; F45-24-11 rotor;25°C). DNA in the aqueous phase was precipitated with 95% ethanol, pelleted bycentrifugation (Eppendorf 5413D centrifuge; F45-24-11 rotor; maximum speed;5 min; 25°C), dissolved in 400 �l TE buffer (pH 8.0) containing 3.0 �l of10-mg/ml RNase A, and incubated at 37°C for 5 min. The RNase-treatedgenomic DNA was precipitated with 95% ethanol, pelleted by centrifugation(Eppendorf 5413D centrifuge; F45-24-11 rotor; maximum speed; 5 min; 25°C),and dissolved in 100 �l ddH2O. Blotting was performed using a standard methodwith some modifications (77). Approximately 20 �g of genomic DNA was di-gested with XbaI overnight at 37°C, separated on a 0.8% agarose gel with1:10,000 SYBR Safe gel stain dye (Invitrogen), transferred onto a positivelycharged nylon membrane (Pall Corporation) by the alkaline transfer method,and fixed by baking it at 80°C for 2 h. The membrane was hybridized with[�-32P]dCTP-labeled HAP43 5� flanking fragments using prehybridization buffer(6� SSC [1� SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 5� Denhardt’sreagent, 0.5% SDS, 100 �g/ml salmon sperm DNA, and 50% formamide) at 42°Covernight. The membrane was then washed and visualized by autoradiography.

TABLE 3. Strains used in confocal microscopy andWestern blotting


CAF2-1 URA3/ura3�::�imm434 SC5314 20CAI4 ura3�::�imm434/ura3�::�imm434 CAF2-1 20CGH43-2 and

CGH43-5ura3�::�imm434/ura3�::�imm434

RPS1/RPS1::�CIpGFP-F-CaHap43-1

CAI4 This study

TABLE 4. Primers and oligonucleotides used in strain constructions

Primer Sequencea Note

Orf19.681UP-1-ApaI 5�-AAGAGGGCCCCAGGATGAGGCTTTCACCAT-3� ApaI, forward, p19.681 5�f UPOrf19.681UP-2-XhoI 5�-TATTCTCGAGGCCGATTACTCGCTGATTTC-3� XhoI, reverse, p19.681 5�f UPOrf19.681DOWN-1-SacII 5�-TAATCCGCGGTTTCTTTTCTTTTCGTTTTGAATTG-3� SacII, forward, pSFS1A-35fCaHap43Orf19.681DOWN-2-SacI 5�-CTATGAGCTCTGTTGTCAAGATTGTCTTGCATT-3� SacI, reverse, pSFS1A-35fCaHap43CaHap43-ORF-2-XhoI 5�-AATGCTCGAGCTAATTATATGCTCTTCT-3� XhoI, reverse, pSFS2A-35fCaHAp43-ORFCaSfu1up-1-ApaI 5�-AATAGGGCCCAATGCTTTGGCATGCTTT-3� ApaI, forward, pSFS1A-5fCaSfu1CaSfu1up-2-XhoI 5�-AATGCTCGAGTGGATTGGAAATTGGAGGAA-3� XhoI, reverse, pSFS1A-5fCaSfu1CaSfu1down-1-SacII 5�-TTATCCGCGGAGTTAATATCAAGGGTTG-3� SacII, forward, pSFS1A-35fCaSfu1CaSfu1down-2-SacI 5�-CTATGAGCTCGCAGCTGGTTCAACTGGACT-3� SacI, reverse, pSFS1A-35fCaSfu1CaSfu1-ORF-2-XhoI 5�-AATGCTCGAGCTATCCATTTAACAACTTCCCAATAG-3� XhoI, reverse, pSFS1A-35fCaSfu1-ORFSaLexA-HindIII-1 5�-GTCAAAGCTTAAAATGAGAGAATTAACAAAACG-3� HindIII, forward, pCIplexA-F1SaLexA-MluI-2 5�-ACCAACGCGTACCCATTTCGCGGTACAAACCAATTAC-3� MluI, reverse, pCIplexA-F1CaGcn4ORF-1-MluI 5�-GGTCCACGCGTGGTGGAGGTCCAGGTGGACCTGCTACTACTC

CTACTAT-3�MluI, polylinker, forward, pCIplexA-F-

CaGCN4-21CaGcn4ORF-2-PstI 5�-GCCCGCCTGCAGTCACTAAAATTGAATACCATT-3� PstI, reverse, pCIplexA-F-CaGCN4-21CaNrg1ORF-1-MluI 5�-GGTCCACGCGTGGTGGAGGTCCAGGTGGACTTTATCAACAAT

CATATCC-3�MluI, polylinker, forward, pCIplexA-F-

CaNRG1-18CaNrg1ORF-2-PstI 5�-GCCCGCCTGCAGTCACTATACTAGGCTCTTGGT-3� PstI, reverse, pCIplexA-F-CaNRG1-18CaHap43ORF-1-MluI 5�-GGTCCACGCGTGGTGGAGGTCCAGGTGGAATGCCCGCAAAA

GGTCCTA-3�MluI, polylinker, forward, pCIplexA-F-

CaHap43-3CaHap43ORF-2-PstI 5�-GCCCGCCTGCAGTCACTAATTATATGCTCTTCTATCTAATTC-3� PstI, reverse, pCIplexA-F-CaHap43-3SaLexA-2(2) 5�-GCAACTGTCAAACGC-3� Forward, integration of pCIplexA F1 derivativesRPS1-1 5�-CGTATTCACTTAATCCCACAC-3� Reverse, integration of pCIplexA F1 derivativesade2hisG-1 5�-CTTTCTCATGCGTTCATGCACCAC-3� Forward, integration of pCR-lacZ derivativespCR-LacZ-vector-2 5�-CTGGTCTATAGTGTCACCT-3� Reverse, integration of pCR-lacZ derivativesOPGCRE-1 5�-TCGACATACGAACAAATATTCGCAATGACTCTGACTCTGACTCTGA

CTCTGACTCCTGCA-3�SalI, SalexA operator, (GCRE)5, PstI, top

strand, pCR-OPGCRElacZOPGCRE-2 5�-GGAGTCAGAGTCAGAGTCAGAGTCAGAGTCATTGCGAATATTTG

TTCGTATG-3�PstI, (GCRE)5, SalexA operator, SalI, down

strand, pCR-OPGCRElacZCaGFP-ORF-HindIII-1 5�-GTCAAAGCCTAAAATGAGTAAGGGAGAAGAACT-3� HindIII, forward, CIpGFP-F-CaHap43-1CaGFP-ORF-MluI-2 5�-ACCAACGCGTACCTTTGTATAGTTCATCCA-3� MluI, reverse, CIpGFP-F-CaHap43-1

a Restriction sites introduced into the primers are underlined and described in the note. S. aureus lexA operator sequences are in boldface. The (GCRE)5 is in italics.



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Virulence assay. Forty female BALB/c mice (7 weeks old) were obtained fromBioLasco Taiwan Co., Ltd., and group housed (5 mice per cage) for 1 weekbefore experiments. The C. albicans cells for inoculations were grown in SCmedium overnight at 30°C and then subcultivated in SC medium to the early logphase. Yeast cells were harvested by centrifugation (Eppendorf 5810R centri-fuge; A-4-62 rotor; 1,500 � g; 5 min; 25°C), washed once with phosphate-buffered saline (PBS), and resuspended in PBS at a density of 5.0 � 107 cells/ml.The cell suspension (100 �l; final, 5.0 � 106 cells) was injected via the lateral tailvein. Ten mice were used for each C. albicans strain. The injected mice weremonitored for clinical symptoms of distress and for survival twice per day for 2weeks. The animal studies were approved by the Institutional Animal Care andUse Committees, National Tsing Hua University and Animal Technology Insti-tute Taiwan, Taiwan. The log rank test was used to assess the differences insurvival between groups of mice. A P value of �0.05 was considered statisticallysignificant.

For kidney sections, organs extracted from the mice on the third day postin-fection were formaldehyde fixed and embedded in paraffin. Tissue sections werestained with periodic acid Schiff (PAS) to visualize C. albicans cells.

One-hybrid analysis. Plasmids and strains used in one-hybrid assays werecreated as described previously (75) with some modifications. BamHI-linearizedpCR-lacZ and pCR-OPlacZ were integrated into CAI8 at the ade2::hisG locus togenerate strains CCR1 and COP1, respectively. The S. aureus LexA operatorlinked with penta-Gcn4 binding sequences [SaLexA-(GCRE)5] was introducedon a double-stranded oligonucleotide (Table 4) between the PstI and SalI sitesin the pCR-lacZ vector to generate pCR-OPGCRElacZ. BamHI-linearizedpCR-OPGCRElacZ was integrated into the CAI8 genome to create theCOPGCRE-5 strain. All C. albicans strains carrying integrated pCR-lacZ and itsderivatives were selected for the plasmid-borne ADE2 marker in SMU medium.

To fuse the lexA sequence of pCIplexA (75) in frame, a HindIII-MluI fragmentcomposed of the LexA coding sequence was amplified from pCIplexA with theprimer pair SaLexA-HindIII-1 and SaLexA-MluI-2. This fragment contains aprimer-introduced GGT sequence at the C terminus and was cloned intopCIplexA between HindIII and MluI sites to generate pCIplexA-F1. In contrastto pCIplexA, the linker sequence 5�-GGTCC-3� located between the LexA cod-ing sequence and the MluI site was changed to 5�-GGT-3� in pCIplexA-F1. TheHAP43, GCN4, and NRG1 ORFs were amplified by PCR (Table 4) from theSC5314 genome and subcloned into pCIplexA-F1 between the MluI and PstIsites to create pCIplexA-F-CaHap43-3, pCIplexA-F-CaGcn4-21, and pCIplexA-F-CaNrg1-18, respectively. Strains CC1, CG21, CN18, and CH43-3 were gener-ated by transforming CCR1 with StuI-linearized pCIplexA-F1, pCIplexA-F-CaGcn4-21, pCIplexA-F-CaNrg1-18, and pCIplexA-F-CaHap43-3, respectively.Strains OC1, OG21, ON18, and OH43-3 were generated by transforming COP1with the four plasmids mentioned above. In addition, the OGRH43 and COGREstrains were generated by transforming COPGCRE-5 with pCIplexA-F-Ca-Hap43-3 and pCIplexA-F1, respectively. Integration of pCIplexA-F1 and itsderivatives into the C. albicans genome at the RPS1 locus was selected by theplasmid-borne URA3 marker using SM medium. Integrations were confirmed byPCR diagnostics (Table 4).

The expression level of the -galactosidase reporter in the one-hybrid strainswas assayed by X-Gal (5-bromo-4-chloro-3-indolyl- -D-galactopyranoside) over-lay assays and liquid -galactosidase ( -Gal) assays as described previously (74)with some modifications. For overlay assays, overnight-grown colonies on agarplates were lysed with chloroform for 5 min, air dried for 10 min after thechloroform was decanted, and overlaid with X-Gal–agarose (1% agarose, 0.25 or0.5 mg/ml X-Gal, 0.1 M sodium phosphate buffer [pH 7.0]). After the gelsolidified, the plates were incubated at 30 or 37°C overnight until the blue colordeveloped. For liquid assays, cells at an optical density at 600 nm (OD600) of �20were resuspended in 300 �l Z buffer with -mercaptoethanol ( -ME). Cells in100 �l of the suspension were lysed by adding 15 �l of 0.1% SDS and 30 �lchloroform and vortexing them for 15 s. Then, the suspensions of lysed cells wereincubated with 200 �l of 4-mg/ml ONPG (o-nitrophenyl- -D-galactopyranoside)(Sigma) in potassium phosphate buffer (pH 7.0) at 37°C for 30 min. The reactionwas stopped by adding 0.5 ml of 1.0 M Na2CO3(aq). The cell debris was spundown, and the absorbance of supernatants at 420 and 550 nm was determinedwith a spectrophotometer. The -galactosidase levels were displayed as Millerunits from at least three independent experiments.

Fluorescence microscopy. To observe the translocation of green fluorescentprotein (GFP)-tagged Hap43 in response to external iron levels, the S. aureuslexA sequence on pCIplexA-F-CaHap43-3 was replaced with the PCR product ofthe Candida-adapted GFP sequence from pNIM1 (62) to generate pCIpGFP-F-CaHap43-1. To generate strain CGH43-2, the StuI-linearized plasmid wasintegrated into the RPS1 locus of CAI4. Integrations were confirmed by PCRusing primers listed in Table 4. The expression of GFP-HAP43 in this system was

driven by the ACT1 promoter, which is constitutively active independently of theiron levels (49, 71). For the induction of GFP-Hap43 nuclear translocation, aniron-depleted culture (YPD plus 400 �M BPS) was prepared at a seeding densityof 0.5 OD600/ml, and the cells were grown to mid-log phase (�5 OD600/ml) at30°C with shaking. The cells were fixed with 3% formaldehyde in the culturemedium at room temperature for 2 h with shaking, washed, and resuspended inPBS. The fixed cells were subsequently stained with DAPI (4�,6-diamidino-2-phenylindole) (Sigma; 10 �g/ml in PBS) at room temperature for 10 min. Thestained cells were mounted on glass slides coated with 1% agarose, covered witha coverslip, and examined under a Carl Zeiss LSM 510 confocal microscopeequipped with HeNe, argon visible-light, and diode lasers. GFP-Hap43 wasexcited by the argon laser at 488 nm, and DAPI was excited at 405 nm.

RNA isolation and quantitative real-time PCR. Total RNA was extracted bythe phenol-chloroform method. Briefly, mid-log-phase cells from subculturesgrown under appropriate conditions were harvested by centrifugation (Eppen-dorf 5810R centrifuge; A-4-62 rotor; 3,000 � g; 5 min; 25°C), washed once withddH2O, and stored at �80°C. Cells (OD600, �20) were thawed on ice andresuspended in 500 �l ice-cold lysis buffer (0.1 M Tris-Cl [pH 7.5], 0.1 M LiCl,2% -ME, 0.01 M EDTA, and 5% SDS). The mixtures were transferred to amicrocentrifuge tube containing 500 �l PCIA (pH 4.5) and 0.3 g glass beads(Sigma). The cells were lysed by vortexing them at maximum speed for 5 min.Supernatants were obtained by centrifugation (Eppendorf 5413D centrifuge;F45-24-11 rotor; maximum speed; 5 min; 4°C), extracted two times with 200 �lPCIA, and transferred into 1 ml of 99% ethanol for precipitation. Nucleic acidpellets were spun down, washed with 70% ethanol, air dried, and then resus-pended in 100 �l nuclease-free water (Ambion). DNA contaminants were re-moved with a Turbo DNase-free kit (Ambion). The overall quality of the RNAwas analyzed by agarose gel electrophoresis.

For cDNA synthesis, 4.0 �g pure RNA was reverse transcribed using 400 UMoloney murine leukemia virus (MMLV) reverse transcriptase (Promega) with1.0 �g oligo(dT)18 primer in a 50-�l reaction mixture according to the manu-facturer’s instructions. Quantitative real-time PCR was carried out with the 7500real-time PCR system (Applied Biosystems). The primers used are listed inTable 5. Briefly, each 20-�l reaction mixture contained 80 ng cDNA, 10 �l PowerSYBR green PCR master mixture (Applied Biosystems), and 300 nM eachforward and reverse primer. The reactions were performed with 1 cycle at 95°Cfor 10 min, followed by 40 repeated cycles at 95°C for 15 s and 60°C for 1 min.

TABLE 5. Primers used in RT-PCR and quantitativereal-time PCR

Primera Sequence

CaHap43-probe1-1 ............5�-ACTCCAGCGTCGAAAAGAAA-3�CaHap43-probe1-2 ............5�-TGATTGGTGAGGGTTGTTCA-3�CaSfu1-probe1-1 ................5�-GAAATCACCGCACACAACAC-3�CaSfu1-probe1-2 ................5�-ACGATGATTGCATTGGTGAA-3�CaEFB1-1 ...........................5�-ATTGAACGAATTCTTGGCTGAC-3�CaEFB1-2 ...........................5�-CATCTTCTTCAACAGCAGCTTG-3�CaAct1-q1-1........................5�-GCCCAATCCAAAAGAGGTAT-3�CaAct1-q1-2........................5�-AGCTTCGGTCAACAAAACTG-3�CaAco1-q1-1.......................5�-TTCCCATTCAACGACTCAAT-3�CaAco1-q1-2.......................5�-GTGGTTCCAAGGTGTTCAAG-3�CaSDH2-q1-1 .....................5�-TGGAACCCAGATACTCCAGA-3�CaSDH2-q1-2 .....................5�-AAGCCAATGTGTTTCTTCCA-3�CaCYC1-q1-1 .....................5�-AAAAAGGTGGTCCACACAAA-3�CaCYC1-q1-2 .....................5�-TTAAACCACCAAAAGCCATT-3�CaCCP1-q1-1......................5�-GGGTGGATCATATGGTGGTA-3�CaCCP1-q1-2......................5�-GTACAGCAGCAACACCTCCT-3�CaYHB1-q2-1.....................5�-TTGGAAGCTTGGACTATTGC-3�CaYHB1-q2-2.....................5�-CGTTCAGGTTTTGGTAATGG-3�CaATM1-q1-1 ....................5�-GCCTCAAGAAACCCCATTAT-3�CaATM1-q1-2 ....................5�-TCCCTCGTTCTCCAACAATA-3�CaISA1-q1-1.......................5�-CCGACCGACAGTAACAAATC-3�CaISA1-q1-2.......................5�-CGAATCAATTTTGGATCAGG-3�CaISU1-q1-1.......................5�-CGCTAAAGAGTTGAGCTTGC-3�CaISU1-q1-2.......................5�-CGGCACTAGCACTTGAACTT-3�CaYAH1-q1-1 ....................5�-ACATTGCGAGCTTTCCATAC-3�CaYAH1-q1-2 ....................5�-ACATGAACCACCACAAGCTC-3�CaFRP1-q1-1......................5�-TTCGGTGGTGATACCTGTCT-3�CaFRP1-q1-2......................5�-CCCCACTTTCAGCAAGACTA-3�

a -1, forward primer; -2, reverse primer.



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The EFB1 transcripts were used as an internal control for RNA input and qualityafter reverse transcription (78), and the ACT1 transcripts were used as anendogenous control for the quantitative PCR.

Protein extraction and immunoblotting. The protein extraction protocol wasadapted from the instructions for the EasySelect Pichia Expression Kit (Invitro-gen). Briefly, cells at an OD600 of about 20 to 30 were harvested by centrifugation(Eppendorf 5810R centrifuge; A-4-62 rotor; 3,000 � g; 5 min; 25°C), washedonce with ddH2O, and stored at �80°C before lysis. Cell pellets were resus-pended in 300 �l ice-cold breaking buffer (1 mM EDTA, 5% glycerol, 1 mMphenylmethylsulfonyl fluoride [PMSF], 1/1,000-diluted protease inhibitor cock-tail [Sigma], 50 mM monobasic sodium phosphate [pH 7.4]) plus 0.3 g glass beads(Sigma). The cells were lysed by vortexing them at maximum speed for 5 min andthen chilled on ice for another 5 min. The supernatants were separated from thebeads and cell debris by centrifugation (Eppendorf 5413D centrifuge; F45-24-11rotor; maximum speed; 5 min; 4°C) and stored at �80°C. Whole-cell extractswere quantified with the Pierce BCA Protein Assay Kit.

Equal amounts of whole-cell extract from each sample were subjected to 8%SDS-PAGE and subsequently transferred to a polyvinylidene difluoride (PVDF)membrane (Pall Corporation) with an enhanced chemiluminescence (ECL)semidry blotter (TE 77; Amersham). The membranes were blocked with 3%nonfat milk in TBST (TBS with 0.1% Tween 20) at room temperature for 2 h.For immunodetection, mouse monoclonal anti-GFP IgG (632380 [Clontech] orNB600-601SS [Novus Biologicals, Inc.]) diluted 1:2,000 in TBST (0.1% Tween20, 1% BSA) was used. After hybridization with primary antibodies, the mem-branes were washed with TBST three times (25°C; 5 min each) and incubatedwith horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbitIgG (Santa Cruz Biotechnology). Chemiluminescence was developed with ECLreagents (Perkin Elmer) and visualized by exposure to X-ray film.

Colorimetric quantification assay for determination of intracellular iron con-tents. Intracellular iron levels of C. albicans were quantified by the BPS-basedcolorimetric method (83) with some modifications. Cells were harvested bycentrifugation (Eppendorf 5810R centrifuge; A-4-62 rotor; 3,000 � g; 5 min;25°C), washed with ddH2O, and resuspended in 500 �l of 3% nitric acid (J. T.Baker). The cell suspensions were boiled for 2 h to digest the cells completely,and cell debris was removed by centrifugation (Eppendorf 5413D centrifuge;F45-24-11 rotor; maximum speed; 5 min; 25°C). The supernatants containingiron (400 �l each) were collected and mixed with 160 �l of 38-mg/ml sodiumascorbate (Sigma), 320 �l of 1.7-mg/ml BPS, and 126 �l of 4 M ammoniumacetate. Chelating reaction mixtures were incubated at room temperature for 5min. The OD535 of the BPS-Fe complex was recorded with a spectrophotometeragainst blanks containing all reagents except the cells. To eliminate the nonspe-cific absorbance, the OD680 was subtracted from the OD535. The values for theiron content were adjusted by normalization according to the number of digestedcells. Cell numbers were indicated by OD600 using the following formula:(OD535 � OD680)/(OD600).

In the 30-min iron uptake assay, the iron uptake activities of C. albicans strainswere evaluated by measuring the increase in intracellular iron content after 30min of iron uptake. Briefly, for pre-iron starvation, C. albicans cells were grownin YPD medium plus 400 �M BPS for at least 24 h, while the cells without thepre-iron starvation treatment were grown in YPD medium without BPS. Thesestationary-phase cells were harvested (Eppendorf 5810R centrifuge; A-4-62 ro-tor; 1,500 � g; 5 min; 25°C), washed with ddH2O, and resuspended in fresh YPDmedium at a cell density of 2.0 OD600/ml. At time zero (T0), cells at an OD600 of�10 were harvested immediately, washed with ddH2O, and stored at �80°C untilthey were used. Uptake reactions were then facilitated at 30°C for 30 min withshaking, and cells at T30 were harvested as described above. Intracellular ironwas measured by the colorimetric method and displayed in arbitrary units (AU).The iron uptake activity of each C. albicans strain was determined by the differ-ence between the intracellular iron levels at T0 (AU0 min) and T30 (AU30 min).Relative increases in intracellular iron within 30 min (�30–0 min) were calculatedby subtracting the average AU0 min from the average AU30 min. The percentagerelative to the wild type without iron starvation (% to �WT) was shown asindicated.

In the growth-dependent iron uptake assay, cells with or without pre-ironstarvation treatment were inoculated at a cell density of 0.5 OD600/ml into freshYPD (high-iron) medium or YPD plus 200 �M BPS (low-iron) medium. Allinoculated cultures were grown at 30°C with shaking for 5 h to allow 3 or 4generations of cell growth. After the incubation, cells were harvested, and theintracellular iron was measured.

2,3,5-Triphenyltetrazolium chloride (TTC) reduction overlay assay. To eval-uate cell surface reductase activity, the overlay assay with TTC (Sigma) wasperformed as described previously (44) with some modifications. Briefly, YPD-grown cells were harvested and diluted with sterile ddH2O to a cell density of 1.0

OD600/ml. Each dilution was spotted onto YPD agar plates (10 �l/spot) andincubated at 30°C for 1 day. Then, the colonies were overlaid with agarose (1.5%in PBS) containing 0.1% TTC. The TTC reduction reactions were performed at30°C, and the development of red color on the colony spots was photographed.To inhibit electron transfer from respiration during the TTC reduction, 20 �g/mlantimycin A (Sigma) was added to the agarose overlay.

Phleomycin sensitivity assay. Phleomycin sensitivity was assessed by spotting10-fold serial dilutions of cells (5 �l/spot) onto YPD agar plates containing 50�g/ml phleomycin (Zeocin; Invitrogen), with incubation at 30°C for 1 day. Torestrict the growth-inhibitory effect of phleomycin on C. albicans, 100 �M BPSwas added to the plates.

Flavin secretion assay. For the induction of flavin secretion, C. albicans cellswere iron starved in NIM at 30°C with shaking for 2 days. Supernatants fromstationary-phase cultures were collected by centrifugation (Eppendorf 5413Dcentrifuge; F45-24-11 rotor; maximum speed; 5 min; 25°C). Two peaks at 360 nmand 450 nm can be observed in the flavin UV-visible spectra (53). The flavincontent of each supernatant was quantified at the absorption maximum of 446.3nm (47). The supernatants were photographed under UV light. Data werecollected from at least three independent experiments.

RESULTS

C. albicans Hap43 contains a region that is highly conservedin the N termini of fungal HapX/Php4. The 634-amino-acidprotein encoded by C. albicans HAP43 was first reported to besimilar to the AP1-like transcription factor (49). We performedBLAST searches using the Candida Genome Database (CGD)(http://www.candidagenome.org) and identified HAP43 as thehomolog of Hap4/Php4/HapX. To elucidate possible func-tional domains conserved in Hap43, we compared the Hap43protein sequence with those of Aspergillus and N. crassa HapX,S. pombe Php4, and S. cerevisiae Hap4 and Yap5. Notably, wecalculated the pairwise identities and similarities with theMatGat program (9) and found that the overall similaritiesamong the proteins were weak. Hap43 and A. nidulans HapXshare 28.7% identity (42.3% similarity), whereas Hap43 andPhp4 share only 17.2% identity (27.6% similarity). Neverthe-less, a highly conserved region was revealed in the N termini ofthese proteins. In this region, Hap43 and A. nidulans HapXshare 42.1% identity (61.7% similarity), whereas Hap43 andPhp4 share 26.1% identity (47.5% similarity). Figure 1A showsalignments of these proteins with residues 1 to 147 of Hap43and highlights the two domains that are particularly well con-served in these N-terminal regions. The first domain is theputative Hap complex-interacting domain that is required forinteraction of Hap4 with the CBC in yeast (5). Interestingly, allproteins but Yap5 possess this domain, implying the presenceof a distinct molecular mechanism between the typical AP1-like bZip factor (i.e., Yap5) and CCAAT-related transcriptionfactors. The second domain within the conserved N-terminalregion is the basic region of the bZip domain. With the excep-tion of S. cerevisiae Hap4, this region exists in all the comparedproteins (Fig. 1A). Notably, all HapX homologs and C. albi-cans Hap43 possess both the putative Hap complex-interactingdomain and the basic region of the bZip structure, whichsuggests that Hap43 may have a conserved function similar tothat of HapX/Php4. In addition, a similar comparison betweenAspergillus HapX and C. albicans Hap43 also highlighted threeconserved cysteine-rich motifs in the central-to-C-terminal re-gion (34).

The hap43� strain is unable to grow under low-iron condi-tions. To determine whether C. albicans Hap43 plays a role iniron homeostasis, we deleted HAP43 using the SAT1 flipper



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method (Fig. 1B) (72). Successful construction of the hap43-null mutants and their reintegrated strains was verified bySouthern blot analysis (Fig. 1C). The cell growth of isogenicmutants was compared with that of the parental wild-typeSC5314 under low-iron conditions. Based on the previous in-vestigation, a synthetic YNB-based medium containing 100�M BPS, an iron chelator, was used as the NIM, and ferrousiron was added to support the growth of C. albicans. Themedium supplied with 10 �M ferrous iron was used as low-ironconditions, whereas supplementation with 100 �M ferrous irongenerated high-iron conditions (49). In contrast to the wild-type and heterozygous strains, hap43 homozygous deletion mu-tants under low-iron conditions (NIM plus 10 �M Fe2�) wereseverely defective in their growth, even though the incubation

was continued for 3 days (Fig. 2A, top) or longer (data notshown). This growth defect was rescued by supplying excessiron (100 �M Fe2�) (Fig. 2A, bottom).

Baek et al. have demonstrated that hap43� mutants cannotgrow on iron-limiting agar plates using a nonfermentable car-bon source (YPG, composed of 1% yeast extract, 2% peptone,and 3% glycerol) and 150 �M BPS (2). We tested the growthof hap43� mutants under similar conditions, but YPD (withglucose as the carbon source) was used instead of YPG. Torestrict the free iron, cells were spotted onto YPD agar plateswith 25, 50, 100, 200, 400, or 800 �M BPS. The defectivegrowth of hap43 mutants on iron-depleted YPD agar plateswas quite similar to that under the NIM-based conditions (Fig.2B). Iron restriction with �100 �M BPS was unable to inhibit

FIG. 1. Construction of hap43-null mutants. (A) The amino acid sequence of C. albicans Hap43 was aligned with those of Aspergillus speciesHapX, N. crassa HapX, S. cerevisiae Yap5, S. pombe Php4, and S. cerevisiae Hap4 using Clustal W (50). The highly conserved N-terminal regionsare boxed. The black bar indicates the putative CBC-interacting domain. The hatched bar indicates the basic domain of the putative bZip structure.The sources for the protein sequences were as follows: NfHapX, NFIA_038200 (Aspergillus Comparative Database); AfHapX, XP_747952 (NCBI);AoHapX, BAE61614 (EMBLCDS); AnHapX, AN8251.3 (Aspergillus Comparative Database); NcHapX, NCU08891.3 (Neurospora crassa Data-base); CaHap43, ORF19.681 (CGD); ScYap5, YIR018W (SGD); SpPhp4, SPBC16E9.01c (Schizosaccharomyces pombe GeneDB); ScHap4,YKL109W (SGD). The amino acid residues are coded as follows: red, small hydrophobic residues; blue, acidic residues; magenta, basic residues;green, hydroxyl and amine basic residues. (B) The HAP43 alleles of C. albicans were knocked out by the homologous-recombination-based SAT1flipper method. The structure of the deletion cassette from pSFS1A-35fCaHap43 (and pSFS2A-35fCaHap43) is shown. The HAP43 coding regionis entirely replaced by the SAT1 flipper cassette. The HAP43 coding region is represented by the white arrow and the upstream and downstreamsequences by the hatched bars. (C) Southern blot analysis of XbaI-digested genomic DNA of the derivative strains of the hap43 mutant and theparental strain, SC5314 (HAP43/HAP43), with the specific DNA probe (black bar). The fragments corresponding to HAP43 (9.1 kb),hap43�::SAT1-FLIP or hap43�::FRT (6 kb), and HAP43-SAT1-FLIP or HAP43-FRT (7.9 kb) alleles are indicated.



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the growth of the tested strains. With 200 �M BPS, the wild-type strain displayed reduced growth, whereas the growth ofthe hap43� strain was severely impeded. On the plates con-taining �400 �M BPS, the growth of all strains was severelydefective in spite of a longer incubation of 3 days (data notshown). These findings allowed us to define YPD (withoutBPS), YPD plus 200 �M BPS, and YPD plus 400 �M BPS ashigh-iron, low-iron, and iron-starved conditions, respectively.The growth defect of the hap43� strain was successfully res-cued by native expression of HAP43 in the reconstituted strain(Fig. 2A and B). Taken together, we concluded that Hap43 isessential for the growth of C. albicans under either acidic(NIM-based; pH 4.2) or near-neutral (YPD-based; pH 6.5)low-iron conditions. Moreover, we also found that YPD-basediron-limiting medium (YPD plus 400 �M BPS) can support theminimal growth of the hap43� strain in liquid culture duringthe early to mid-log phase but is unable to sustain the growthof this mutant strain to the stationary phase (data not shown).Thus, we could easily collect sufficient hap43� cells at themid-log phase in YPD-based iron-limited culture without theprolonged incubation that is needed for synthetic medium(NIM based) (data not shown). As a result, YPD-based con-ditions were used in most of our subsequent experiments forconvenience.

Loss of HAP43 impedes virulence in a mouse model of dis-seminated candidiasis. To resist microbial infections, the hu-man body develops natural resistance by storing iron and main-taining an extremely low level of free ionic iron (10�18 M) intissue fluids (8). This tightly regulated low-iron environmentnot only supports the normal function of the immune system,but also inhibits the growth of invading or resident pathogens.Because Hap43 is essential for C. albicans growth under iron-restricted conditions, we speculated that Hap43 might play arole in the virulence of C. albicans. We tested this hypothesis in

a mouse dissemination model of candidiasis (Fig. 3A). Afterthe intravenous injection of the tails of mice with 5 � 106 yeastcells in PBS, nearly all mice displayed symptoms of illness, suchas ruffled hair, shivering, and lethargy, on the third day postin-fection (data not shown). The wild-type and the hap43 het-erozygous strains exhibited the highest virulence, leading tothe rapid death of the mice beginning on the third and fourthdays postinfection, and all mice in these two groups died within8 days. In contrast, mice infected with the hap43 homozygousmutant began to die on the seventh day, and 60% of the miceremained alive 2 weeks postinfection. Moreover, all survivingmice recovered from the illness by the 15th day postinfection(data not shown). In addition, all mice that had been infectedwith the HAP43 reconstituted strain succumbed to infectionwithin 13 days. Although the death rate of mice injected withthe reconstituted strain was lower than that of mice injectedwith wild-type cells (P � 0.00527), the reintegration of HAP43did restore the attenuated virulence of the hap43-null mutant(P � 1.25E�05) to a level that was not significantly differentfrom the heterozygous (hap43�/HAP43) mutant (P � 0.0581).

Kidneys were also collected on the third day postinfectionfrom both wild-type-infected and hap43�/hap43� strain-in-fected mice for histological examination. In the kidney sectionsexamined, the wild-type cells were observed as filamentousstructures (Fig. 3C and D). In contrast, no cells from thehap43-null mutant strain could be visualized (Fig. 3B), imply-ing that there is a slow invasion of the mutant cells or that theyare eliminated from the host tissues. These results support ourhypothesis that Hap43 is essential for growth under iron-re-stricted conditions, such as those within the host, and contrib-utes to C. albicans virulence.

The hap43 deletion mutant is not defective in iron acquisi-tion. To understand the nature of growth defects in the hap43�mutant under iron-restricted conditions, two possible hypoth-

FIG. 2. Deletion of C. albicans HAP43 causes growth defects under low-iron conditions. (A) Cells of C. albicans wild-type, hap43 heterozygousand homozygous deletion, and HAP43 reconstituted strains were serially diluted and spotted onto NIM-based iron agar plates with 10 �M (LIM)or 100 �M (HIM) ferrous ammonium sulfate (Fe2�). Two independent lineages of isogenic mutant strains from SC5314 were examined for someof the constructs. (B) Cells from the same strains were spotted onto YPD-based agar plates. To restrict free iron, the plates were supplementedwith 0, 100, 200, or 400 �M BPS. Cell numbers are shown at the top of each panel. Deletion of both HAP43 alleles led to growth defects underiron-deficient conditions (�200 �M BPS).



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eses were considered. One was that C. albicans is unable toacquire sufficient iron from extracellular environments in theabsence of Hap43. The other was that an irregular distributionof iron occurs within the cell when HAP43 is deleted. Todistinguish these two possibilities, we used a 30-min iron up-take assay to quantify the net increase in intracellular iron inthe hap43� mutant. Wild-type, sfu1�, and ftr1� strains wereused as controls, as iron uptake genes are derepressed in thesfu1� mutant under high-iron conditions (49) and the ftr1�mutant is defective in iron uptake (71). For the cells withoutpre-iron starvation (i.e., cells grown in YPD plus 400 �M BPSfor 24 h), both sfu1� and hap43� mutants exhibited a 40 to50% increase in iron uptake in fresh YPD medium comparedwith the wild type, whereas the ftr1� mutant showed extremelylow iron uptake activity (Fig. 4A). Moreover, although ironstarvation enhanced the iron uptake activity in all strains, in-cluding the ftr1� mutant, there were no significant differencesin intracellular iron among iron-starved hap43�, sfu1�, and

wild-type strains. This result implies that the hap43� mutantpossesses a functional iron acquisition activity even when cellshave been iron starved compared with the wild type.

The sfu1� mutant exhibits an iron overload phenotype un-der high-iron conditions, and this is possibly due to its consti-tutive expression of iron uptake-related genes, including cellsurface ferric reductases (49). Elevated ferric reductase activityas determined by the TTC reduction assay is also demon-strated in S. pombe fep1� (64) and C. neoformans cir1� (44)mutants. Therefore, to further study the iron uptake-relatedactivity, we performed the TTC reduction assay to determinethe ferric iron reduction activities of C. albicans strains. Thesfu1� strain displayed a high level of cell surface reductaseactivity, as indicated by the dark-red formazan precipitate thatformed on top of the colony spot (Fig. 4B). In contrast, thehap43� and ftr1� mutants showed no obvious differences incolony color compared with the wild type, indicating that highintracellular iron in the hap43� strain is not caused by elevated

FIG. 3. Deletion of HAP43 attenuates C. albicans virulence. (A) Ten female BALB/c mice were injected via the tail vein with 5 � 106 C. albicanscells, including SC5314 (orange), a heterozygous hap43 deletion mutant (black), a homozygous hap43 deletion mutant (blue), and a HAP43reconstituted strain (red). The number of surviving mice was plotted against time (in days). Notably, one mouse in the group infected with thehap43-null mutant died soon after injection, possibly because of operational error or some unknown shock. A representative result is shown (logrank test, wild type versus HAP43/hap43�, P � 0.647; wild type versus hap43�/hap43�, P � 1.25E�05; reconstituted HAP43 versus hap43�/hap43�, P � 3E�04; reconstituted HAP43 versus HAP43/hap43�, P � 0.0581; reconstituted HAP43 versus wild type, P � 0.00527). The hap43-nullmutant had the lowest virulence in comparison with the wild-type, HAP43/hap43�, and HAP43 reconstituted strains. (B) Kidney section of hap43�strain-infected mice showing no fungal colonization (�100 magnification). (C) Multiple sites (arrows) of fungal colonization were observed inwild-type-infected kidneys (�100 magnification). (D) The boxed area in panel C enlarged (�400 magnification) to show the filamentous forms ofwild-type C. albicans. The cells of C. albicans were visualized by PAS staining. The cell walls of C. albicans were stained purple, whereas the kidneytissues were stained blue, as shown.



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FIG. 4. The hap43� strain is not defective in iron acquisition under iron-depleted conditions. (A) The iron uptake activities of C. albicans wereevaluated in a 30-min uptake assay in fresh YPD medium. Normal and iron-starved stationary-phase cells were used. The wild type (WT) was takenas a normal control, whereas the sfu1� and ftr1� strains were used as a control of iron overload strain and defective-uptake strain, respectively.Intracellular iron as measured by the colorimetric assay is represented as the mean � standard deviation (SD) in AU (left). All data were collectedfrom at least three independent experiments. (B) C. albicans colony spots were overlaid with agarose containing TTC and incubated at 30°C. Ared color on the colony spot indicates increased cell surface reductase activity. Antimycin A is a mitochondrial electron transport inhibitor. (C) AllC. albicans cultures (initial cell density, 0.5 OD600/ml) were incubated at 30°C for 5 h in high-iron (YPD) or low-iron (YPD plus 200 �M BPS)medium. Iron starvation before the growth assay increased final intracellular iron in all strains (right), in contrast to the control samples withoutpre-iron starvation (left). The inset is an enlarged illustration indicating the level of intracellular iron from iron-starved cells grown in the low-ironmedium. The iron-starved ftr1� mutant showed no growth in the low-iron medium, and thus its intracellular iron content was not measured. Thehap43� cells accumulated higher levels of intracellular iron, as did sfu1� cells, after 5 h of growth in the high-iron medium compared with the wildtype, and the hap43� cells contained a level of iron similar to those of the wild type and the sfu1� mutant when grown in the low-iron medium.(D) Cells were serially diluted and spotted onto YPD plates containing the iron-dependent free-radical generator phleomycin and incubated at30°C for 1 day. Cell numbers are shown at the top of each image. Only the sfu1� mutant was hypersensitive to phleomycin, and addition of 100�M BPS partially inhibited this effect.



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activity of ferric reductases. The contribution of ferric reduc-tases in the TTC reduction was further confirmed by the ad-dition of a respiration inhibitor, antimycin A, which eliminatesthe effect of mitochondrial electron transport. Antimycin A didnot affect the pattern of formazan formation (Fig. 4B). Thisresult is consistent with the previous finding that C. albicansferric reductase activity is independent of mitochondrial res-piration (46).

We also measured the intracellular iron levels of all strainsusing a growth-dependent iron uptake assay, as described inMaterials and Methods. After cell growth in the high-ironmedium for several generations, both the hap43� and sfu1�mutants accumulated higher levels of intracellular iron thandid the wild type (Fig. 4C, gray bar). When cells were grown inthe low-iron medium, however, they exhibited no significantdifferences from the wild type in the level of intracellular iron.In addition, cells without and with pre-iron starvation showedthe same result. Although the iron uptake activity of the ftr1�mutant was quite low in the 30-min assay (Fig. 4A), the ftr1�strain gained a normal iron content compared with the wildtype (Fig. 4C), unless it was pre-iron starved followed bygrowth in the low-iron medium. This is because Ftr1 is essen-tial only under low-iron conditions, and the ftr1� strain cangrow well under iron-sufficient conditions (71). Therefore, theftr1� strain was omitted from the assay under iron-starvedconditions (i.e., cells were pre-iron starved and then grown inthe low-iron medium) because it did not grow after 5 h ofincubation in this assay.

To further determine the intracellular iron contents, tests ofcell sensitivity to phleomycin were also performed. Phleomycinbelongs to an antibiotic family that requires iron to generatefree radicals. Sensitivity to phleomycin reflects the level of freeintracellular iron (44, 64). The accumulation of excess free ironwithin the sfu1� mutant led to its hypersensitivity to phleomy-cin, and this phenotype was alleviated by iron restriction withBPS (Fig. 4D). The hap43� strain, however, was not hypersen-sitive to phleomycin compared with the sfu1� strain, suggestingthat excess iron within hap43� cells may not exist as a freelyaccessible form to react with phleomycin to cause cytotoxicity.Taken together, these studies not only substantiate the role ofC. albicans Hap43 in iron homeostasis, but also exclude thepossibility that the growth defect of the hap43� mutant underiron-depleted conditions (Fig. 2) is due to impaired iron ac-quisition.

Hap43 is responsible for the repression of iron utilizationgenes under iron-deprived conditions. To test the second pos-sibility mentioned above, we performed quantitative gene ex-pression analyses to determine whether Hap43 can function asa regulator of the expression of genes involved in iron ho-meostasis, especially when the extracellular iron levels are dis-turbed. S. cerevisiae utilizes multiple transcriptional and post-transcriptional mechanisms to optimize iron usage in responseto iron deprivation (45) by inducing the expression of genesencoding iron acquisition apparatuses and of Cth1/Cth2, twoproteins required for mRNA degradation. Cth1 and Cth2 areresponsible for the downregulation of genes encoding iron-dependent proteins that engage in metabolic pathways, such asrespiration, the TCA cycle, lipid synthesis, heme synthesis,biotin synthesis, and Fe/S cluster assembly. Similar iron-depen-dent metabolic remodeling occurs in S. pombe and A. nidulans

(34, 57), and key negative regulators that participate in thisprocess are Php4 and HapX, respectively. The existence of asimilar remodeling system and the possible role of Hap43 inthis iron-responsive regulatory mechanism have not, however,been investigated in C. albicans.

Accordingly, we selected several genes that are differentiallyexpressed in response to iron limitation based on genome-widemicroarray data (49) and examined their expression in thehap43 deletion background. We first confirmed that expressionof HAP43 is upregulated in the sfu1� mutant under high-ironconditions (49) and that expression of SFU1 is also upregu-lated in the hap43� mutant independently of iron levels (datanot shown); this suggests that there is reciprocal transcriptionalregulation between the expression of HAP43 and that of SFU1.Interestingly, expression of HAP43 was higher under low-ironconditions than under high-iron conditions (data not shown),implying its essential role in iron-deprived states. This differ-ential expression of C. albicans HAP43 has not been previouslyreported. A similar upregulation in response to low-iron con-ditions was, however, reported for S. pombe php4� and A.nidulans hapX (34, 57).

Deletion of HAP43 led to the increased expression ofACO1, SDH2, CYC1, CCP1, and YHB1 under iron-deprivedconditions (Fig. 5A). These genes are all downregulated inresponse to iron deficiency (49). ACO1 and SDH2 encodeaconitase and succinate dehydrogenase, respectively, andare both Fe-S cluster proteins involved in the TCA cycle.CYC1, CCP1, and YHB1 encode heme-containing proteins.Cyc1 is the cytochrome c responsible for transferring elec-trons from mitochondrial complex III to complex IV. Theamino acid sequence of Ccp1 is similar to the N terminus ofcytochrome c peroxidase, and Yhb1 is predicted to be anitric oxide oxidoreductase. Our quantitative gene expres-sion analysis was also applied to genes required for Fe-Scluster assembly. We analyzed four genes (ATM1, ISA1,ISU1, and YAH1) whose functions were predicted based ontheir gene homologs in S. cerevisiae (15, 51, 52, 73). Thesegenes were all derepressed in the cells lacking Hap43 underlow-iron conditions (Fig. 5B). ATM1 encodes a mitochon-drial inner membrane ABC transporter that is possibly in-volved in exporting mitochondrially synthesized precursorsof Fe-S clusters to the cytosol. ISA1 and ISU1 gene productsare predicted to be a mitochondrial matrix protein and ascaffold protein required for the biogenesis of the Fe-Scluster, respectively. YAH1 encodes a ferredoxin homolo-gous to S. cerevisiae Yah1, which has electron transfer ac-tivity involved in the Fe-S cluster biosynthesis. The 5� up-stream regions of theses potential Hap43 target genes(ACO1, SDH2, CYC1, CCP1, YHB1, ATM1, ISA1, ISU1, andYAH1) all contain multiple CCAAT motifs (data notshown), suggesting that Hap43/CBC may directly regulatethe expression of theses genes.

Furthermore, other genes encoding iron-dependent proteinssuch as CYT1, CAT1, and BIO2, and one gene encoding anon-iron-carrying protein, Hem1, were analyzed (data notshown). The CYT1 gene product, cytochrome c1, is a heme-containing protein that constitutes a subunit of complex III inthe electron transport chain. The CAT1 product is a catalaseresponsible for the breakdown of H2O2 to protect cells fromoxidative stress, especially when iron is overloaded (89). BIO2



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encodes an Fe-S cluster protein and is predicted to be a biotinsynthase that participates in biotin biosynthesis. Hem1 is de-scribed as a 5-aminolevulinate synthase that catalyzes the firststep of the heme biosynthetic pathway. A. nidulans hemA, thehomolog of the C. albicans Hem1 gene, is repressed by HapXunder low-iron conditions (34). Interestingly, no obvious dif-ferential expression of these four genes was observed in theabsence of C. albicans Hap43 in response to iron deprivation(data not shown).

The expression of a ferric reductase gene, FRP1, was alsoquantified. A previous report suggested that expression ofFRP1 is regulated by CBC factors and that Hap43 may beinvolved in this regulation (2). In contrast to the expressionpattern of genes encoding iron-dependent proteins, FRP1 isupregulated (by 22-fold) in response to iron deficiency,whereas the deletion of HAP43 decreased the low-iron-in-duced activation (to only 10-fold) (Fig. 5C), implying a positiverole for Hap43 in controlling some iron-responsive genes.

FIG. 5. Hap43 represses the transcription of genes encoding iron-dependent proteins under iron-deprived conditions. Quantitative real-timePCR was performed for selected iron-responsive genes. Cells were inoculated into high-iron (YPD) or low-iron (YPD plus 400 �M BPS) medium,incubated at 30°C for 5 h, and used for RNA isolation. The threshold cycle (CT) value of each gene was derived from the average of three technicalrepeats in each experiment. The �CT value was determined by subtracting the average CT of endogenous ACT1 from the average CT of targetgenes. The ��CT of each target gene was calculated by subtracting the �CT value of the corresponding calibration value (from a wild-type samplegrown under high-iron conditions). The average ��CT and SD were determined from at least triplicate experiments. The relative fold change ofeach gene is shown as 2��CT. The de-repression of each gene in the hap43� mutant in response to iron depletion (YPD plus 400 �M BPS) ishighlighted by an asterisk.



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Iron levels affect the transcriptional activity of Hap43. Thedata suggested that Hap43 plays a role in iron homeostasis asa repressor to downregulate the expression of genes encodingiron-dependent proteins when iron is not sufficient (Fig. 5Aand B). The sequence homolog of Hap43 in budding yeast,Hap4, acts as an activator to regulate respiratory gene expres-sion (21). Php4 in fission yeast is a negative regulatory subunitof the CBC complex under low-iron conditions (57). Similarrepression activity of HapX, together with CBC factors, hasalso been reported (34). However, Hap43 plays a positive rolein the regulation of FRP1 expression (Fig. 5C). Accordingly, tofurther investigate the role of C. albicans Hap43 in transcrip-tional regulation, the one-hybrid system was used (75). Wefused the sequence of the Staphylococcus aureus LexA DNA-binding domain to the N terminus of Hap43 and constitutivelyexpressed the fusion protein in the lacZ reporter-carryingstrains COP1 and CCR1. The COP1-derived strains use aLexA operator and an ADH1 basal promoter to drive the lacZreporter, whereas CCR1-derived strains lack the LexA opera-tor. Both the X-Gal overlay assay and the quantitative -ga-lactosidase assay were performed. Strains that express LexA-Gcn4 and LexA-Nrg1 were included as controls for theactivator and repressor, respectively. Although not as strong asthe activity of LexA-Gcn4, the LacZ activity generated byLexA-Hap43 was about 3-fold higher than that of basal con-trols (strains without the LexA operator) in the YPD medium(Fig. 6A). Because YPD medium represents an iron-sufficientcondition (Fig. 2B), we then examined the activity of Hap43 viathe X-Gal overlay assay under low-iron conditions by adding200 �M BPS or under the excess-iron condition by adding 50�M Fe2� to YPD agar plates. The activation of the lacZreporter by Hap43 completely disappeared when cells weregrown on the low-iron agar plate, even when the plate wasincubated at 37°C to enhance the development of the bluecolor (Fig. 6B). This result suggests that environmental ironcan regulate the transcriptional activity of Hap43.

Moreover, we proposed that the availability of iron mightalter Hap43 activity in a dose-dependent manner. To test thishypothesis, different concentrations of BPS from 25 to 800 �Mwere added to YPD to restrict iron availability, and -galac-tosidase assays for expression of the reporter were conducted.The transactivation activity of Hap43 was maximal in the ab-sence of the iron chelator BPS and gradually decreased in thepresence of increasing levels of BPS (Fig. 6C). Interestingly,Hap43 activity declined to the basal level under low-iron con-ditions (200 �M BPS) and appeared lower than basal activityunder iron-starved conditions (400 and 800 �M BPS).

From the expression analyses (Fig. 5) and one-hybrid assays(Fig. 6A to C), we were still unable to decide the biological roleof Hap43 under high-iron conditions; however, they raised astrong possibility of Hap43 serving as a transcriptional repres-sor under low-iron conditions. To assess this possibility, wedetermined the repression activity of Hap43 in a more precisesystem. By introducing (GCRE)5, a penta-general controlresponse element sequence, into the reporter strains betweenthe S. aureus LexA operator and the ADH1 basal promoter, theexpression of the lacZ reporter can be enhanced in a Gcn4-dependent manner (85). We adapted this idea to generate newreporter strains that exhibited basal levels of lacZ higher thanthat of the original LexA operator-only strain, allowing more

sensitive and accurate evaluations of gene repression mediatedby Hap43. As shown in Fig. 6D, the LexA-only strains contain-ing lexAOP-(GCRE)5-lacZ showed a 30-fold increase in thebasal level of reporter gene expression in comparison with theLexA operator-only strains (Fig. 6A) under either low- orhigh-iron conditions. Cells expressing the LexA-Hap43 fusionprotein did, however, have a dramatic reduction in lacZ ex-pression under low-iron conditions (Fig. 6D and E). Further-more, the presence of Hap43 also resulted in LacZ activityhigher than that in the LexA-only strains in YPD. This result isconsistent with the data shown in Fig. 6A to C, which implythat Hap43 potentially plays a positive role in transcriptionalregulation only under iron-rich conditions. Taken together,these data suggest that Hap43 regulates iron homeostasis byturning into a repressor in response to iron deficiency.

Iron limitation induces nuclear accumulation of Hap43. Toelucidate the possible mechanism by which Hap43 is regulatedin response to external iron levels, we examined the cellularlocalization of Hap43 by confocal microscopy. To correlatethese results with the data from the one-hybrid assays, wemodified the Hap43 one-hybrid plasmid by replacing the LexAcoding sequence with the sequence from Candida-adaptedGFP. This construct was transformed into CAI4 to create theGFP-Hap43-expressing strains. The strains were grown inYPD overnight and subcultured in iron-depleted and iron-sufficient media (YPD with and without 400 �M BPS, respec-tively). After a 5-h culture period, GFP-Hap43 proteins accu-mulated in the nucleus only under iron-depleted conditions(Fig. 7A). Similar results were seen when 400 �M BPS wasused to treat cells for 2 h (data not shown), indicating that thenuclear accumulation of Hap43 is a process regulated by aresponse to environmental iron rather than by a growth-de-pendent mechanism. In addition, we excluded the possibilitythat the expression of GFP-HAP43 is regulated at the tran-scriptional level by using a constitutive promoter-driven ex-pression system similar to the one used in the one-hybridstrains (see Materials and Methods). Thus, we focused on theregulation of Hap43 at the protein level. To evaluate the ex-pression of GFP-Hap43 under low- or high-iron conditions,immunoblotting was used to detect GFP fusion proteins withmonoclonal anti-GFP (Fig. 7B). The results indicated that thefusion proteins were expressed under both iron conditions.However, an unexpected supershift of the bands correspondingto GFP-Hap43 was observed on SDS-PAGE (from the pre-dicted position at 97 kDa to �120 kDa) independent of ironconditions. Moreover, more GFP-Hap43 was present underlow-iron conditions. In summary, the iron-responsiveness ofHap43 is in part regulated by the cytonuclear localization.

Iron starvation induces production of flavin molecules onlyin the presence of Hap43. C. albicans can secrete flavin inresponse to iron limitation. This activity is controlled by thegeneral transcriptional corepressor Tup1. Flavin production bythe tup1� strain is elevated in spite of the increased iron in theYPD medium (47). Because Tup1 is also a negative regulatorof genes involved in iron homeostasis, we proposed that otheriron-responsive repressors might also participate in the processof flavinogenesis. To test this hypothesis, the regulatory rolesof Sfu1, Hap43, and Tup1 in low-iron-induced flavinogenesiswere examined. For the convenience of observational andspectrometric analyses, transparent YNB-based NIM and SC



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medium were used instead of a YPD-based medium to in-duce the production of flavin. Sfu1 had no effect on low-iron-induced flavinogenesis compared with the wild-typestrain (Fig. 8). Hap43 was, however, essential for the iron-responsive process of flavin production. In addition, Tup1was required to sustain the production of flavin under iron-depleted conditions (NIM). Deletion of TUP1 led to a 50%reduction in flavin production (Fig. 8). These findings sug-gest that Hap43 may cooperate with Tup1 to regulate tran-scriptional and metabolic remodeling, especially under con-ditions of iron depletion.

DISCUSSION

Iron is essential for almost all organisms. Both iron defi-ciency and iron overload cause deleterious consequences forliving cells. C. albicans maintains iron homeostasis by re-modeling transcriptional programs to regulate iron-respon-sive genes. Besides the high-iron-specific repressor Sfu1(49), we have shown that the low-iron-specific repressorHap43 is also important for the regulation of C. albicansiron homeostasis.

Hap43 contains a conserved N-terminal region composed of

FIG. 6. One-hybrid analysis demonstrated that Hap43 is an activator under iron-rich conditions and a repressor under iron-deficient conditions.LexA-Hap43 binds to the LexA operator (LexAOP) upstream of the basal promoter of the lacZ reporter gene and modulates the expression oflacZ. LacZ activity was measured by a liquid -galactosidase assay and was also shown with the X-Gal overlay assay. The known activator Gcn4and the repressor Nrg1 fused with LexA were used as controls for the assays. The strains expressing LexA protein only and strains without the LexAoperator upstream of the lacZ reporter gene generated a basal expression level of lacZ. (A) C. albicans Hap43 activity was assayed by the -galactosidase method. In the mid-log phase cells grown in YPD (i.e., iron-rich medium), Hap43 displayed activation activity in cells carryingLexAOP. (B) Dilutions of YPD overnight cultures (5 �l; 5.0 OD600/ml) were spotted on YPD agar plates supplemented with 50 �M ferrousammonium sulfate (Fe2�) or 200 �M BPS. The plates were incubated at 30°C overnight. An X-Gal overlay assay was performed on the agar plates,and the plates were incubated at 30 or 37°C for 21 h. The basal strains developed a light-blue color because of the basal level of lacZ expression;activator-expressing strains generated a darker blue color. Repressor-expressing strains produced completely white colonies. (C) The iron-dependent conversion of Hap43 activity was determined with the -galactosidase assay using cells grown in YPD under iron restriction by adding0, 25, 50, 100, 200, 400, or 800 �M BPS. (D) The repression activity of Hap43 under iron-deprived conditions was assayed using reporter strainsthat included (GCRE)5 (85) between the LexA operator and the basal promoter of lacZ. These strains possess higher levels of basal expressionand were used to assay the repression activity of Hap43 in response to iron depletion. The strains expressing LexA only (without Hap43 fusion)were used as basal controls. Two independent clones of each strain were tested. (E) The LacZ activities of the LexAOP-(GCRE)5 strain were alsodisplayed by X-Gal overlay assay.



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a putative CBC-interacting domain and a bZip domain. Itfunctions more similarly to HapX/Php4 than to yeast Hap4 andanother bZip transcription factor, Yap5. Both Hap4 and Yap5lack either one of the conserved structures described above.Apart from Hap43, another two possible Hap43 homologs

(Hap41 and Hap42) exist in C. albicans (43). Interestingly, C.albicans Hap41 and Hap42 contain the putative CBC-interact-ing domains, like S. cerevisiae Hap4, but lack the bZip structure(Fig. 1A). Hap41 and Hap42 display lower identity/similarity toA. nidulans HapX (19.1%/33.7% and 14.2%/26.7%, respec-

FIG. 7. Iron deficiency induces nuclear accumulation of Hap43. The C. albicans CAI4 strain was transformed with a GFP-fused Hap43 underthe control of the ACT1 promoter. Cells expressing GFP-Hap43 were grown in YPD overnight and inoculated into iron-depleted YPD medium(400 �M BPS) or iron-sufficient medium (YPD without BPS). (A) Mid-log-phase cells were fixed, washed, and examined with differentialinterference contrast optics. Nuclei were visualized by DAPI staining. DAPI (red) and GFP (green) signals were viewed by laser scanning confocalmicroscopy, and the images were superimposed. GFP-Hap43 was dispersed throughout the cell when iron was sufficient and accumulated in thenucleus in response to iron depletion. (B) For protein extractions, samples were prepared as for panel A but without fixation. Whole-cell extracts(80 �g each) were resolved by SDS-PAGE, and GFP-Hap43 was detected with anti-GFP. Protein molecular mass standards are indicated on theleft (kDa). The CAF2-1 prototroph was used as the empty control. Two independent GFP-HAP43 clones are shown. GFP-Hap43 was expressedunder both high-iron (no BPS) and low-iron (400 �M BPS) conditions. The black arrow indicates the predicted molecular mass of GFP-Hap43,whereas the white arrow indicates the exact migration of GFP-Hap43. These Western blots were repeated several times with anti-GFP fromdifferent manufacturers; only one set of data is presented.



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tively) than does Hap43. Moreover, based on the work of Baeket al. (2), Hap41 is not required for the limiting iron-restrictedbinding of the FRP1 promoter and makes no contribution tolow-iron-dependent growth (YPG medium plus 100 �M BPS).Both Hap5 and Hap43, however, do play essential roles inlow-iron cell growth. Therefore, we doubt that Hap41 andHap42 are required for adaptation to iron-deficient environ-ments. An analysis of the functions of Hap41/Hap42 is cur-rently under way.

Accumulated evidence has suggested that iron is stronglycorrelated with microbial infections, including those caused byzoopathogenic and opportunistic fungi (35, 36, 82). In addi-tion, iron-restricting therapies are effective in treating fungalinfections in mouse models (39–42). Several iron-related pro-teins contribute to the virulence of C. albicans and otherpathogenic fungi (31, 32, 44, 71, 79, 80). In our study, weprovided the first example in C. albicans of an iron-responsivetranscription factor, Hap43, which is essential under low-ironconditions and can contribute to virulence in the disseminatedinfection (Fig. 3). Notably, most of the gene deletions de-scribed above lead to defects in iron uptake, and this lack ofcapability may consequently decrease the fitness of C. albicansto survive within the host. Furthermore, cryptococcal Cir1 isreported to regulate many virulence factors, such as capsuleand melanin production and the capability to survive at 37°C,as well as iron acquisition (24). Nevertheless, in C. albicans, itseems that the administration of iron uptake is not the majorcausative factor of Hap43-mediated virulence, based on ourresults (Fig. 4). Instead, hap43� cells had a higher intracellulariron content under iron-sufficient conditions, as did the sfu1�cells (Fig. 4C), and after overnight iron starvation than the wildtype and all other strains tested (Fig. 4A, 0 min). In addition,the hap43� mutant showed no increased ferric reductase ac-

tivity and phleomycin sensitivity, as did the sfu1� strain, im-plying that different molecular and biochemical events occur inthe absence of Hap43 and consequently cause the accumula-tion of a higher intracellular iron content.

Among fungal HapX/Php4 homologs, apart from C. albicansHap43, only A. fumigatus HapX has been shown to be requiredfor virulence so far (79). The attenuated virulence in thehapX� strain is possibly caused by the general dysregulation ofgene expression essential for metabolic adaptation to iron de-ficiency, the accumulation of toxic metabolites, and/or the de-creased expression of possible virulence determinants. In C.albicans, we found that the deletion of HAP43 does not affectthe morphogenesis of C. albicans in hypha-inducing spidermedium (data not shown), but the contribution of Hap43 tothe regulation of other virulence factors, such as secreted pro-teases, lipases, adhesins, and biofilm formation, is still unclear.Nevertheless, we demonstrated that Hap43 is essential for therepression of many genes that encode iron-dependent pro-teins, especially those involved in the TCA cycle, respiration,and Fe-S cluster assembly (Fig. 5A and B), indicating thatHap43 possesses general functions similar to those of Aspergil-lus HapX (34) and S. pombe Php4 (57).

According to our hypothesis and experimental evidence(Fig. 4), we have de-emphasized the regulation of iron uptakegenes in the hap43� mutant and have instead focused on therole of Hap43 in the transcriptional repression of genes en-coding iron-dependent proteins. Hap43 acted as a positivetranscription factor under iron-rich conditions, and this acti-vating activity decreased with the decline of iron availability(Fig. 6A, B, and C). We also provided evidence that low-ironstatus converts Hap43 to a transcriptional repressor (Fig. 6D).The iron responsiveness of Hap43 can be interpreted in twopossible ways. One is that Hap43 loses its ability to be anactivator under low-iron conditions, and the other is based ona molecular conversion of Hap43 from an activator to a re-pressor in response to iron deficiency. Some bacterial regula-tors of metal homeostasis also possess this contrary activity toactivate or repress metal assimilation or consumption (16, 37).S. pombe Php4 does not, however, exhibit activation activityunder high-iron conditions (57). Therefore, we cannot excludethe possibility that the activating activity of Hap43 is an artifactthat results from the constitutive expression of HAP43 as con-trolled by the ACT1 promoter or from the possible absence ofCBC (Hap2, Hap3, and Hap5 [Hap2/3/5]) factors that werereplaced by the LexA DNA-binding domain in the one-hybridassays. Furthermore, although the N-terminal LexA seems tohave no influence on the activity of Hap43 under low-ironconditions, we do not know whether LexA affects the activity,stability, structure, or transcriptional partners of Hap43 underhigh-iron conditions, even though the level of Hap43 is lowerthan that under low-iron conditions. Deletion of HAP43 onlypartially decreased the expression of FRP1 in response to low-iron conditions (Fig. 5C). In the study of Baek et al. (2), theinteraction between the FRP1 promoter and the protein ex-tracts from the hap43� strain were assayed by electrophoreticmobility shift assay (EMSA). They showed that a band wasonly slightly reduced (not completely absent) in the iron-starved hap43� cells compared with its expression in the wildtype. Our explanation for this finding is that Hap43 may playan indirect role by negatively regulating an unidentified repres-

FIG. 8. Hap43 is essential for the flavinogenesis induced by ironstarvation. (A) The absorbance at 446.3 nm of each supernatant fromthe stationary-phase culture in NIM (Fe�) or SC medium (Fe�) isdisplayed. This measurement is directly correlated with the levels offlavin in the supernatants. Quantitative data were calculated from atleast three independent experiments. The error bars indicate SD.(B) Supernatants prepared as indicated for panel A in NIM or SCmedium were collected and photographed under UV light. Flavinmolecules in the supernatants emitted fluorescence by UV excitation.The result from one of the replicates is displayed. The absorbance andfluorescence values of supernatants from the wild-type, hap43 deletion,and HAP43 reconstituted strains and sfu1- and tup1-null mutants werecompared.



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sor that controls the expression of FRP1. Therefore, the ab-sence of Hap43 leads to the elevation of the unidentified re-pressor and results in reduced FRP1 induction in response toiron deprivation. Moreover, the deletion of HAP43 led to ac-cumulation in the cell of phleomycin-inaccessible iron (Fig.4D). To explain this observation, we hypothesize that the ab-sence of Hap43 elevated the level of iron-dependent proteinsby derepressing gene expression (Fig. 5A and B), leading to anincrease in iron binding by these resultant proteins and a de-crease in phleomycin-inaccessible iron. This hypothesis is ableto explain only the higher intracellular iron content in theiron-starved hap43� mutant (Fig. 4A, at 0 min) but cannotexplain why hap43� cells maintain a high level of iron whengrown under iron-sufficient conditions (Fig. 4C).

Although the detailed mechanism for the regulation ofHap43 activity is not clear, Hap43 dispersed throughout thecell under conditions of iron sufficiency and accumulated in thenucleus when iron levels were low. A. nidulans HapX (34) andS. pombe Php4 (55) display similar nucleocytoplasmic shuttlingin response to iron levels. Some evidence suggests that themechanisms that regulate HapX/Php4/Hap43 translocalizationand their activities in target gene repression are much morecomplicated. For example, the iron-induced export of S. pombePhp4 from the nucleus relies on a physical interaction with amonothiol glutaredoxin, Grx4. Deletion of grx4� leads to thenuclear retention of Php4 and sustained repression of its targetgene, even when iron is sufficient (55). In the case of yeastAft1, iron-mediated inhibition requires two glutaredoxins,Grx3 and Grx4 (61, 69). Loss of both Grx3 and Grx4, but notloss of just one of them, causes the sustained nuclear localiza-tion of Aft1 and constitutive expression of Aft1 target genes. InC. albicans, there are at least four putative glutaredoxin genes(GRX1, GRX3, TTR1, and GRX5) annotated in the CGD, butthe expression of only GRX1 is induced in response to low-ironconditions (49). We generated a C. albicans grx1� strain butfound that there was no significant effect on the expression ofISA1, ATM1, CCP1, and YHB1 when GRX1 was deleted (datanot shown). Therefore, determining the role of each glutare-doxin in the iron-responsive regulation of Hap43 activity re-quires further investigation. Furthermore, translocation ofPhp4 depends on the Leu-rich nuclear export signal (93LLEQLEML100) and the exportin Crm1 (55). In C. albicans, a puta-tive Leu-rich nuclear export signal (129LVNTINKLKV138) waspredicted in Hap43. This signal overlaps with part of theleucine zipper sequence (129LVNTINKLKVENQFLVKNLEQL150 [structural leucines are underlined]) of Hap43 (Fig. 1A),and it provides a potential domain for the iron-responsiveregulation of Hap43.

There is a link between iron metabolism and flavinogenesisin eukaryotic cells (17). For example, in the riboflavin-over-producing yeast Pichia guilliermondii, flavinogenesis is coregu-lated with reductive iron assimilation (19). In addition, ironcan transcriptionally repress riboflavin synthesis in Pichia (4).A deficiency in mitochondrial frataxin, which is involved in irontrafficking and storage, leads to dysregulation of riboflavinbiosynthesis (70). In the case of bacteria, secreted flavins maybe advantageous for iron acquisition by acting as electron shut-tles to facilitate the reduction of insoluble ferric ions in thesurrounding environment or to assist in the release of ironfrom ferrisiderophores (14, 87). However, the physiological

function of inducible flavinogenesis remains to be elucidated.Recently, flavinogenic Candida species have been character-ized (47, 88, 91). In C. albicans, the iron starvation-inducedproduction of flavin is regulated by Tup1, which was originallyidentified as a corepressor and functions as a global regulatorof morphogenesis, metabolism, phase switching, and mating (6,59, 63, 92). Deletion of TUP1 causes an elevation of flavinproduction that is independent of iron levels in YPD medium(47). Interestingly, in our study, we found that Tup1 exerted apositive effect on low-iron-induced flavinogenesis in the acidicdefined medium (YNB based). The iron-responsive inductionof flavinogenesis is present in the tup1� strain, but this induc-tion shows a 50% reduction in comparison with the wild-typestrain. Taken together, these findings suggest potential coop-erative roles for Hap43 and Tup1 in the regulation of iron-dependent metabolism, such as flavinogenesis.

To summarize, a simplified model of Hap43-mediated reg-ulation in response to iron availability is proposed (Fig. 9). Inthis model, Sfu1 represses excess iron assimilation in coordi-nation with the corepressor Tup1 by repressing the expressionof genes encoding iron uptake proteins under iron-rich condi-tions. Under iron-deficient conditions, nuclear-accumulatedHap43 represses the expression of SFU1 to relieve the depres-sion of iron acquisition and also represses the expression ofiron-dependent genes to reapportion the usage of restrictediron. Furthermore, the expression of HAP43 is partially re-pressed by Sfu1 when iron is sufficient to activate the expres-sion of genes encoding iron-dependent proteins. Hap43 alsopositively regulates low-iron-induced flavinogenesis indepen-dently or cooperates with Tup1-mediated regulation. Thesecomplicated and interacting functions may contribute to the

FIG. 9. A simple model of Hap43-mediated iron metabolism isproposed. When cells encounter a shift from iron sufficiency to irondeficiency, the expression of HAP43 is released from repression bySfu1. In turn, Hap43 is induced to repress the expression of SFU1,leading to depression of many iron uptake genes and elevated ironassimilation. In contrast, Hap43 is also responsible for the attenuationof excess iron consumption by repressing the expression of genesencoding iron-dependent proteins. The function of Hap43 depends onits low-iron-induced nuclear accumulation and controls the normalgrowth, virulence, and flavinogenesis in iron-limiting states. The Tup1corepressor acts as a coregulator of the expression of some iron uptakegenes and flavinogenesis, possibly cooperating with Hap43 and Sfu1.The CCAAT-binding complex (Hap2/3/5) may take part in this com-plicated iron metabolism, together with Hap43, but its precise roleremains to be elucidated.



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normal growth and virulence of C. albicans within the host.Although Hap43 appeared to cooperate with CBC factors in astudy of FRP1 transcription regulation (2), deletion of the CBCsubunit HAP2, HAP3, or HAP5 generated some phenotypesthat are inconsistent with that observed in the hap43� strain(33). For example, hap2� and hap5� strains are hyperresistantto rapamycin and caffeine, in contrast to the wild-type, hap43�,and hap3� strains (33). Additionally, the growth defect ofhap43� under low-iron conditions was more severe than thatof hap2�, hap3�, and hap5� in Homann et al. (33) and basedon our unpublished data. These inconsistencies indicate thatnot all cell functions mediated by Hap43 rely on cooperationwith CBC factors. Therefore, more investigations are requiredto understand the role of each CBC factor in Hap43-mediatediron homeostasis.

ACKNOWLEDGMENTS

This work was supported by grants NSC95-2311-B-007-022-MY3,NSC98-2627-B-007-015, and NSC99-2627-B-007-007 from the Na-tional Science Council, Taiwan (to C.-Y.L.).

We thank Alistair J. P. Brown for the kind gift of one-hybrid mate-rials (pCIplexA, pCR-lacZ, pCR-OPlacZ, and CAI8). We also thankJoachim Morschhauser for the generous gifts of SAT1-FLIP genedeletion cassettes (pSFS1A and pSFS2A) and the GFP template frompNIM1. We thank Yu-Ting Chen for technical assistance with confocalmicroscopy.

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