Molecular Microbiology (2004)

53

(3), 969–983 doi:10.1111/j.1365-2958.2004.04185.x

© 2004 Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2004

? 2004

53

3969983

Original Article

C. albicans CaAce2 knock-out affects morphogenesisM. T. Kelly

et al

.

Accepted 16 April, 2004. *For correspondence. E-mailGeraldine.Butler@ucd.ie; Tel. (+353) 1 716 6885; Fax (+353) 1 2837211.

The

Candida albicans CaACE2

gene affects morphogenesis, adherence and virulence

Mary T. Kelly,

1

Donna M. MacCallum,

2

Susanne D. Clancy,

1

Frank C. Odds,

2

Alistair J. P. Brown

2

and Geraldine Butler

1

*

1

Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.

2

Aberdeen Fungal Group, Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Aberdeen AB25 2ZD, UK.

Summary

Morphogenesis between yeast and hyphal growth isa characteristic associated with virulence in

Candidaalbicans

and involves changes in the cell wall. In

Sac-charomyces cerevisiae

, the transcription factor pairAce2p and Swi5p are key regulators of cell wallmetabolism. Here, we have characterized the

CaACE2

gene, which encodes the only

C. albicans

homologueof

S. cerevisiae ACE2

and

SWI5

. Deleting

CaACE2

results in a defect in cell separation, increased inva-sion of solid agar medium and inappropriate pseudo-hyphal growth, even in the absence of externalinducers. The mutant cells have reduced adherenceto plastic surfaces and generate biofilms with dis-tinctly different morphology from wild-type cells. Theyare also avirulent in a mouse model. Deleting

CaACE2

has no effect on expression of the chitinase gene

CHT2

, but expression of

CHT3

and the putative cellwall genes

CaDSE1

and

CaSCW11

is reduced in bothyeast and hyphal forms

.

The CaAce2 protein is local-ized to the daughter nucleus of large budded cells atthe end of mitosis.

C. albicans

Ace2p therefore playsa major role in morphogenesis and adherence andresembles

S. cerevisiae

Ace2p in function.

Introduction

Candida albicans

is the most prominent fungal pathogenin humans. Despite being a component of the normalmicroflora in the oral cavity, gastrointestinal tract andvagina of healthy individuals,

C. albicans

can cause a

range of infections from superficial thrush to septicaemia(Odds, 1988). The ability of

C. albicans

to act as anopportunistic pathogen is seen primarily, although notexclusively, in immunocompromised hosts. In thecurrent climate, with increasing numbers of individualswith immune dysfunction,

C. albicans

poses an ever-increasing threat and is one of the four most commoncauses of bloodstream infections (Haynes, 2001; Pfaller

et al

., 2001). At present, the most commonly used anti-fungal drugs are fluconazole, flucytosine and amphotericinB. However, there are problems with acquired resistanceto azoles and considerable toxicity with flucytosine andamphotericin B (Pfaller

et al

., 2001). As a result, there isan urgent need to identify novel antifungal drug targets.

There are many factors associated with virulence in

C.albicans

. The ability to switch between yeast and hyphalgrowth is likely to be one of the most important. Non-filamentous

C. albicans

mutants are avirulent in mousemodels (Lo

et al

., 1997), and it has been suggested that,although yeast-form cells are necessary for dissemina-tion, hyphae may be required to invade host tissue. Mor-phogenesis is triggered by a number of environmentalfactors, but perhaps most significantly can be induced byboth human serum and human macrophages (Shepherd

et al

., 1980).The transition from yeast form to hyphal form involves

changes in cell wall structure and composition, includingan increase in chitin levels in hyphae (Munro

et al

., 1998).Chitin provides skeletal support to the yeast cell wall andis regulated using both chitin synthase and chitinaseenzymes. In

C. albicans

, there are four chitin synthase(

CHS

) genes (Au-Young and Robbins, 1990; Sudoh

et al

.,1993; Gow

et al

., 1994; Munro

et al

., 2003). CHS3p activ-ity is responsible for the majority of cell wall chitin in bothyeast and hyphal cells, and its disruption causes a fivefoldreduction in chitin levels and attenuated virulence in mice(Bulawa

et al

., 1995). The

C. albicans

genome also con-tains three chitinase (

CHT

) genes, with gene products thatare responsible for chitin degradation.

CHT2

and

CHT3

have increased levels of expression during growth of theyeast form, while

CHT1

is constitutively expressed at lowlevels (McCreath

et al

., 1995; Nantel

et al

., 2002). Aschitin is not found in the mammalian cell membrane, it haslong been considered a potential target for antifungalagents such as nikkomycin Z, a chitin synthesis inhibitor(Chapman

et al

., 1992). The chitinase enzyme has also

970

M. T. Kelly

et al.

© 2004 Blackwell Publishing Ltd,

Molecular Microbiology

,

53

, 969–983

been proposed as a drug target, but this is complicatedby the fact that there are chitinases in human cells (Hu

et al

., 1996). Therefore, it may be more effective to targetthe regulators of chitinase activity in pathogenic yeastrather than the enzyme itself. The regulation of both chitinsynthase and chitinase genes in

C. albicans

is poorlyunderstood however, despite having a potentially criticalrole in virulence.

In contrast, the regulation of chitin levels in

Saccharo-myces cerevisiae

has been studied extensively.

S. cere-visiae

contains three chitin synthase genes and a singlegene encoding chitinase,

CTS1

(Kuranda and Robbins,1991; Henar Valdivieso

et al

., 1999). Transcription of

CTS1

is regulated by Ace2p, and deletion of Ace2pcauses an increase in pseudohyphal growth andincreased invasion of solid agar (King and Butler, 1998;O’Conallain

et al

., 1999).

ACE2

is expressed during theG2 phase of the cell cycle, and the protein enters thenucleus only in daughter cells (Colman-Lerner

et al

.,2001; Weiss

et al

., 2002).

CTS1

expression peaks dur-ing late M/early G1 phase. Ace2p and the related tran-scription factor Swi5p control the expression of anumber of genes expressed at a similar stage in mito-sis, including several involved in cell wall metabolism(Doolin

et al

., 2001; Simon

et al

., 2001; Lee

et al

.,2002).

We identified a single homologue of

ACE2

and

SWI5

inthe

C. albicans

genome sequence (Tzung

et al

., 2001).This gene (

CaACE2

) encodes a putative product with 24%identity to Ace2p and 21% identity to Swi5p (Fig. 1).Despite the low overall sequence identity,

CaACE2

andthe

S. cerevisiae

gene pair are reciprocal best

BLAST

hitsand show extensive sequence conservation in the zincfinger region (Fig. 1).

Here, we report the construction of mutant strains inwhich both alleles of

CaACE2

were deleted. As in

S.cerevisiae

, the deletion strain cells fail to separate follow-ing cell division, have raised rough colonies on solid agarand have an increased tendency to form pseudohyphae.The strains also show altered adherence to plastic sur-faces and form defective biofilms. The

CaACE2

knock-outresults in reduced expression of one chitinase gene andof other genes with a potential role in cell wall metabolism.Finally, the mutants are avirulent when introduced intomice.

Results

Cloning of the

C. albicans ACE2

gene

CaACE2

was identified by a

BLAST

search with the

S.cerevisiae

Ace2p sequence. The

C. albicans

genome con-tains only one Ace2-like open reading frame (ORF6.5105), and it is equally distantly related to Ace2p and

Swi5p. Because of the cell separation defect describedbelow and the presence of a SGTAIF motif towards the N-terminus of the protein (Fig. 1), we called this ORF

CaACE2

. The gene was amplified by polymerase chainreaction (PCR) from the wild-type strain SC5314 usingoligonucleotides containing

Hin

dIII restriction sites andcloned into the vector CIp10 (Murad

et al

., 2000). Restric-tion analysis showed that the cloned allele was missingone

Eco

RI site compared with the

CaACE2

sequencefrom the Stanford Sequencing Project. Subsequent ream-plification and sequence analysis confirmed that

C. albi-cans

SC5314 contains two alleles of

CaACE2

that differat only one base (T to C at position +1281) resulting inthe loss of an

Eco

RI site.

Construction of

C. albicans ace2

mutants by targeted gene disruption

Candida albicans

is an asexual diploid organism so genedeletion requires the sequential knock-out of each allele.

CaACE2

gene disruption was achieved using mycophe-nolic acid resistance (MPA

R

) as a selective markerwithin a flipping cassette (Wirsching

et al

., 2000). Twonested cassettes were constructed containing differentsequences from the 5

¢

and 3

¢

ends of the

CaACE2

ORFflanking the MPA

R

cassette (Fig. 2A) and used sequen-tially to disrupt both

CaACE2

alleles. The wild-type strainSC5314 was first transformed with the external cassetteconstruct isolated from the plasmid pMTK2 by digestionwith

Apa

I and

Sac

I. Homologous recombination of thiscassette into the genome deleted the entire ORF of one

CaACE2

allele, as verified by PCR and restriction analysisof MPA

R

transformants. The

Eco

RI polymorphism in thetwo alleles was exploited during the verification analysis(Fig. 2B). Of 11 transformants checked, four contained theexternal cassette integrated correctly at

CaACE2

. TheMPA

R

marker was removed in one of these strains byinducing

FLP

-mediated recombination to generate strainMK89, an MPA

S

heterozygote mutant verified by PCR.MK89 was then transformed with the internal cassetteconstruct (Fig. 2A) isolated from the plasmid pMTK4 bydigestion with

Apa

I and

Sac

I. The internal cassette con-tained

CaACE2

flanking regions that were present only inthe remaining intact allele to eliminate the possibility ofintegration at the disrupted allele. Homologous recombi-nation of this cassette into the genome deleted the second

CaACE2

allele from 22 bases downstream of the startcodon to 7 bases upstream of the stop codon. Integrationof this cassette was again verified by PCR analysis. TheMPA

R

cassette was excised from one transformant asbefore and confirmed by PCR analysis. In the resul-ting strain, MK106 (Caace2::FRT/Caace2::FRT), bothCaACE2 alleles have been deleted and replaced with asingle FLP recognition site.

C. albicans CaAce2 knock-out affects morphogenesis 971

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 969–983

We also used a second disruption approach in whichthe two CaACE2 alleles were deleted by sequentialreplacement with lox-CaHIS1-lox and lox-CaURA3-loxcassettes (Fig. 2C). These cassettes were amplified from

plasmids pLHL2 and pLUL2, respectively, with primersthat tagged the cassettes with 70 bp of flanking sequencecomplementary to sequence either side of the CaACE2ORF. The strain RM1000, a derivative of SC5314, was first

Fig. 1. Comparison of yeast Ace2 sequences. The S. cerevisiae Swi5p (ScSwi5), S. cerevisiae Ace2p (ScAce2) and C. albicans Ace2p (CaAce2) protein sequences were aligned using CLUSTALW (Thompson et al., 1994) and visualized using BOXSHADE. Black shading indicates identical residues and grey shading similar residues.

ScSwi5 ----MDTSNSWFDASKVQSLNFDLQTNSYYSN-----------ARGSDPSSYAIEGEYKTScAce2 ---MDNVVDPWYIN--PSGFAKDTQDEEYVQH-----------HDNVNPTIPPPDNYILNCaAce2 MHWKFSNFRKYHLSFHLNLFDLSLFFISFYCFPILYICFFNQVHSFRSTQPSLIMNKFDL

ScSwi5 LATDD-LGNILNLNYGETNEVIMN-EINDLNLPLGP------LSDEKSVKVSTFSELIGNScAce2 NENDDGLDNLLGMDYYNIDDLLTQ-ELRDLDIPLVPSPKTGDGSSDKKNIDRTWNLGDENCaAce2 FDDYSTKGSTIPLPNENFDQLFLSSEANDMEFLFNET---LMGLQDLDVPSGYGIPQNTI

ScSwi5 DWQSMNFDLENNSREVTLNATSLLNENRLNQDSGMTVYQKTMSDKPHDEK------KISMScAce2 NKVSHYSKKSMSSHKRGLSGTAIFGFLGHNKTLSISSLQQSILNMSKDPQ------PMELCaAce2 NNDFQHTPNKSKSHSRQYSGTAIFGFADHNKDLSINGVNNDLCKQSNKAINTQSVSPGEL

ScSwi5 ADNLLSTINKSEINKGFDRN---------LGELLLQQQQELREQLRAQQEANKKLELELKScAce2 INELGNHNTVKNNNDDFDHIRENDGENSYLSQVLLKQQEELRIALEKQKEVNEKLEKQLRCaAce2 LKRSRGSQTPTPTSALPDTAQDILDFNFEEKPILLLEEDELEEEKHKQQQRMMTQSSPLK

ScSwi5 QTQYKQQQ--LQATLENSD-------------GPQFLSPKRKISPASEN-----------ScAce2 DNQIQQEK--LRKVLEEQEEVAQKLVSGATNSNSKPGSPVILKTPAMQNGRMKDNAIIVTCaAce2 RVTTPSQSPFVQQPQTMKQRKPHKKTNEYIVANENPNSYKFPPSPSPTAKRQQYPP---S

ScSwi5 ---VEDVYANSLSPMISPPMSNTSFTGSPSRRNNRQKYCLQRKNSSGT------------ScAce2 TNSANGGYQFPPPTLISPRMSNTSINGSPSRKYHRQRYPNKSPESNGLNLFSSNSGYLRDCaAce2 SPIPYNPKSDSVGGNSYSAKYLQSLNKTQQIEYVDDIEPLLQEDNNNMKYIPIPVQEPMS

ScSwi5 --VGPLCFQELNEGFNDSLISPKKIRSNPNENLSSKTKFITPFTPKSRVSS-ATSNSANIScAce2 SELLSFSPQNYNLNLDGLTYNDHNNTSDKNNNDKKNSTGDNIFRLFEKTSPGGLSISPRICaAce2 YQKQKPVTPPLQSQNDSQQLEPLKTPQPQPKQQQQQQQPNNEQDKEFTANINFNTFLPPP

ScSwi5 TPNNLRLDFKINVE-DQESEYSEKPLG--LGIELLGKPGPSPTKSVSLKSASVDIMPTIPScAce2 NGNSLRSPFLVGTDKSRDDRYAAGTFTPRTQLSPIHKKRESVVSTVSTISQLQDDTEPIHCaAce2 TPPNLINGSPDWNSSPEPHSPSPGRLQPPQQISPIHQNLGAMGNNINFYTPMYYELPVQA

ScSwi5 GSVNNTPSVNKVSLSSSYIDQYTPRGKQLHFSSISENALGINAATPHLKPPSQQARHREGScAce2 MRNTQNPTLRNANALASSSVLPPIPGSSNNTPIKNSLPQKHVFQHTPVKAPPKNGSNLAPCaAce2 EQPQPQPQPHQQQHQQ---QQHQPELQNTYQQIKHIQQQQQMLQHQFHNQNNQLRQQHPN

ScSwi5 VFNDLDPNVLTKNTDNEGDDNEENEPESR----------------FVISETPSPVLKSQSScAce2 LLNAPDLTDHQLEIKTPIRNNSHCEVESY----------------PQVPPVTHDIHKSPTCaAce2 QFQNQNQNQNQNQTKTPYSQQSQFSPTHSNFNLSPAKQLNSNVGSMHLSPLKKQLPNTPT

ScSwi5 KYEGRSPQFGTHIKEIN------TYTTNSPSKITRKLTTLPRGSIDKYVKEMP-DKTFECScAce2 LHS-------TSPLPDE------IIPRTTPMKITKKPTTLPPGTIDQYVKELP-DKLFECCaAce2 KQPPVTIEWSPVISPNSKQPLHKQIKESSPRRRIKKTSLLPPGELDNYWTGPDEDKIYTC

ScSwi5 LFPGCTKTFKRRYNIRSHIQTHLEDRPYSCDHPGCDKAFVRNHDLIRHKKSHQEKAY-ACScAce2 LYPNCNKVFKRRYNIRSHIQTHLQDRPYSCDFPGCTKAFVRNHDLIRHKISHNAKKY-ICCaAce2 TYKNCGKKFTRRYNVRSHIQTHLSDRPFGCQF--CPKRFVRQHDLNRHVKGHIEARYSKC

ScSwi5 PCGKKFNREDALVVHRSRMICSGGKKYENVVIKRSPRKRGRPRKDGTSSVSSSPIKENINScAce2 PCGKRFNREDALMVHRSRMICTGGKKLEHSINKKLTSPK---KSLLDSPHDTSPVKETIACaAce2 PCGKEFARLDALRKHQDRNICVGGN---KNVISKPTKKK------GTNNTQQQLLKTDTV

ScSwi5 KDHNGQLMFKLEDQLRRERSYDGNGTGIMVSPMKTNQR-----------ScAce2 RDKDGSVLMKMEEQLRDDMRKHGLLDPPPSTAAHEQNSNRTLSNETDALCaAce2 VERIEKQLLQEDKSVTEEFLMLQ--------------------------

972 M. T. Kelly et al.

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 969–983

transformed with the targeted lox-CaHIS1-lox cassette,and transformants were screened for histidine prototrophy.Homologous recombination results in deletion of theentire ORF of one CaACE2 allele, as confirmed by PCR.One of the heterozygotes (MK37) was then transformedwith the lox-CaURA3-lox cassette, and the transformantswere screened for both histidine and uridine prototrophy.PCR confirmed the correct integration of the second cas-sette and retention of the first cassette. The resultingstrain, MK62, is a Caace2 null mutant (Caace2::HIS1/Caace2::URA3). Using both approaches, a total of 11heterozygote knock-out strains were constructed.

Deleting CaACE2 affects cell separation and invasion

Deletion of a single CaACE2 allele had no effect on thegrowth phenotype, and the heterozygote strains wereindistinguishable from the wild type. The Caace2 nullmutants, however, showed a number of very distinctgrowth phenotypes. For the Caace2::FRT/Caace2::FRTmutant, these phenotypes were evident both before andafter excision of the MPAR cassette. The null mutant cellsfailed to separate following cell division, leading to theformation of cell clumps and flocculation during growth inliquid media even when shaken at 200 r.p.m. Treating the

Fig. 2. Construction of CaACE2 knock-outs.A. The flipper cassette shown on the top includes flanking FLP recombination targets (FRT), the promoter of the C. albicans SAP2 gene (PSAP2) driving expression of the CaFLP gene, the transcription termination sequence of the C. albicans ACT1 gene (ACTIT) and the gene encoding resistance to mycophenolic acid (MPAR). Restriction sites unique in the original cassette construct are indicated. Disruption constructs were generated by cloning CaACE2-specific regions on either side of the cassette. The extremities of the external construct are indicated by solid lines and the internal construct by dashed lines. The primers used to generate and test the constructs are indicated and listed in Table 2. The first CaACE2 allele was replaced with the external cassette and the second allele with the internal cassette. The regions between the FRT sites were subsequently removed by recombination as described in the text.B. The two CaACE2 alleles were amplified from SC5314 and digested with EcoRI (lane 2). The 2029 bp fragment represents the allele missing one EcoRI site. Lane 3 shows a similar amplification for the heterozygote strain MK89. Lane 1 contains a 1 kb size ladder.C. CaACE2 was also knocked out in C. albicans RM1000 by replacing one allele with a HIS1 cassette and the second with a URA3 cassette. The cassettes were amplified by PCR using identical primers homologous to regions surrounding the start and stop codons of CaACE2. The constructs were verified by PCR using the indicated primers.

CAF1 CAF2CAF3 CAF4

CAR1 CAR2CAR3 CAR4

FRT P CaFLP ACTIT MPA FRT

FLP1

P1 P2 P3 P5P4

CaACE2

A B

341 bp

946 bp1083 bp2029 bp

ApaI XhoI SacIISacI

SAP2

R

CaACE2

CaURA3

CA5CASS CA3CASS

loxP loxPURA3-1

CaHIS1

loxP loxPHIS1-2R

C

C. albicans CaAce2 knock-out affects morphogenesis 973

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 969–983

cells with Calcofluor white (to stain chitin) showed thatthey remained attached at the mother–daughter junction(Fig. 3). It was also clear that the null mutant formedpseudohyphae under conditions (YPD at 30∞C) that do notinduce filamentous growth in the wild-type strain and arecapable of producing true hyphae when induced withserum (Fig. 3). Null mutants generated by both knock-outapproaches had the same phenotype, while the heterozy-

gotes are indistinguishable from wild type (not shown).Disrupting ACE2 in S. cerevisiae also results in increasedpseudohyphal growth and altered invasion of agar media(King and Butler, 1998). We therefore tested the invasionproperties of the C. albicans Caace2 knock-outs. On solidagar at 30∞C, the null mutants formed clumped, raisedcolonies that were distinctly different from those seen forthe wild-type strain or a heterozygote mutant (Fig. 4).

YEAST HYPHAE

SC5314

MK106

Fig. 3. Deleting CaACE2 causes defects in cell separation and pseudohyphal growth. WT (SC5314; wild type) and Caace2 null (MK106; Caace2::FRT/Caace2::FRT) were grown in YPD (yeast) cells at 30∞C or YPD supple-mented with 10% FCS at 37∞C (hyphae) and the chitin stained by treatment with Calcofluor white.

Fig. 4. Deleting CaACE2 increases invasion of agar media. Equal volumes of overnight cultures from the wild-type strain SC5314 (WT), a heterozygote (HZ) MK89 (CaACE2/Caace2::FRT) and two Caace2 null mutants (MK106 Caace2::FRT/Caace2::FRT and MK62 Caace2::HIS1/Caace2::URA3) were spotted on to agar plates containing YPD or YPD plus 10% FCS and incubated at 30∞C or 37∞C for 4 days. Cells on the surface were subsequently removed by washing under running water.

SC5314 MK89 MK106 MK62

30∞C 37∞C

SC5314 MK89 MK106 MK62

YPD

YPD AFTER WASH

YPD + 10% FCS

YPD + 10% FCSAFTER WASH

WT HZCaace2 null

WT HZCaace2 null

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© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 969–983

When these plates were washed with sterile water, thecell mass left behind embedded in the agar by the nullmutants was consistently denser than that left behind byeither the wild-type or heterozygote strains (Fig. 4). Thiscould be seen as early as 24 h but, for illustration pur-poses, is shown after 4 days of growth. This enhancedinvasiveness may be linked to the ability of the Caace2null mutants to form pseudohyphae under all growth con-ditions tested. When grown under hypha-inducing condi-tions (YPD + 10% FCS at 37∞C), the colony morphologyof the wild-type and heterozygote strains was rougherthan at 30∞C, but the colony morphology of the Caace2null mutants was again raised, clumpy and distinctivecompared with the other strains (Fig. 4). When theseplates were washed, the outline of hyphae from all strainscould be seen, but again the cell mass left behind by theCaace2 null mutants was denser and so does not showthe same detail (Fig. 4).

CaACE2 affects biofilm formation

The cell washing experiments described above suggestthat deleting CaACE2 affects invasion of agar media. Wealso noted that, despite extensive flocculation in liquidmedia, neither Caace2 null mutant formed tight pelletsafter centrifugation. This suggested that the adherenceproperties of the strains may also be affected. We choseto examine the adherence to plastic surfaces, as this isan important step in the development of biofilms and maycontribute to the virulence properties of the organism(Kumamoto, 2002). As the cell separation defect made itdifficult to determine accurate absorbance measure-ments, we used dry weight measurements to calculateequivalent cell numbers for wild-type and knock-outstrains. Initially, equal numbers of wild-type andCaace2::FRT/Caace2::FRT null mutant cells were allowedto adhere to 96-well polystyrene plates for up to 2 h. Theextent of adhesion was determined using a tetrazolium(XTT) assay and measuring the absorbance at 490 nm.There was a dramatic difference in the ability of the wild-type and the homozygote Caace2 knockout strains toadhere (Fig. 5A), resulting in approximately twofold lessadherent mutant cells (P < 0.0001). When adherent cellswere incubated in fresh media for up to 24 h, biofilms wereformed by both mutant and wild type (Fig. 5B). In thisexperiment, the yeasts were left to adhere to the platesfor 60 min, after which non-adhered cells were washedaway. Biofilms were then allowed to develop over 24 h.The mass of the biofilms formed by the Caace2::FRT/Caace2::FRT mutant cells was lower than wild type (notshown), and microscopic examination showed that thestructures are very different (Fig. 5B). By 24 h, the wild-type biofilms were almost confluent on the base of thewell, whereas the mutant strains never became confluent

even at much later times. The mutant cells also hadmuch longer filaments (Fig. 5B). The Caace2::HIS1/Caace2::URA3 mutant phenotype is identical (not shown).

CaAce2p regulates expression of cell wall genes

The cell separation defect, the alteration in invasion andadhesion and the increased pseudohyphal growthdescribed here for the CaACE2 knock-out are similar tophenotypes of ACE2 knock-outs in S. cerevisiae (King andButler, 1998; O’Conallain et al., 1999). The ScAce2 pro-

Fig. 5. Deleting CaACE2 reduces adherence to polystyrene surfaces.A. Equal quantities of cells from WT (SC5314; wild type) and Caace2 null (MK106; Caace2::FRT/Caace2::FRT) were suspended in 100 ml of RPMI and incubated in 96-well plates for the times indicated. Non-adherent cells were then removed by washing, and adherent cells were measured using an XTT assay. Error bars show standard devi-ations of eight replicates of three independent cultures. All time points show a significant difference in adherence (P < 0.0001).B. WT (SC5314; wild type) and Caace2 null (MK106; Caace2::FRT/Caace2::FRT) cells were allowed to adhere for 1 h before washing and then incubated for 24 h to allow biofilm formation. Adherent cells were stained with crystal violet and photographed at 10¥ and 40¥ magnification using an inverted microscope.

WT Caace2 nullB

A

A49

0

Time (min)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

5 20 35 45 65 80 95 105 120

SC5314 (WT)MK106 (Caace2 null)

10X

40X

C. albicans CaAce2 knock-out affects morphogenesis 975

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 969–983

tein regulates the expression of chitinase and severalother cell wall genes that are expressed predominantly indaughter cells (Colman-Lerner et al., 2001; Doolin et al.,2001). We therefore determined the effect of CaACE2 onthe expression of chitinase and other homologous genesfrom C. albicans identified by BLAST analysis. The strainswere grown under yeast-inducing conditions (YPD at30∞C) or under hypha-inducing conditions (YPD + 10%FCS at 37∞C). Expression of CHT2, CHT3, CaSCW11(ORF 6.2346) and CaDSE1 (ORF 6.2710) was deter-mined using reverse transcription (RT)-PCR (Fig. 6).CHT1 expression was not detectable under either growthcondition, as reported previously (McCreath et al., 1995),and is not shown. Deleting CaACE2 had no effect onexpression of CHT2 under yeast- or hypha-inducing con-ditions (Fig. 6). Expression of CHT3, CaSCW11 andCaDSE1, however, is greatly reduced in both knock-outstrains in both conditions. Expression of CaDSE1 is higherin yeast-inducing conditions than in hypha-inducing con-ditions and is not completely abolished in the Caace2knock-outs (Fig. 6). CaACT1 expression was used to nor-malize all expression patterns.

CaAce2p is localized to daughter yeast cells

In S. cerevisiae, the Swi5 protein is localized to thenuclei of mother and daughter cells towards the end ofcell division (Moll et al., 1991), whereas Ace2p ispredominantly localized to the nuclei of daughter cells(Colman-Lerner et al., 2001; Weiss et al., 2002). Todetermine the localization of CaAce2p, we generatedstrain SCAY1 by inserting a green fluorescent protein(GFP) tag at the 3¢ end of one of the endogenousCaACE2 alleles in strain CAI-4. Cells were arrested in

early M phase with nocodazole arrest and then released.Samples were examined every 15 min until the popula-tion consisted of predominantly large budded cells with anucleus in each cell. CaAce2p-GFP was seen in ª20%of cells and, when present, the protein was found only inthe daughter cell nucleus (Fig. 7A). CaAce2p localizationwas also determined in cells exposed to FCS for120 min. Staining with DAPI confirmed that mitosis hadoccurred by this point, and nuclei are visible in both themother cell and the daughter hypha. CaAce2p is foundpredominantly in the hyphal nucleus (Fig. 7B). To ensurethat the GFP-tagged protein is functional, we alsotagged the single intact CaACE2 allele in the heterozy-gote strain MK67 (CaACE2/Caace2::HIS1). CaAce2p isalso localized to the daughter nuclei in this strain(MK127, Fig. 7C). MK127 has the same growth pheno-

Fig. 6. Analysis of expression of cell wall genes. Total RNA was isolated from cells grown in YPD at 30∞C (YEAST) or in YPD supple-mented with 10% FCS at 37∞C (HYPHAE). RNA (3 mg) from each strain, WT (SC5314; wild type), HZ (MK89; CaACE2/Caace2::FRT) and Caace2 null mutants (MK62; Caace2::HIS1/Caace2::URA3 and MK106; Caace2::FRT/Caace2::FRT), was used in separate RT-PCRs, using gene-specific oligonucleotides listed in Table 2. The expression of actin is shown as an internal control.

CHT3

SCW11

ACT1

DSE1

CHT2

YEAST HYPHAE

WT HZCaace2 null

WT HZCaace2 null

Fig. 7. Localization of CaAce2p. Cells expressing GFP-tagged CaAce2p were visualized using phase-contrast and fluorescence microscopy.A. SCAY1 (CaACE2/CaACE2-GFP::URA3) cells were taken from cul-tures 30 min after release from nocodazole arrest, when most cells had large buds and two nuclei. The cells were not fixed.B. CaAce2p is localized to the nuclei of SCAY1 cells, formed in response to induction with serum for 120 min.C. CaAce2p is also localized to daughter nuclei in strain MK127, in which the only intact allele of CaACE2 is tagged with GFP.D. Staining with Calcofluor white shows that tagging CaAce2p with GFP does not affect cell separation, even when no other intact CaACE2 is present (MK127). The growth phenotype of MK127 is identical to that of its parent, MK67.

GFPDAPI

A

B

C

D

MK127 MK67

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types as all other heterozygote strains including its par-ent MK67 (Fig. 7D), confirming that the GFP tag doesnot interfere with cell separation and is therefore likely tobe fully functional.

Animal models

To assess the virulence of the Caace2::FRT/Caace2::FRTmutant strain, female mice were injected intravenouslywith a challenge dose of each C. albicans strain at1.5 ¥ 103 cfu g-1 body weight via the lateral tail vein. Thewild-type and heterozygote strains caused fatality within5–7 days (Fig. 8A). In stark contrast, the animals infectedwith the homozygous mutant strain survived the full28 days of the experiment and were humanely terminated.On the day of death or termination, both kidneys and thebrain from each animal were removed, and the tissueburdens of C. albicans were determined. The tissueburdens consistently reflected the lower virulence ofthe Caace2/Caace2 mutant. The virulence of theCaace2::HIS1/Caace2::URA3 mutant strain wasassessed in a separate experiment. The knock-out wascompared with infection with C. albicans strain CAF2-1(URA3/ura3D::limm434) to avoid any difficulties causedby uracil auxotrophy. CAF2-1 caused fatality within 5–7 days, whereas the animals infected with the null mutantsurvived the full 28 days of the experiment (Fig. 8B). Thetissue burdens were determined in both kidneys of theseanimals and, once again, reflected the reduced virulenceof a Caace2 null mutant.

Discussion

We report here on the isolation and characterization ofthe CaACE2 gene. The predicted protein is equally relatedto both Ace2p and Swi5p from S. cerevisiae. As theseproteins arose from a genome duplication event thatoccurred after the split from the C. albicans lineage (Wolfeand Shields, 1997; Wong et al., 2002), it is not surprisingthat the C. albicans genome contains only one homolo-gous ORF. It is likely that this carries out the roles of boththe S. cerevisiae proteins. We have called this CaACE2because of the cell separation defects of the null mutants.

We chose the MPAR system to delete the ORF becausethe resulting Caace2 null mutant is otherwise isogenic tothe wild-type strain, except for the addition of one shortrecognition site for the FLP recombinase. Several studieshave shown that gene disruption using URA3 is associ-ated with phenotypes that reflect the expression of URA3rather than the deletion (Lay et al., 1998; Bain et al., 2001;Sundstrom et al., 2002; Cheng et al., 2003; Staab andSundstrom, 2003). However, to ensure that the pheno-types that we report are caused by deleting CaACE2 andare not artifacts, we also generated constructs in whichthe two CaACE2 alleles in C. albicans RM1000 werereplaced with functional URA3 and HIS1 genes. Fourhomozygous knock-outs were generated using thisapproach, and one was introduced into animals. All phe-notypes tested are identical to those described usingMPAR.

The mother and daughter cells of the Caace2 nullmutant strains fail to separate during growth in liquid

Fig. 8. CaACE2 affects virulence in a systemic animal model.A. Female DBA/2 mice were infected intravenously with SC5314 (wild type), MK89 (CaACE2/Caace2::FRT) and MK106 (Caace2::FRT/Caace2::FRT). Survival was determined over the time indicated. The post-death tissue burdens in brain and kidneys of infected animals are shown below the graph.B. In a separate experiment, mice were similarly infected with CAF2-1 (wild type) and MK62 (Caace2::HIS1/Caace2::URA3) cells. The associated post-death tissue burdens are shown below.

0 5 10 15 20 25

100

80

60

40

20

0

Days post challenge

% s

urvi

vors

SC5314MK89MK106

SC5314 (WT)

MK89 (HZ)

MK106 (null)

3.7±1.1

3.6±0.5

28.0±0.0

0.0

0.0

57.1

6.9±0.2

6.7±0.2

2.1±0.9

0.0

0.0

57.1

6.8±0.4

6.8±0.1

2.2±1.3

71.4

71.4

71.4

4.2±1.2

4.7±0.4

2.1±1.4

7

7

7

Strain N=Survival (days)

Left kidney Right kidney Brain

%neglog

CFU/g %neg %neglog

CFU/glog

CFU/g

A B

Strain N=Survival (days)

Left kidney Right kidney

%neglog

CFU/g %neglog

CFU/g

CAF2-1 (WT)

MK62 (null)

5

6

5.2±0.0

28.0±0.0

0.0

100.0

7.1±0.0 0.0

100.0

4.8±0.2

0 5 10 15 20 25 0

% s

urvi

vors

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CAF2-1

MK62

100

80

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20

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media or on solid agar, form pseudohyphae under condi-tions that do not induce filamentous growth in the wild-type strain and show enhanced invasion on solid agarcompared with the wild-type cells. These phenotypes arevery similar to those seen when ACE2 is deleted in S.cerevisiae and suggest that the resulting transcription fac-tor plays a similar regulatory role in both organisms.Ace2p is the major regulator of expression of CTS1 (chiti-nase) and other daughter cell-specific genes in S. cerevi-siae. We showed that, in C. albicans, expression of onechitinase gene (CHT2) is unaffected by deleting CaACE2.However, expression of CHT3 and CaSCW11 is almostcompletely abolished, and CaDSE1 expression is greatlyreduced in both Caace2::FRT/Caace2::FRT andCaace2::HIS1/Caace2::URA3 knock-outs under yeast- orhypha-inducing growth conditions. SCW11 from S. cere-visiae encodes a glucan 1,3-beta-glucosidase (Cappel-laro et al., 1998), while DSE1 is also predicted to play arole in the cell wall (Doolin et al., 2001). In Schizosaccha-romyces pombe, a homologue of ACE2 regulates theexpression of eng1, which encodes a beta-glucanase(Martin-Cuadrado et al., 2003). It is therefore very likelythat Ace2p functions by regulating cell wall metabolism inseveral different fungi.

The changes in pseudohyphal growth and the defectsin cell separation and invasion of agar seen in the nullmutants is also compatible with altered cell wall biosyn-thesis. This is apparent from the reduced ability of theCaace2 null mutant to adhere to polystyrene and themorphology of the resulting biofilms (Fig. 5). The mutantstrains have much longer hyphae and never reached con-fluence on the plates (Fig. 5). The ability of fungal patho-gens to adhere to surfaces and form biofilms has beenstrongly correlated with filamentation, virulence and resis-tance to antifungal agents (Chandra et al., 2001; Donlanand Costerton, 2002; Douglas, 2003). Changes in mor-phogenesis are probably required for stable biofilm forma-tion, as both yeast- and hypha-defective mutants haveabnormal biofilm architecture (Baillie and Douglas, 1999).Biofilms are also associated with changes in the expres-sion of both drug resistance (Ramage et al., 2002a) andcell wall genes (Chandra et al., 2001). However, the onlybiofilm regulator identified in C. albicans to date is Efg1p,which also controls filamentation and cell wall metabolism(Lewis et al., 2002; Ramage et al., 2002b). Our resultssuggest that CaAce2p also plays a role.

The effect of the Caace2 null mutants on adherenceand biofilm formation is consistent with our finding thatthey have reduced virulence and reduced burdens in theorgans of infected animals. The effect on virulence maybe related to the role of CaAce2p in pseudohyphal growth,as mutants that either fail to form hyphae or grow consti-tutively in hyphal forms are also avirulent in mouse models(Lo et al., 1997). Alternatively, the reduction in virulence

could be caused by changes in the cell wall, which affectthe adherence and interaction with host cells.

We have shown that a CaAce2p–GFP fusion is local-ized to the daughter nuclei only of cells at the end ofmitosis (Fig. 7). It is also found in the hyphal nucleus. Thisis similar to S. cerevisiae, where Ace2p enters the nucleiof yeast cells at the end of mitosis and rapidly disappearsfrom the mother cell nucleus (O’Conallain et al., 1999;Colman-Lerner et al., 2001; Weiss et al., 2002). Itremains only in the nuclei of daughter cells. Localizationof Ace2p is dependent on the activities of Cbk1 and othermembers of the RAM network (Colman-Lerner et al.,2001; Weiss et al., 2002; Nelson et al., 2003). It will beinteresting to determine the roles of homologues of thesegenes in the localization of CaAce2p, particularly as adisruption of the CaCBK1 gene results in decreasedexpression of two chitinase genes (McNemar and Fonzi,2002).

Disrupting CaACE2 abolishes virulence in a mouse model

This is a dramatically different phenotype from a disrup-tion of an ACE2 homologue in Candida glabrata, whichwe reported recently (Kamran et al., 2004). In C. glabrata,an ACE2 disruption results in hypervirulence associatedwith severe sepsis. It is highly likely that the differencebetween the C. albicans and the C. glabrata knock-outphenotypes is related to species-specific differences inthe targets of the transcription factors. CgAce2p regulatesexpression of the sole chitinase gene in C. glabrata,whereas CaAce2p regulates expression of only one of thethree chitinase genes in C. albicans. There are likely tobe other significant differences between the species, andwe are therefore currently identifying the downstream tar-gets of CaAce2p.

Experimental procedures

Strains and media

Candida albicans strains (Table 1) were grown routinely inYPD medium (1% yeast extract, 2% peptone, 2% glucose)at 30∞C and maintained on YPD agar (2% agar). Hyphalgrowth was induced at 37∞C in YPD medium or on YPD agarsupplemented with 10% FCS (Gibco). MPA-resistant strainswere selected and maintained on Synthetic Complete (SC)agar (0.67% yeast nitrogen base, 2% glucose, 0.075% allamino acids mix, 2% agar) at 30∞C with MPA at a finalconcentration of 10 mg ml-1. Excision of the MPA cassettewas achieved by growing resistant strains in YCB-BSA media(2.34% yeast carbon base, 0.4% bovine serum albumin,pH 4.0) and plating on SC agar supplemented with 0–15 mg ml-1 MPA at 30∞C. Uridine and histidine auxotrophswere grown on SD medium (0.67% yeast nitrogen base with-out amino acids, 2% glucose) supplemented with 0.008%uridine and/or 0.02% histidine, while prototrophic transfor-

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mants were selected on SD agar in the absence of one orboth supplements. Biofilms were grown in RPMI-1640 cellculture media (Gibco) supplemented with 2 mM L-glutamine(Gibco). Strains used for fluorescent microscopy were grownin SC media or YPD supplemented with 10% or 20% FCS asindicated.

Cloning of CaACE2

The oligonucleotides P1 and P4 (Table 2) amplify a 3113 bpproduct from SC5314 genomic DNA that includes the entireCaACE2 ORF plus 657 bp of upstream sequence and 104 bpof downstream sequence. This product was digested withHindIII and cloned into the vector CIp10 (Murad et al., 2000)also digested with HindIII. EcoRI, EcoRV and HindIII restric-tion digests were used to verify the orientation of the insertsin the vector. The Agowa Sequencing Service, Germany,sequenced a number of the clones.

Construction of CaACE2-targeted MPAR cassettes

The plasmid pSFI1, kindly provided as a gift by GerwaldKohler, University of California San Francisco, is an 8505 bpplasmid carrying the MPAR flipper cassette in the vectorpBluescript-II KS (Wirsching et al., 2000). The 5.6 kb cas-sette is flanked upstream by unique ApaI and XhoI restrictionsites and downstream by unique SacII and SacI restrictionsites, which were used to design two CaACE2-targeted ver-sions of the cassette. PCR primers (Table 2) were designedfrom the Stanford Candida Sequencing Project contig 6-2388, in which CaACE2 spans the bases 14416–12065. Theprimers CAF1, upstream of a naturally occurring ApaI site,and CAF2, including the start codon of CaACE2 and anengineered XhoI site, were used to PCR amplify a 392 bpproduct from SC5314 genomic DNA. This upstreamsequence product was digested and cloned into pSFI1digested with ApaI and XhoI to make plasmid pMTK1. Prim-ers CAR1, including two bases from the CaACE2 stop codonand an engineered SacII site, and CAR2, including an engi-neered SacI site, were used to PCR amplify a 275 bp productfrom SC5314 genomic DNA. This downstream sequenceproduct was digested and cloned into pMTK1 digested withSacII and SacI to make plasmid pMTK2. Plasmid pMTK2 wasdigested with ApaI and SacI to release the 6267 bp fragmentof the external cassette. Primers CAR3, which contains anengineered SacII site, and CAR4, which contains a SacI site

and a sequence around the seventh from last base ofCaACE2, were used to PCR amplify a 225 bp product fromSC5314 genomic DNA. This product was digested andcloned into pSFI1 digested with SacII and SacI to make

Table 1. C. albicans strains used in this study.

Strain Characteristics Reference

SC5314 Wild type Gillum et al. (1984)CAF2-1 URA3/ura3D::limm434 Fonzi and Irwin (1993)CAI-4 ura3::l imm434/ura3::limm434 Fonzi and Irwin (1993)RM1000 ura3D::limm434/ura3D::limm434 his1::hisG/his1::hisG) Negredo et al. (1997)MK89 As SC5314, but CaACE2/Caace2::FRT This studyMK106 As SC5314, but Caace2::FRT/Caace2::FRT This studyMK37 As RM1000, but CaACE2/Caace2::HIS1 This studyMK62 As RM1000, but Caace2::HIS1/Caace2::URA3 This studySCAY1 As CAI-4, but CaACE2-GFP::URA3 This studyMK67 As RM1000, but CaACE2/Caace2::HIS1 This studyMK127 As RM1000, but CaACE2-GFP::URA3/Caace2::HIS1 This study

Table 2. Oligonucleotide primers used for cloning CaACE2, verifica-tion of cassette constructs and RT-PCR analysis.

Primer name Primer sequence (5¢ to 3¢)

P1 ATATGGTAAGCTTCAGTTCGP2 TCACTAAATATGCATTGGAAAP3 GGCAGAAGTCTATTGCAACATP4 ATAGTATAAGCTTGAAAAGGP5 ATGGATGATGCTTCAGGATCAF1 TATATGGTGTAAATCTCACTGCAF2 TTTCCAATGCATCTCGAGTGAAGGCAF3 TTTCGAAAGGGCCCTCTTTCTTTCAF4 ATCATTGGCCTCGAGACTTAAAAACAR1 AGACTTCTGCCGCGGGTTGGTTTTCAR2 ATTTGTTGGGAGCTCCACAACACTCAR3 GCAAATGTCCGCGGGGTAAAGACAR4 CAACATTAAGAGCTCCTCAGTAACACE2F CAAGTATTTTATTTACCATTTTTTCTTTTTCTC

ATTTACTTTCCCAACTTTTGCACCTCCTTCACTAAATGGTAACGCCAGGGTTTTCCC

ACE2R ACTGAAATGCAATCTTCTCTCCCACTCGAGCAAACACTCTCTCCATGAAAAACCAACTTGTGGCAGAAGTAAACAGCTATGACCATGATT

CA5CASS TACAATCAATCCTAAAAAGGCA3CASS TATAATTTTGAAAAGGGTAAURA3-1 AATTCCTTATCGGATTTAGCHIS1-2 TTACACAAGACATATTCCACE2GFP-F GGTTGAGAGGATAGAAAAACAGTTGCTACAG

GAAGATAAGAGTGTTACTGAGGAGTTTTTAATGTTGCAAGGTGGTGGTTCTAAAGGTGAAGAATTAT

ACE2GFP-R ACTGAAATGCAATCTTCTCTCCCACTCGAGCAAACACTCTCTCCATGAAAAACCAACTTGTGGCAGAAGTTCTAGAAGGACCACCTTTGATTG

GFP TCACCATCTAATTCAACCCAACE5 TTCCATGCATTTATCACCACT1FWD GACGGTGAAGAAGTTGCTGCACT1REV CAAACCTAAATCAGCTGGTCCHT2FWD ATTAGCTGCTGCAGTTGTAGCHT2REV TGGGGCAACACAAGCATTTTCHT3FWD TGTTGCTGTTTATTGGGGACHT3REV ATACCGCCAAAATACTTGCTDSE1FWD AGTAGCAGAACTAGAAGTAATDSE1REV CAGAAATCTGTAAACGATTCFLP1 TTCCGTTATGTGTAATCATCCSCW11FWD GTTCAATACCGTATGGACCTGSCW11REV ATTGTGTACTCACCATATGC

Sequences derived from cassettes are underlined.

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plasmid pMTK3. Primers CAF3, which contains an engi-neered ApaI site and is aligned with the 22nd base ofCaACE2, and CAF4, which contains an engineered XhoI site,were used to PCR amplify a 245 bp product from SC5314genomic DNA. This product was digested and cloned intopMTK3 digested with ApaI and XhoI to make plasmid pMTK4.Plasmid pMTK4 was digested with ApaI and SacI to releasethe 6072 bp fragment of the internal cassette.

Transformation of CaACE2-targeted MPAR cassettes

Strains were transformed by electroporation (Staib et al.,2001). Cells grown overnight in YPD were diluted to A600 of0.2 in 50 ml of fresh YPD and grown to A600 of 1.6–2.0. Cellswere harvested by centrifugation, resuspended in 10 ml ofTE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) containing100 mM lithium acetate and incubated at 30∞C for 1 h withshaking at 150 r.p.m. Dithiothreitol was added to a final con-centration of 25 mM, and the cells were incubated for afurther 30 min. Water (40 ml) was then added to the cellsuspension, and the cells were harvested by centrifugationand then washed sequentially in 50 ml of ice-cold water and10 ml of ice-cold 1 M sorbitol. Finally, the cells were centri-fuged and resuspended in 500 ml of 1 M sorbitol and kept onice. The plasmids pMTK2 or pMTK4 were digested asdescribed above to release their respective targeted cas-sette, and the DNA for transformation was purified from a 1%agarose gel using the Concert rapid gel extraction system(Gibco). Competent cells (100 ml) were transformed with 1 mgof DNA by electroporation in an Invitrogen electroporator II(1500 V, 50 mF, 150 W). Cells were plated on SC agar with10 mg ml-1 MPA at 30∞C for 5–8 days. Transformants wererestreaked on to the same media to verify stability. GenomicDNA was then isolated from a number of individual MPA-resistant colonies, and integration of the cassette was verifiedby PCR.

Excision of CaACE2-targeted MPAR cassettes

MPA-resistant colonies were grown for 48 h in YCB-BSAmedia at 30∞C to induce the SAP2 promoter and then diluted1:1000 and plated on SC agar supplemented with 0–15 mg ml-1 MPA at 30∞C. After 4–5 days, two colony sizeswere seen, and the smaller colonies, usually from the 1–3 mg ml-1 MPA plates, were restreaked on SC agar with10 mg ml-1 MPA and YPD agar to verify MPA sensitivity.Genomic DNA was then isolated from a number of individualMPA-sensitive colonies, and excision of the cassette wasverified by PCR.

PCR verification of MPAR cassette integration and excision

A combination of five primers (Table 2) was used to verify theintegration or excision of the cassettes in the genome. P2and P3 amplify a 2370 bp product from a SC5314 genomictemplate. EcoRI digestion of this CaACE2 PCR productreveals the presence of both alleles with a digestion patternof 341 bp and 2029 bp for one allele, and a pattern of 341 bp,946 bp and 1083 bp for the other allele. This PCR amplifica-tion followed by an EcoRI digest was used to verify the

presence of two (wild-type SC5314), one (heterozygoteknock-out) or no (homozygote knock-out) CaACE2 allelesrespectively (Fig. 2C). Integration of a targeted cassettewithin a CaACE2 allele was verified using primer P1, whichbinds upstream of the start codon of CaACE2, and primerFLP1, which binds within the FLP1 gene of the MPAR cas-sette, yielding a 1.9 kb product. Subsequent excision of acassette means that these two primers can no longer amplifya product. A positive control for excision used the primers P1and P5, which binds downstream of CaACE2 beyond thesites of cassette integration/excision. These primers amplifya 3356 bp product in the presence of a wild-type CaACE2allele, a 6589 bp product when a cassette is integrated intoan allele and a 989 bp product after excision of a cassette.All the oligonucleotides used in this study were designed forthis study with the exception of FLP1, which was a gift fromGary Moran of Trinity College Dublin, and the ACT1FWD andACT1REV oligonucleotides taken from Li et al. (2002).

Construction and integration of CaACE2-targeted HIS1 and URA3 cassettes

The plasmids pLHL2 and pLUL2 were kindly provided by PaulDennison, University of Aberdeen. This pair of pRS315-based plasmids contains lox-CaHIS1-lox and lox-CaURA3-lox gene disruption cassettes respectively (Fig. 2C). The cas-settes were targeted for integration at the CaACE2 ORF byPCR amplification using the primers ACE2F and ACE2R,which each contain 70 bp of sequence directly flanking thestart and stop codons of CaACE2 and 20 bp of sequencefrom the pRS315 plasmid backbone immediately flanking thecassettes (Table 2). The PCR product amplified from plasmidpLHL2 was transformed into lithium acetate-treated RM1000cells. Transformants were selected on SD agar supplementedwith uridine only. Integration of the cassette at the correctposition was verified by PCR. Primers CA5CASS andCA3CASS amplify a pair of 2552 bp products containing theCaACE2 alleles. Integration of the HIS1 cassette at oneCaACE2 allele reduces the size of one of the products to1934 bp. In addition, successful amplification of a productusing CA5CASS in combination with primer HIS1-2 con-firmed the location of the cassette. One of the resultingCaACE2/Caace2 heterozygotes (MK37) was transformedwith the PCR product amplified from plasmid pLUL2. Trans-formants were selected on SD medium in the absence of anysupplements. CA5CASS and CA3CASS amplified 1934 bpand 1828 bp products from the resulting Caace2/Caace2homozygote (MK62) and no 2552 bp products. Location ofthe second cassette was confirmed by successful amplifica-tion of a product using CA5CASS and the primer URA3-1.

Fluorescence microscopy

The CaAce2p was tagged with GFP at the C-terminus usingthe method of Gerami-Nejad et al. (2001). The GFPsequence was isolated by PCR from plasmid pGFP-URA3provided by Dr J. Berman, using oligonucleotidesACE2GFP-F and ACE2GFP-R. The PCR product was thenintroduced into lithium acetate-treated C. albicans CAI-4.One construct (called SCAY1) containing one tagged copy

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of the CaACE2 ORF was confirmed by PCR, using an oligofrom within GFP and oligo CAACE5 from within CaACE2.In the same way, a GFP tag was introduced into theremaining intact CaACE2 allele of the heterozygote strainMK67 to generate strain MK127 (CaACE2-GFP::URA3/Caace2::HIS1). To observe GFP fluorescence, cells weregrown in SC media for yeast-inducing conditions, washedcopiously with 1¥ PBS and resuspended in an appropriatevolume of 1¥ PBS. Logarithmically grown cells werearrested by treatment with a final concentration of 50 mMnocodazole in 1% DMSO for 2 h at 30∞C, filtered throughcellulose nitrate and washed with PBS. Cells were intro-duced into fresh SC media and grown for an additional30 min after release. Cells were not fixed before microscopy.Nuclei were stained by the addition of 1 mg of DAPI ml-1 cellsuspension, allowed to incubate at room temperature for10 min and then mounted. An aliquot of 4 ml of the cell sus-pension was mounted on glass slides in the presence of 5 mlof antifade mounting solution (Vectashield; Vector Laborato-ries). Slides were stored at 4∞C. Phase-contrast microscopyand epifluorescence microscopy were performed using anOlympus BX60 fluorescence microscope, and images werecollected using an F-View 2 digital camera from Soft Imag-ing Systems. To study hyphal growth, cells were grown over-night in SC media at 30∞C. Approximately 1 ¥ 104 cells wereplaced on polylysine slides and allowed to adhere for 20 minat room temperature. Unadhered cells were removed byaspiration, and the slides were submerged in a sterile Petridish containing SC media supplemented with 20% FCS for120 min at 37∞C. The slides were then washed several timeswith 1¥ PBS, followed by a final wash in distilled water andleft to air dry in the dark. A sample of 10 ml of 1 mg ml-1

DAPI was added to each slide and allowed to air dry. Theslides were viewed in the presence of Vectashield asdescribed above. To observe chitin localization, cells werestained with Calcofluor white as described previously (Lordet al., 2002). Briefly, cells were grown overnight in YPD at30∞C, washed twice in 1¥ PBS, resuspended in 100 ml of1 mg ml-1 Calcofluor white solution and incubated for 5 minat room temperature. Cells were washed again twice with 1¥PBS and finally resuspended in 100 ml of 1¥ PBS andmounted on polylysine-coated slides for microscopicexamination.

Dry weight measurements

Cells grown overnight in YPD at 30∞C were diluted to an A600

of 0.2 into fresh 50 ml of YPD and grown for 6 h at 30∞C withshaking. Each culture (30 ml) was filtered through 0.45 mmGN-6 Metricel Grid filters (PALL Gelman Sciences) that werepredried at 37∞C for 241/2 h and preweighed. The filters werethen dried at 37∞C for 24 h, and the dry weight was calculatedfor each strain. Each assay was performed in triplicate.

Biofilm formation

Cells were grown overnight in YPD at 30∞C. Dry weightassays were used to equalize cell numbers between SC5314and the MK106 homozygote Caace2 knock-out culture. Thediluted cultures were washed twice in PBS and then resus-

pended in the same volume of RPMI 1640 supplementedwith L-glutamine. Biofilms were grown in 96-well polystyreneplates (Nalge Nunc International) in a method adapted fromRamage et al. (2001). Cell suspension (100 ml) was added toeach well and incubated at 37∞C for up to 2 h. At each timepoint, the RPMI media and any non-adherent cells wereremoved, and the cultures were washed twice with 100 mlof PBS. Biofilm formation was measured using a semi-quantitative XTT reduction assay. XTT [2,3-bis(2-methoxy-4-nitro-5-sulpho-phenyl)-2H-tetrazolium-5-carboxanilide;Sigma] was dissolved in PBS at 0.5 g l-1, filter sterilized andstored at -70∞C. For each assay, the XTT solution wasthawed and supplemented with a final concentration of 1 mMmenadione (10 mM stock dissolved in acetone; Sigma). analiquot of 100 ml of the XTT-menadione solution was addedper well, and the plates were incubated in the dark at 37∞Cfor 2 h. A sample (50 ml) was then transferred from each wellinto a fresh 96-well plate (to eliminate interference of cellswith colorimetric readings), and the colorimetric changeresulting from XTT reduction was measured at 490 nm(Ramage et al., 2001; Kuhn et al., 2002). All biofilm cultureswere grown in triplicate, and each assay was performedeight times. For longer incubations, the cells were firstallowed to adhere for 1 h, the wells were washed twice with100 ml of fresh growth medium to remove non-adhered cells,and then 100 ml of fresh growth medium was added to thewells, and the plates were returned to the incubator. Forphotographs, the biofilms were stained with crystal violet(Reynolds and Fink, 2001).

RNA isolation and RT-PCR

Strains were cultured overnight in YPD medium, diluted to anA600 of 0.2 in 50 ml of YPD at 30∞C or in YPD supplementedwith 10% FCS at 37∞C and grown for 4 h with shaking. TotalRNA was extracted immediately and treated with amplifica-tion-grade DNase I (Invitrogen) to remove genomic contam-ination, which was verified by PCR. Treated RNA (3 mg) wasincubated with 291 ng of oligo dT-15 primer (Promega) in afinal volume of 15 ml with diethyl pyrocarbonate (DEPC)-treated H2O for 10 min at 70∞C and then chilled on ice. A45 ml cocktail containing 0.6 mM dNTPs, 120 units of RNasinribonuclease inhibitor (Promega), 600 units of Moloneymurine leukaemia virus reverse transcriptase (M-MLV RT;Promega) and 1¥ Promega RT-PCR reaction buffer was thenadded. The reaction was incubated for 1 h at 37∞C followedby 2 min at 95∞C, and the cDNA was stored at 4∞C. For eachindependent PCR, 5 ml of the cDNA pool was used as tem-plate in a 50 ml reaction mixture containing 0.25 mM dNTPs,1¥ reaction buffer supplemented with 1.5 mM MgCl2, 25 pmolof each PCR primer and 2.5 units of Taq DNA polymerase(Invitrogen). The PCR regime used included an initial 1.5 minhot start at 94∞C, a 30 cycle programme with a 35 s denatur-ation step at 94∞C, a 2 min annealing step at 55∞C and a2 min elongation step at 72∞C, followed by a single 7 minelongation step at 72∞C. PCR products were electrophoresedin a 1% agarose gel and then stained with ethidium bromide.The products were visualized under UV light and photo-graphed using GRAB IT software. All PCRs were tested atvarying cycle numbers to verify that 30 was within the linearrange of amplification.

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Virulence study

The experiments were carried out at the University of Aber-deen and were approved by the local ethics committee anddone under the terms of the UK Home Office licences forresearch on animals. Female DBA/2 mice (Harlan) with aweight range from 17 to 23 g were maintained under condi-tions specified by the Heath and Safety Executive for level 2biohazard containment. The animals were supplied with foodand water ad libitum. C. albicans isolates were grown for 24 hat 30∞C in NGY medium, comprising 0.1% neopeptone(Difco), 0.4% glucose and 0.1% yeast extract. Cultures wereincubated with gyratory shaking at 200 r.p.m. The cultures ofthe mutants were briefly sonicated to disperse clumps. Forstrains SC5314, MK89 and MK106, cell concentrations weredetermined by spectrophotometry and adjusted to suitablelevels in physiological saline. For CAF2-1 and MK62, ATPassays were used. Samples of inoculum suspensions wereplated on Sabouraud agar to confirm the accuracy of thechallenge dose. Mice were infected intravenously with a chal-lenge dose of each strain at 1.5 ¥ 103 cfu g-1 body weight viathe lateral tail vein. The body weight of each mouse wasrecorded daily, and the condition of each mouse wasassessed twice daily. The day of death of each animal wasrecorded. Animals that became incapable of reaching thefood or drinking fluid were humanely terminated andrecorded as having died on the following day. The experimentwas terminated 28 days after C. albicans challenge. On theday of death or termination, both kidneys from each animalwere removed with full aseptic precautions, homogenized in1 ml of water with an Ultra-Turrax apparatus, and tissue bur-dens of C. albicans were determined by viable counting with100 ml samples from a 10-fold dilution series. Tissue burdensin the brain of SC5314-, MK89- and MK106-infected micewere also determined.

Acknowledgements

We are grateful to Paul Dennison of the University of Aber-deen for providing the HIS1 and URA3 cassettes. We thankGerwald Kohler of the University of California San Franciscoand Gary Moran of Trinity College Dublin for providing plas-mid pSFI1and their advice on the MPA flipper system, andDr Judith Berman for the GFP plasmids. Thank you to MrSean Hogan, Veterinary Microbiology and ParasitologyDepartment, UCD, Dr Peter Sudbery, Sheffield, and Dr Car-oline Barelle, Aberdeen, for their assistance with microscopy.This work was supported by the Health Research Board,including a post-doctoral fellowship awarded to M. T. Kelly.Sequence data for C. albicans were obtained from the Stan-ford Genome Technology Centre website at http://www-sequence.stanford.edu/group/Candida. The BOXSHADE facilitywas used online at the Biology Workbench at the Universityof California, San Diego, at http://workbench.sdsc.edu.

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