In vitro expression of Candida albicans alcohol dehydrogenase genes involved in

acetaldehyde metabolism

Bakri MM1, Rich AM2, Cannon RD2, Holmes AR2

1Department of Oral Biology, Faculty of Dentistry, University of Malaya, Kuala Lumpur,

Malaysia; 2Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago,

Dunedin, New Zealand

Running title: Candida albicans alcohol dehydrogenase genes

Keywords: Candida albicans, alcohol dehydrogenase, acetaldehyde

Content category: Genes and genome

Word count: 5863

Number of Tables: 3

Number of Figures: 4

Supplementary materials: 2 Tables and 2 Figures

Corresponding author e-mail: marinab@um.edu.my

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SUMMARY

Alcohol consumption is a risk factor for oral cancer, possibly via its conversion to acetaldehyde,

a known carcinogen. The oral commensal yeast Candida albicans may be one of the agents

responsible for this conversion intra-orally. The alcohol dehydrogenase (Adh) family of enzymes

are involved in acetaldehyde metabolism in yeast but, for C. albicans, it is not known which

family member is responsible for the conversion of ethanol to acetaldehyde. In this study we

determined the expression of mRNAs from three C. albicans Adh genes (CaADH1, CaADH2 and

CaCDH3) for cells grown in different culture media at different growth phases by Northern blot

analysis and qRT-PCR. CaADH1 was constitutively expressed under all growth conditions but

there was differential expression of CaADH2. CaADH3 expression was not detected. To

investigate whether CaAdh1p or CaAdh2p can contribute to alcohol catabolism in C. albicans,

each gene from the reference strain C. albicans SC5314 was expressed in Saccharomyces

cerevisiae. Cell extracts from an CaAdh1p-expressing S. cerevisiae recombinant, but not an

CaAdh2p-expressing recombinant, or an empty vector control strain, possessed ethanol utilising

Adh activity above endogenous S. cerevisiae activity. Furthermore, expression of C. albicans

Adh1p in a recombinant S. cerevisiae strain in which the endogenous ScADH2 gene (known to

convert ethanol to acetaldehyde in this yeast) had been deleted, conferred an NAD-dependant

ethanol utilising, and thus acetaldehyde producing, Adh activity. We conclude that CaAdh1p is

the enzyme responsible for ethanol utilisation under in vitro growth conditions, and may

contribute to the intra-oral production of acetaldehyde.

INTRODUCTION

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Tobacco use and alcohol consumption are risk factors associated with oral cancer (Franceschi &

La Vecchia, 1990, Poschl & Seitz, 2004, Pelucchi et al., 2008). Alcohol may contribute to oral

cancer via its conversion to acetaldehyde, a known carcinogen, which is also a product of

Candida albicans metabolism (Collings et al., 1991, Marttila et al., 2013a). The adverse effects

of acetaldehyde have been shown in many cell culture studies as well as in animal models

(Poschl & Seitz, 2004, Brooks & Theruvathu, 2005, Seitz & Stickel, 2009). Attributes of C.

albicans that may influence oral cancer development have been reviewed (Sitheeque &

Samaranayake, 2003, Meurman & Uittamo, 2008, Mohd Bakri et al., 2010) and the production of

acetaldehyde by C. albicans has been implicated as one of the possible mechanisms involved

(Tillonen et al., 1999, Gainza-Cirauqui et al., 2013, Marttila et al., 2013b). Acetaldehye

accumulation may reflect both expression of the producing enzymes (pyruvate decarboxylase

(Pdc11p) and the alcohol dehydrogenase (Adh), family of enzymes and repression of the

catabolising enzymes (aldehyde dehydrogenase (Ald6p) and acetyl-CoA synthetases (Acs1p and

Acs2p)) (Marttila et al., 2013a). In the model yeast Saccharomyces cerevisiae, Adh2p expression

is known to contribute directly to acetaldehyde production from ethanol (Ciriacy, 1975a, 1975b,

Denis et al., 1981). Although there are five genes that encode alcohol dehydrogenases in this

yeast, four (ScAdh1p, ScAdh3p, ScAdh4p, and ScAdh5p), reduce acetaldehyde to ethanol during

glucose fermentation, with Adh2p being the sole acetaldehyde-producing enzyme (Thomson et

al., 2005). In contrast, pathways of acetaldehyde metabolism in C. albicans have not been

elucidated, and although seven members of the ADH gene family have been identified in the C.

albicans genome (http://www.candidagenome.org/), only three (ADH1-3) are thought to encode

functional proteins (Swoboda et al., 1994, Bertram et al., 1996, Lan et al., 2002, Kusch et al.,

2008). It is important to identify the genes involved in acetaldehyde production in order to

determine whether there is a link between their expression and oral cancer progression in vivo.

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We hypothesised that production of acetaldehyde by C. albicans and, by inference, expression of

the ADH genes responsible for its production, varies depending on environmental conditions in

the host. Previous studies have reported that the expression of CaADH1 mRNA is regulated by

the carbon source and also varies according to growth phase (Swoboda et al., 1994, Bertram et

al., 1996). More recently, it has been reported that CaAdh2p has been detected only in the

stationary phase of cells grown in YPD medium (Kusch et al., 2008).

In this study, the expression of the three CaADH genes was measured at different growth phases

in different media representing minimal and replete nutritional conditions. Furthermore, in order

to determine whether CaAdh1p or CaAdh2p contributed to alcohol catabolism in C. albicans,

each gene was expressed in S. cerevisiae. Recombinant protein production was monitored by

immunoblotting, and Adh enzyme activity by following ethanol utilisation in cell extracts. The

background ethanol utilising activity in the host S. cerevisiae strain was eliminated by deletion of

the ScADH2 gene.

METHODS

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Strains, media and culture conditions

C. albicans strains SC5314 (Jones et al., 2004) and ATCC 10261 (American Type Culture

Collection, Manassas, VA, USA) were used in this study. C. albicans strains were maintained on

yeast extract peptone (YPD) agar, which contained (per litre): 10 g yeast extract, 20 g Bacto

peptone (Becton Dickinson, Sparks, MD, USA), 20 g glucose and 20 g agar. Cultures of C.

albicans cells were grown at 30°C in liquid cultures of three media with shaking (250 rpm):

YPD, yeast nitrogen base without amino acids (YNB; (Becton Dickinson, Auckland, New

Zealand) or glucose salts biotin (GSB). YNB medium contained (per litre): 6.7 g YNB and 20 g

glucose. GSB contained (per liter): 1 g (NH4)2SO4, 2 g KH2PO4, 50 mg MgSO4·7H2O, 50 mg

CaCl2·2H2O (0.34 mM), 0.05 mg biotin, and 20 g glucose (Holmes & Shepherd, 1988).

All S. cerevisiae strains created in this study were based on AD1-8u− (Decottignies et al., 1998,

Nakamura et al., 2001) Strain ADΔ is identical to strain AD1-8u− except that the entire

chromosomal URA3 locus, which corresponds to the URA3 marker of plasmid pABC3 (Lamping

et al., 2007), was deleted by replacing the ura3 gene of strain AD1-8u− with the 422-bp repeat

region of the CaURA3 blaster cassette (Wilson et al., 2000). Yeast transformants were selected on

complete synthetic medium without uracil (CSM-ura) plates which contained (per litre): 6.7 g

YNB, 0.77 g CSM-ura (Bio 101, California, USA.) 20 g glucose and 20 g agar. Plasmids were

maintained in Escherichia coli strain DH5α. E. coli cells were grown in Luria-Bertani medium

which contained (per litre): 5 g yeast extract, 10 g Bacto peptone, 10 g NaCl, pH 7.4 and

ampicillin (100 µg/ml).

RNA isolation

Total RNA was isolated from cell suspensions (107 cells/ml) of early-exponential, mid-

exponential and late-exponential phase C. albicans. Time points for each growth phase in the

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different media were established from triplicate growth curve experiemnts and were as follows

for GSB, YNB and YPD respectively: early exponential sample time-points were 12 h, 4 h and 4

h; mid-exponential sample time-points were 18 h, 7 h and 7 h, and stationary phase sample time-

points were 30 h, 30 h and 24 h. Cells were harvested by centrifugation (2,000 x g, 5 min), and

the cell pellet was stored at -80°C until ready for use. RNA was extracted by the hot phenol

method as previously described (Schmitt et al., 1990). RNA samples (5 mg) were treated to

remove contaminating DNA using a DNAfreeTM kit (Ambion, Austin, TX) in a total volume of 10

ml.

Northern blot hybridizations

RNA samples (20 - 30 µg) were denatured using formamide and formaldehyde at 55ºC for 15

min followed by quenching on ice. Samples were electrophoresed on a denaturing agarose gel

before vacuum blotting (TransVacTM, Hoefer, Holliston, MA) onto Hybond-™XL nylon

membrane (GE Healthcare, Little Chalfont, UK). The membranes were hybridized for 1 h at

65°C in Church and Gilbert (Church & Gilbert, 1984) solution with DNA probes that had been

labelled with α-³²P-dCTP using the RadPrime DNA Labeling System (Invitrogen, Carlsbad, CA).

Probes were PCR products generated from a template of C. albicans ATCC 10261 genomic

DNA. Primers used to generate the PCR products (Table 1) were based on highly divergent N-

terminal or 5’ sequences such that the probes generated were specific for each ADH mRNA with

minimal similarity between the products. An alignment of the probes generated for each C.

albicans ADH gene with each other gene (from C. albicans SC5314) is shown in Fig. S1. The

ADH1 probe was completely unique. There was a 75% similarity between the probe for ADH2

and the ADH1 gene, and a greater mis-match between the probe for ADH3 and the ADH1 (54%

similarity) and ADH2 (49% similarity) genes. Although the ADH2 reverse primer was very

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similar to the ADH1 sequence, there was a mismatch at the 3’ end and a large mismatch between

the ADH2 forward primer and the cognate region of ADH1. Therefore amplification of ADH1

sequence with the ADH2 primer pair was considered as unlikely at the annealing temperatures

used for PCR generation of the probes. Autoradiography was carried out at -80°C for 1–7 days

using BioMax Film (Kodak).

RT-PCR and qRT-PCR

RNA samples (2 ml) treated as described above to remove contaminating DNA were used as

templates for cDNA production using oligo dT (Invitrogen) and random primers (Applied

Biosystems). For some experiments unique primers were used (Beggs et al., 2004) but these were

not employed in the comparison of ADH gene expression by qRT-PCR, as described below. RT-

PCRs were carried out using the primers listed in Table 1 and reverse transcriptase (SuperScript

III; Invitrogen, Life Technologies, Auckland) in total volumes of 20 µl. RT negative controls

were prepared in the same way but without the Superscript III enzyme. Quantitative real-time

PCR (qPCR) was performed using the 7500 Fast Real-Time PCR system (Applied Biosystems,

Life Technologies, Auckland). The qPCR reactions were undertaken using Fast SYBR Green

Master Mix (Applied Biosystems) in 20 l volumes. Thermal cycling was performed with an

initial enzyme activation step at 95°C for 20 s, followed by 40 cycles of denaturation at 95°C for

3 s, annealing at 60°C for 15 s and extension at 60°C for 15 s.

Expression of C. albicans ADH1 and ADH2 genes in S. cerevisiae

The C. albicans ADH1 and ADH2 genes were cloned using overlap extension PCR into S.

cerevisiae strain ADΔ as described previously (Lamping et al., 2013). Briefly, genomic DNA

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was isolated from C. albicans strain SC5314 using a Y-DER kit (Pierce, Rockford, IL) and used

as a template for amplification of the C. albicans ADH1 and ADH2 ORFs using N-terminal and

C-terminal primers and the high-fidelity KOD+ DNA polymerase (Novagen, San Diego, CA).

The primers contained sequences that were overlapped with the target region (the PDR5 locus) of

the host strain genome, allowing the generation of cloning cassettes by overlap extension PCR

(Lamping et al., 2013). For ADH1 expression, the primers were based on the upstream ATG as

reported for the immunogenic alcohol dehydrogenase within sequence NW_139432.1 encoded by

the GenBank sequences XP_721905.1 (strain SC5314) and EEQ46516.1 (WO strain). The ORF

included an N-terminal extension relative to the ADH1 gene annotated in Candida genome

database (CGD) and encodes a polypeptide of 434 amino acids with a predicted mass of 46.2

kDa. Although the CaADH1 transcription start site has been mapped to be 5’ to the shorter ORF

encoding 349 amino acids with an expected mass of 36.8 kDa (Bertram et al., 1996), we felt that

there may also be alternative expression of the longer ORF. Indeed it has been shown in a 2-D gel

electrophoresis analysis of whole cell extracts from the database strain C. albicans SC5314

(Kusch et al., 2008) that Adh1p was 46 kDa in size whereas Adh2p was found to resolve at 37

kDa. Therefore we cloned the longer ORF and it was functional when expressed in S. cerevisiae.

For ADH2 expression, the primers were based on the sequence XM_712482.1. Transformants of

the host strain ADΔ were selected on CSM–Ura agar plates by incubation at 30°C for 48-72 h. In

each experiment, 24 transformants were selected and genomic DNA was extracted for PCR and

sequencing to check for the correct integration of the complete transformation cassette at the

chromosomal PDR5 locus. DNA samples for sequencing were sent to the Micromon DNA

Sequencing Facility (Monash University, Melbourne, Australia).

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For certain experiments, the S. cerevisiae ADH2 gene was disrupted using a recyclable URA3

cassette (Wilson et al., 2000). The first step was to create a S. cerevisiae strain expressing C.

albicans Adh1p using a cassette that contained the HIS1 marker (pABC5) (Lamping et al., 2007).

The strain created was denoted ADΔ/CaADH1(pABC5). The pABC5 cassette is similar to the

pABC3 cassette except that pABC5 has the HIS1 marker instead of the URA3 marker (Lamping

et al., 2007). The methods used for the insertion of CaADH1(pABC5) into the S. cerevisiae

PDR5 locus of the host strain ADΔ were similar to those described above for cloning CaADH1

and CaADH2. Use of the HIS1 marker allowed the recyclable URA3 cassette (Wilson et al.,

2000) to be used for disruption of ScADH2 in this strain (Fig. S2) to create ADΔ

/CaADH1(pABC5)/ScADH2.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblot

analysis and chemiluminescence detection

Proteins were separated on 8% gels by SDS-PAGE (Laemmli, 1970) and stained with Coomasie

blue. HybondTMECLTM nitrocellulose membranes (GE Healthcare) were used for Western transfer

(Towbin et al., 1979). Immunodetection using chemiluminescent detection was performed as

described proviously (Holmes et al., 2008). Primary antibodies used included mouse monoclonal

anti-polyhistidine antibodies (Sigma) at a 1:1,000 dilution or rabbit polyclonal anti-S. cerevisiae

Adh1p antibodies (Abcam, Cambridge, UK) at a 1:20,000 dilution. Appropriate horseradish

peroxidase (HRP)-linked secondary antibodies were obtained from Dako, Glostrup, Denmark.

Preparation of yeast cell extracts

Yeast cells were disrupted as described previously (Niimi et al., 2004). Following differential

centifugation to remove cell debris and glass beads, the supernatant fraction was recovered for

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further centrifugation at 30,000 x g at 4°C for 45 min. His-tagged proteins were purified from S.

cerevisiae culture supernatants by nickel column chromatography, using nickel-nitrilotriacetic

acid (Ni-NTA) agarose (Qiagen, Germantown, MD).

Alcohol dehydrogenase enzyme assay

Alcohol dehydrogenase enzyme activity was measured by following the production of NADH

spectrophotometrically, adapting a previously described method (Crichton & Moore, 2007).

Assays were carried out using ethanol as a substrate in a final volume of 1 ml, containing 100 µl

250 mM Tris-HCl (pH 8.5), 20 µl of 100 mM β-nicotinamide adenine dinucleotide (NAD+),

enzyme extract (approximately 100 μg protein) and 20 µl of 17 mM ethanol. The reaction was

started by adding ethanol and the reduction of NAD+ was measured at 340 nm using a Shimadzu

UV-240 spectrophotometer (Shimadzu Co., Tokyo, Japan). One unit of enzyme activity was

defined as the amount required to form 1 µmol of NAD+ (NADH) per min, respectively. Specific

Adh activity was expressed as units (U) per mg of protein. All assays were carried out in

triplicate and the Student’s t-test was used to compare the Adh enzyme activities. A P value of <

0.05 was considered statistically significant.

Bioinformatic analysis

All sequence data for C. albicans and S. cerevisiae were obtained from the CGD

(http://www.candidagenome.org/) and Saccharomyces Genome Database (SGD)

(http://www.yeastgenome.org/). Analysis of nucleic acid and protein sequences was carried out

using on-line software such as the ClustalW2 web-based alignment tool

(http://www.ebi.ac.uk/Tools/clustalw2/index.html). DNA and amino acid homology between C.

albicans and S. cerevisiae ADH genes was measured using software available on the Australian

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National Genomic Information Service (ANGIS) or the the EMBOSS Needle alignment

programme available at EMBL-EBI (http://www.ebi.ac.uk/).

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RESULTS

Northern blot analysis of C. albicans ADH1, ADH2 and ADH3 expression

C. albicans ADH gene expression was measured in three media and at three phases of growth by

Northen blot analysis of mRNA levels. For all blots, equivalent RNA loading in each lane was

confirmed by comparing the intensity of ethidium bromide-stained rRNA bands (Fig. 1).

Triplicate blots were probed with one of the three 32P radiolabelled ADH gene-specific PCR

products. No expression of ADH3 was detected depite lengthy (2 weeks) exposure of the X-ray

film to the blots (results not shown). There was greater expression of CaADH1 mRNA compared

to CaADH2 mRNA (Fig. 1). Interestingly, the ADH2 signal appeared to be growth phase-specific

for the mRNAs from cells grown in YPD; a signal was only observed for stationary phase cells,

not with extracts from the early and mid-exponential phase cells.

Quantification of CaADH mRNAs in yeast cells grown under different nutritional

conditions

In initial experiments, RT-PCRs were performed using gene-specific primers to establish

appropriate dilution ranges for each of the template mRNAs to be detected (Fig. S3). Specifc

primers were used to generate cDNAs for Ca18S rRNA, CaACT1, CaADH1 and CaADH2

cDNAs. There was a large range in the detection limits for the different genes (Table 2). 18S

rRNA was the most abundant transcript (a 3 x 10-5 fold template dilution gave detectable product,

Table 2 and Fig. S3), wheras the CaADH2 template was not detectable at a dilution greater than

10 fold (Table 2 and Fig. S3).

However, in order to be able to compare expression of ADH1 and ADH2 mRNAs directly, under

different yeast growth conditions, and relative to a housekeeping gene (CaACT1), a universal

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cDNA template was synthesised using oligo dT combined with random primers for use in qRT-

PCRs for each mRNA. Due to the cellular abundance of Ca18S rRNA, it was not included in the

analysis so that a single template dilution could be used to generate the common template for

CaACT1, CaADH1 and CaADH2 cDNA quantification. The qRT-PCR results (Table 3) are

presented as ∆Ct values which have been normalized relative to CaACT1 Ct values. Regardless

of the growth phase (early exponential, mid-exponential or stationary) or type of media (GSB,

YPD, or YNB), there was greater expression of CaADH1 than either CaACT1 or CaADH2 (Table

3). In general, Ct values above 30 are not considered significant (Schmittgen et al., 2000, Skern

et al., 2005, Karlen et al., 2007). By this criterion, CaADH2 expression was not detectable in

YPD, with CaADH2 expression in YNB being borderline. There was more expression of

CaADH2 during mid-exponential and stationary growth phase in GSB media relative to the early

exponential phase. In contrast, expression of CaADH1 was relatively consistent for all conditions

but was greatest relative to the CaACT1 control when cells were grown in the GSB medium.

Expression of C. albicans ADH genes in S. cerevisiae

Cloning and expression of the C. albicans ADH1 and ADH2 genes in S. cerevisiae was carried

out by overlap extension PCR as described in the Methods section. A hexahistidine affinity tag

(His) allowed for affinity purification and immunodetection of the heterologously expressed

proteins. Western blotting and detection with an anti-His tag antibody (Fig. 2) revealed that

neither CaAdh1p nor CaAdh2p were detectable in the crude extracts from the recombinant strains

constructed (AD/CaADH1-his and AD/CaADH2-his). However, expression of recombinant

proteins of expected sizes (Adh1p ~ 46 kDa and Adh2p ~ 37kDa) could be detected in affinity

purified cell extracts from these strains (Fig. 2). A cross-reacting protein band of ~65 kDa was

present in affinity-purified samples from all strains including the AD/pABC3-his control strain

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(results not shown). This ~65 kDa band was assumed to be a his-rich contaminating polypeptide

present in the extracts.

Alcohol dehydrogenase (Adh) enzyme activities in extracts of S. cerevisiae strains

expressing recombinant C. albicans Adh proteins

Adh activity with ethanol as substrate was determined using partially purified cell-free extracts

from recombinant S. cerevisiae strains AD/CaADH1-his, AD/CaADH2-his and the control

AD/pABC3-his (Fig. 3). Samples from the strain expressing CaAdh1p possessed the highest

Adh activity (~2-fold greater than activities of the control or CaAdh2p-expressing strains)

Enzyme activity in the preparation purified from the strain expressing CaAdh2p was similar to

that from the AD/pABC3-his control strain (Fig. 3). This would suggest that expression of

CaAdh2p did not confer additional ethanol utilising activity above background enzyme activity in

the host S. cerevisiae AD strain, assumed to be due to the presence of co-purified S. cerevisiae

Adh2p. However, elimination of the endogenous activity was considered necessary to confirm

the apparent ethanol-utilising activity of C. albicans Adh1 when expressed in S. cerevsiae.

Expression of C. albicans Adh1p in an S. cervisiae strain in which the endogenous ethanol

utilising enzyme (ScAdh2) was disrupted

To confirm the ethanol utilising activity of CaAdh1p, ScADH2 was disrupted in the CaAdh1-

expressing strain, AD/CaADH1(pABC5). Transformants were designated as ADΔ

/CaADH1(pABC5)/ScADH2. Strain ADΔ/CaADH1(pABC5)/ScADH2 possessed greater

NAD-dependent ethanol utilising activity (0.13 U/mg protein) than the control ScADH2

disruptant strain (<0.001 U/mg protein) or the S. cerevisiae ADΔ strain with intact ScADH2 (Fig.

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4). This would suggest that the background ethanol utilising activity in strain ADΔ was indeed

due to ScAdh2p and that in the strain AD/CaADH1(pABC5)/ScADH2, the cloned CaAdh1p

conferred ethanol utilising activity on the host yeast.

DISCUSSION

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The expression of CaADH mRNAs in C. albicans was determined by both Northern blot analysis

and by applying RT and qRT techniques. Northern blot analysis is highly sensitive and can give

an indication of relative mRNA expression, but this can be misleading as the specific activity of

the probes, and hence signal strengths, can vary. The Northern analysis indicated that CaADH1

was consistently expressed in cells grown in all three media. In contrast, the expression of

CaADH2 was found to vary according to the media used. CaADH2 was expressed in minimal

(GSB) and defined (YNB) media at all growth phases; but in the rich medium, YPD, CaADH2

was only expressed during the stationary phase. This is consistent with a proteomic analysis of C.

albicans proteins present in extracts from cells grown to either exponential or stationary growth

phases in YPD medium which found that CaAdh2p was only detected in the stationary phase

(Kusch et al., 2008). In contrast, CaAdh1p was present in both exponential and stationary phase

extracts. No expression of ADH3 mRNA was detected. This was in agreement with other

published information for this gene; expression of CaADH3 mRNA has only been reported in a

study of the opaque/white switching of C. albicans (Lan et al., 2002). The C. albicans ADH1 and

ADH2 genes have been annotated as alcohol dehydrogenases in the CGD, whereas ADH3 is

annotated as a putative NAD-dependent (R,R)-butanediol dehydrogenase. A bioinformatic

analysis (Tables S1 and S2) revealed significant nucleotide homologies between the C. albicans

ADH1 and ADH2 genes and three S. cerevisiae ADH genes (ADH1, 2 and 3). At the predicted

protein sequence level, CaAdh1p and CaAdh2p both show equivalent homologies to ScAdh1p,

ScAdh2p as well as to ScAdh3p. Therefore it was not possible to predict any functional

homologies for CaAdh1p or CaAdh2p by comparison with the S. cerevisiae Adh protein

sequences. Adh3p had low homology to CaAdh1p or CaAdh2p and also little homology to any of

the S. cerevisiae Adh polypeptides (Table S2). Also, as CaADH3 mRNA could not be detected in

C. albicans cells, CaAdh3p was not studied further. The Northern blot results (Fig. 1) showed

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that expression of CaADH2 was apparently unaffected by the growth phase of the cells in GSB or

YNB cultures, although CaADH2 expression was absent under conditions of glucose excess in

rich YPD medium, possibly indicating glucose repression of CaADH2 in this medium. However,

there was no evidence for change of expression of CaADH1 mRNA in the three growth phases

examined for any medium. Therefore our data would suggest that CaADH1 is not subject to

glucose repression, as is the case for the S. cerevisiae ethanol utilising enzyme, ScAdh2p

(Walther & Schüller, 2001, Young et al., 2008). Indeed, Siikala and colleagues have also found a

remarkably high level of CaADH1 gene expression in C. albicans cells grown in YPD containing

2% glucose (Siikala et al., 2011).

When expression of CaADH1 and CaADH2 was analysed using qRT-PCR under different growth

conditions, there was more expression of CaADH1 than that of the housekeeping gene CaACT1

at all time points and regardless of which medium was used. This is consistent with previous

observations that C. albicans Adh1p is a major component of the cell proteome (Chaffin, 2008,

Kusch et al., 2008, Martínez-Gomariz et al., 2009) as well as being an immunodominant protein

(Shen et al., 1991, Ishiguro et al., 1992, Swoboda et al., 1993). A decline in the level of CaADH1

expression between mid-exponential phase and stationary phase cultures was observed for cells

grown in GSB media (∆Ct increase from -5.7 to -3.5; Table 3). In contrast, CaADH2 expression

in GSB and YNB, as measured by qRT-PCR, was less than that of the housekeeping gene

CaACT1 at all time points, indicating that expression of CaADH2 was less than that of CaADH1.

Similar to CaADH1, expression of CaADH2 in GSB and YNB was also less in stationary phase

cultures than in mid-exponential phase cultures. The expression of CaADH2 in YPD, as

measured by qRT-PCR, was considered insignificant because the Ct values for CaADH2

exceeded 30. However, Northern analysis showed that CaADH2 mRNA was present in the

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stationary phase cells grown in YPD. This may reflect the greater sensitivity of Northern

analysis.

Having established that CaADH1 and CaADH2 mRNAs are expressed in C. albicans cells grown

under a variety of conditions, both genes were cloned and expressed in S. cerevisiae (Fig. 2).

Assays of enzyme activity in affinity-purified extracts from Adh1p- or Adh2p-expressing cells

demonstrated only the CaAdh1p-expressing strain exhibited greater activity than the empty

cassette vector pABC3-his control (Fig. 3). Although the presence of CaAdh2p in the purified

cell extract from the AD/CaADH2-his strain was confirmed by Western blot using an anti-His

antibody (Fig. 2), extracts from this strain showed enzyme activities similar to those in extracts

from the control empty vector strain (Fig. 3). This would suggest that CaAdh2p is inactive under

the conditions tested, and that the activity observed for the AD/CaADH2-his strain represents the

background Adh activities of the host AD strain.

In order to confirm that the ethanol-utilising activity in extracts from the AD/CaADH1-his

recombinant strain was mediated by the cloned CaAdh1p rather than contaminating ScAdh2p,

CaAdh1p was expressed in a strain in which ScADH2 was disrupted. ScADH2 was successfully

disrupted in the parental AD strain. In this recombinant strain (AD/ScADH2) no significant

NAD reduction was detected in extracts when ethanol was given as the substrate (Fig. 4)

confirming that ScAdh2p catalyses the ethanol utilising activity in S. cerevisiae (Ciriacy 1975a).

When ScADH2 was disrupted in a recombinant strain expressing CaAdh1p, ethanol utilising

activity was present (Fig. 4) confirming that CaAdh1p catalyses the utilisation of ethanol which,

under the in vitro conditions tested, would result in production of acetaldehyde.

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We have previously shown that acetaldehyde is produced in vitro by C. albicans (Collings et al.,

1991) and more recent studies have suggested that oral colonisation with C. albicans and other

yeast can contribute to the presence of acetaldehyde in saliva (Nieminen et al., 2009, Gainza-

Cirauqui et al., 2013, Marttila et al., 2013a, Marttila et al., 2013b). Intriguingly, Marttila et al

(2013a) reported that under low oxygen tension in vitro there was no correlation between

acealdehyde concentration and ADH1 RNA expression, but rather with expression of ADH6 RNA

(encoding an acetaldehyde utilising enzyme). However, as noted by these authors, Adh1p levels

do not correlate closely with ADH1 mRNA levels (Bertram et al., 1996), possibly due to post-

translational or post-transcriptional control. Indeed, Adh1p is upregulated in biofilm formation

(Martinez-Gomariz et al., 2009) and oral biofilms can contain C. albicans (Zijnge et al., 2010).

We conclude that mRNAs of two members of the ADH gene family (ADH1 and ADH2) were

expressed under growth conditions replicating both replete and minimal nutritional conditions.

Furthermore expression of C. albicans Adh1p, but not Adh2p, conferred ethanol utilisation

activity when heterologously expressed in S. cerevisiae. Although regulation of other enzymes

involved in acetaldehyde metabolism, such as Adh6p, may be involved in the accumulation of

acetaldehyde in cultures or in vivo, we suggest that the high expression of Adh1p by C. albicans,

and its ethanol utilising activity, can contribute to the generation of the carcinogen acetaldehyde

in the saliva of colonised individuals.

ACKNOWLEDGEMENTS

MM Bakri was supported by a postgraduate scholarship from the University of Malaya and this

work was also supported by the New Zealand Dental Association Research Foundation.

We are grateful for the free availability of the Candida and Saccharomyces genome databases

(CGD; http://www.candidagenome.org/ and SGD; http://www.yeastgenome.org/).

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Table 1. PCR primers

Northern blotsCaADH1 For a AGTCTCACCACTACCACTGCCaADH1 RT a GCACAGTGGATCCAGACAATGCaADH2 For a TGTTATTTTCGAAACCAACGGTCaADH2 RT a

CaADH3 For a

CaADH3 RT a

RT-PCR

AACGTTGATTAACAATTCATTTGCATCAATTAGTGCTGTTGGTGACCACAAGCTTGAACAATTTGTAAA

Ca18S rRNA For GGATTTACTGAAGACTAACTACTGCa18S rRnA rev GAACAACAACCGATCCCTAGTCaACT1 For ATGGACGGGAAGAAGTTGCCaACT1 reverse CCCATACCAACCATGATACCCaADH1 For CACTCACGATGGTTCATTCGCaADH1 reverse AAGATGGTGCGACATTGGCaADH2 For CACCCACGATGGTTCTTTCCCaADH2 reverse AAGATTGGTGCAACATGGGpABC cloningADHIforPac GGTTTAATTAAAAAATGCAAGCAAGCTTATTCAGAATTADH1revNot ATGCGGCCACTGGTGTCCAATACGTATCTACT

ADH2forPac GGTTTAATTAAAAAATGTCTGTCCCAACTACTCAAAAAGCTG

ADH2revNot ATGCGGCCGCCTTTGTCGTTGTCCAAGACATATCTAC

MutantsURA3For ACAAAAAGCATACAATCAACTATCAACTATTAACTATA

TCGTATACACACACTATAGGGCGAATTGGGGAGCTCURA3Rev GGCATACTTGATAATGAAAACTATAAATCG

TAAAGACATAAGAGATCCGCAGGTCGACGGTATCGATAAGCTTCA

Up500For GCCTTATGATCCGTCTCTCCGGTTACUp500Rev TGTGTATTACGATATAGTTAATAGTTGATAGTTGDwn500For GCGGATCTCTTATGTCTTTACGATTDwn500Rev GAGGGTGTGTACATTGCAGTGCATCCHECKFOR GATCAAAGGGGC AAAACGTAGGGGCHECKRev GGAAGTCGTTCGTACTAGCAACATGGpABC3-For b TTGGCAACTAGGAACTTTCGPDR5downstream b TATGAGAAGACGGTTCGCCATTCGGACAG

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a (38)b(Lamping et al., 2007)Restriction enzyme sites are in bold.

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Table 2. Ct values for amplification of Ca18S rRNA, CaACT1, CaADH1 and CaADH2 mRNAs by qRT-PCR using gene specific primers. The template dilution for each of the template mRNAs were determined in initial RT-PCRs experiments, using gene-specific primers.

cDNA template Template dilution Ct

Ca18S rRNA1 x 10-5

3 x 10-527.229.2

CaACT11 x 10-3

3 x 10-327.8

29.5

CaADH13 x 10-2

1 x 10-3

2627.5

CaADH2 Neat 1 l1 x 10-1

22.728

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Table 3. Detection of CaADH mRNAs* by qRT-PCR using a cDNA template generated with

oligo dT and random primers.

Growth Medium

Growth Phase

Ct

ACTI ADH1 ∆Ct ADH2† ∆Ct

GSB E 25.5 21.0 -4.5 28.0 2.5M 26.2 20.5 -5.7 26.5 0.3S 25.0 21.5 -3.5 26.5 1.5

YNB E 23.5 22.5 -1.0 (30.0) NR†M 25.0 23.8 -1.2 29.8 4.8S 28.0 26.4 -1.6 (30.0) NR

YPD E 26.5 25.8 -0.7 (33.5) NRM 25.0 23.2 -0.7 (33.2) NRS 26.0 23.5 -2.5 (33.2) NR

*RNA was extracted from C. albicans ATCC 10261 cells grown in GSB, YNB and YPD. Cells

were harvested during early exponential (E), mid exponential (M) or stationary phase (S).

Template cDNA was prepared using oligo dT and random primers. A single dilution of the

cDNA template (1:10) was used in each of the PCRs.

† NR = Not Relevant. Ct values greater than 30 (contained in parentheses) were considered to be

insignificant and therefore a calculation of ∆Ct was not relevant.

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FIGURE LEGENDS

Figure 1. Northern blot analysis of CaADH gene expression. C. albicans strain ATCC 10261

was cultured in GSB, YNB or YPD at 30°C. The CaADH1 and CaADH2 blots were exposed to

X-ray film for 2 h. (a) Blot hybridised with CaADH1-specific DNA probe. (b). Blot hybridised

with CaADH2-specific DNAprobe. (c) Ethidium bromide stained gels used to make replicate

blots. E: early exponential phase, M: mid-exponential phase; and S: stationary phase.

(c)

GSB YPD

GSB YNB YPD

(b) CaADH2

(a) CaADH1

E M S E M S E M S

1.3 kb

1.2 kb

rRNA bands

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Figure 2. Purification of his-tagged CaAdh1p and CaAdh2p by nickel chromatography. Soluble

cellular fractions obtained from recombinant S. cerevisiae strains AD/CaADH1-his and

AD/CaADH2-his were used for affinity purification. Eluates of both unpurified and purified

column fractions were electrophoresed on polyacrylamide gels and electroblotted on

nitrocellulose membrane. Immunodetection was carried out using anti-His antibodies. Neither

CaAdh1p nor CaAdh2p were detectable in the crude (unpurified) extracts as applied to the

column, but following affinity purification, both proteins were detected.

AD

/CaA

DH

1-hi

s

AD

/CaA

DH

2-hi

s

50-

36-

148-

64-

AD

/CaA

DH

1-hi

s

AD

//CaA

DH

2-hi

s

CaAdh1pCaAdh2p

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Figure 3. Adh enzyme activities of Ni-NTA-purified cell-free extracts of recombinant S.

cerevisiae strains AD/CaADH1-his, AD/CaADH2-his and the control strain AD/pABC3-his.

Enzyme activities were determined with ethanol as a substrate and the assays were carried out at

pH 8.5 with 2 mM NAD and 0.3 mM ethanol. All assays were carried out in triplicate. *P < 0.01.

*

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Figure 4. Adh enzyme activities of cell-free extracts of S. cerevisiae strains

AD∆/CaADH1(pABC5)/∆ScADH2 and the controls AD∆ and AD∆/∆ScADH2. Enzyme

activities were determined with ethanol as a substrate and the assays were carried out at pH 8.5

with 2 mM NAD and 0.3 mM ethanol. All assays were carried out in triplicate. *P < 0.001.

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