R E S E A R C H A R T I C L E

ADH1expression inverselycorrelateswithCDR1andCDR2 inCandidaalbicans fromchronicoral candidosis inAPECED (APS-I) patientsEmilia Siikala1,2,3, Paul Bowyer4, Malcolm Richardson5, Harri Saxen6, Dominique Sanglard2 & RiinaRautemaa1,3,4

1Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland; 2Institute of Microbiology, University of

Lausanne and University Hospital Center, Lausanne, Switzerland; 3Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital,

Helsinki, Finland; 4The University of Manchester, Manchester Academic Health Science Centre, Translational Research Facility, School of Translational

Medicine, and University Hospital of South Manchester, Wythenshawe Hospital, Manchester, UK; 5Mycology Reference Centre, University Hospital of

South Manchester, Wythenshawe Hospital and The University of Manchester, Manchester Academic Health Science Centre, Translational Research

Facility, School of Translational Medicine, Manchester, UK; and 6Hospital for Children and Adolescents, Helsinki University Central Hospital, University of

Helsinki, Helsinki, Finland

Correspondence: Emilia Siikala, Department

of Bacteriology and Immunology, Haartman

Institute, University of Helsinki,

Haartmaninkatu 3, PO Box 21, FI-00014

Helsinki, Finland. Tel.: 1358 919 126 377;

fax: 1358 919 126 382; e-mail:

emilia.siikala@helsinki.fi

Received 28 October 2010; revised 28 January

2011; accepted 9 May 2011.

Final version published online 16 June 2011.

DOI:10.1111/j.1567-1364.2011.00739.x

Editor: Richard Calderone

Keywords

resistance; ADH1; CDR1; CDR2; acetaldehyde.

Abstract

Expression of the alcohol dehydrogenase gene ADH1, which converts ethanol into

carcinogenic acetaldehyde, significantly inversely correlated with the expression of

CDR1 and CDR2, genes linked to azole resistance in Candida albicans isolated

from chronic oral candidosis in autoimmune polyendocrinopathy–candidosis–

ectodermal dystrophy (APECED, APS-I) patients. This is a novel link between

candidal two-carbon metabolism genes and azole resistance.

Introduction

Autoimmune polyendocrinopathy–candidosis–ectodermal

dystrophy (APECED), also called autoimmune polyendo-

crine syndrome type I (APS-I), is a rare autosomal recessive

disease causing T-cell-mediated dysfunction of the immune

system (Husebye et al., 2009). Most patients suffer from

chronic mucocutaneous candidosis of the oral and oesopha-

geal mucosa, which may result in the development of

squamous cell carcinoma (Rautemaa et al., 2007a). A high

oral and oesophageal carcinoma prevalence of 10.3% has

been reported among APECED patients over the age of 25

years (Rautemaa et al., 2007a).

Acetaldehyde is a highly toxic and mutagenic product of

alcohol fermentation and metabolism and it is especially

linked to cancers of the digestive tract (Secretan et al., 2009;

Uittamo et al., 2009). The latest consensus meeting of the

International Agency for Research on Cancer of the WHO

reclassified acetaldehyde as a class I carcinogen in association

with alcohol consumption (Secretan et al., 2009). According

to our previous results, Candida albicans isolates from

APECED patients can produce carcinogenic levels of acetalde-

hyde in vitro in both ethanol and glucose incubation (Uittamo

et al., 2009). Oxidation of ethanol into acetaldehyde is

catalysed by the alcohol dehydrogenase enzyme (Adh), which

is encoded by a single ADH gene. In C. albicans, only one Adh

isoenzyme has been identified, suggesting that one enzyme,

Adh1, operates in a bidirectional manner in both ethanol

fermentation and metabolism (Bertram et al., 1996).

Most APECED patients receive repeated courses of azole

antifungals and many have developed resistance, mainly to

fluconazole (Rautemaa et al., 2007b). Decreased fluconazole

susceptibility has been found to correlate with a high num-

ber of prophylactic and therapeutic courses of fluconazole,

ketoconazole and topical miconazole (Siikala et al., 2009).

The main molecular mechanism behind resistance in

FEMS Yeast Res 11 (2011) 494–498c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

YEA

ST R

ESEA

RC

H

C. albicans isolates from APECED patients is upregulation

of CDR1 and CDR2, the genes linked to azole resistance,

followed by gain-of-function mutations in the mutual

activator, the transcriptional activator TAC1 (Siikala et al.,

2010). Multidrug efflux transporters of the ATP-binding

cassette (ABC) superfamily and of the major facilitator class

play a key role in the low level of accumulation of azoles in

the yeast cell (Sanglard et al., 1995). CDR1 and CDR2 and

genes for ABC transporters have been shown to be upregu-

lated in resistant strains, leading to an enhanced efflux of the

drug (Sanglard et al., 1995). CDR2 expression is absent in

sensitive C. albicans strains, but its level of expression is

known to increase during the development of azole resis-

tance (Sanglard et al., 1997). Therefore, it can be seen as a

useful marker of CDR-mediated resistance.

In order to determine whether ADH1, the catalyst of

ethanol oxidation, is regulated in C. albicans isolated from

APECED patients with variable minimum inhibitory concen-

trations (MICs) to fluconazole, we analyzed the transcript

levels for ADH1. We also wanted to determine whether there

was a correlation between levels of expression of ADH1 and

fluconazole MICs as well as the expression of CDR1 and

CDR2, genes linked to azole resistance. As overexpression of

ADH1 is likely to lead to increased levels of carcinogenic

aldehyde; we also wanted to analyse whether CDR-mediated

fluconazole resistance correlated with ADH1 expression and

the possible risk for carcinogenicity of the infection.

Materials and methods

Candida albicans isolates

Candida albicans strains isolated during 1995–2007 from the

oral cavities of nine APECED patients suffering from CMC

were used in this study (one to three isolates/patient).

Strains that had been typed using multilocus sequencing

typing were selected (Siikala et al., 2010). Isolates with

identical diploid sequence type (DST) and MICs were

excluded to ensure that all isolates were independent.

However, isolates with similar DSTs and different MICs

were included to distinguish the role of different MICs in a

genetically closely related strain. A total of 19 strains

consisting of five resistant (R) strains (fluconazole

MICZ64 mg L�1), 11 susceptible dose-dependent (DD)

strains (fluconazole MIC 16 � MIC � 64 mg L�1) and three

susceptible (S) strains (fluconazole MIC � 8 mg L�1) were

included (National Committee for Clinical Laboratory

Standards, 2002). The small number of susceptible isolates

is due to decades of azole treatment leading to azole

resistance and lack of susceptible isolates in the APECED

patient population.

The C. albicans isolates had been identified from patient

samples using conventional culture and identification

methods at the Clinical Microbiology Laboratory of the

Helsinki University Central Hospital. The identification of

C. albicans was based on colony morphology on

CHROMagars Candida medium (CHROMagar, Paris,

France) and a negative Bichro-Dublis latex coagglutination

test result for Candida dubliniensis (Fumouze Diagnostics,

Levallois-Perret, France). The strains had been stored in

milk–glycerin at � 70 1C.

Growth media

The C. albicans strains were subcultured on YEPD agar [1%

Bacto peptone (Difco Laboratories, Basel, Switzerland),

0.5% yeast extract (Difco Laboratories) and 2% glucose

(Fluka, Buchs, Switzerland)].

Susceptibility testing

The susceptibility profiles of the isolates for fluconazole

were reanalysed by Etest (AB Biodisk, Sweden) according to

the manufacturer’s instructions and as described previously

(Arendrup et al., 2001). Quality control for susceptibility

testing was performed using C. albicans strains ATCC 90028

and ATCC 24433 and Candida glabrata strain ATCC 90030.

Northern blotting

Small-scale isolation of total RNA was performed from cells

grown to the logarithmic growth phase in YEPD medium at

30 1C with constant shaking. Northern blotting was per-

formed for CDR1, CDR2 and ADH1 as described previously

(Sanglard et al., 1995). RNA samples were separated by

agarose gel electrophoresis and transferred to a nitrocellu-

lose membrane using the Vacuum Blotting System (Hoefer

Scientific Instruments, San Fransisco, CA). Probes were

labelled with [a-32P]dATP with random priming using the

MegaPrime DNA Labelling System dNTP Kit (GE Health-

care, Waukesha, WI) according to the manufacturer’s in-

structions. CDR1, CDR2 and ACT1 probes were prepared

by PCR as described earlier (Sanglard et al., 1995). The

primers for the PCR-amplified ADH1-probe were forward,

50-GCAAGCTTATTCAGAATTTTCAGAGGTGC-30, and

reverse, 50-CAACTGGTGTCCAATACGTATCTACTCAAG-

30. Radioactive signals were revealed by exposure to a Kodak

BioMax MR film (GE Healthcare). Signals obtained in

blotted membranes were quantified by counting of radio-

activity (Typhoon Trio, GE Healthcare). As a control for the

evaluation of the expression levels of CDR1 and CDR2, the

membranes were hybridized with ACT1 and the amount of

RNA was normalized according to the expression of ACT1.

The strain with the lowest ADH1 expression was chosen as a

baseline control strain and the expression levels of the other

isolates were quantified relative to this strain.

FEMS Yeast Res 11 (2011) 494–498 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

495ADH1 and CDR correlate in azole-resistant Candida

Statistical analysis

Data were analysed using GRAPHPAD PRISM version 5.00

(GraphPad Inc., San Diego, CA). Geometric means and

range were used for the analyses of MICs and the mean

and range for the analysis of gene expression levels. Spear-

man’s rho (rS) was used for the analyses of correlations.

Correlations are presented with a 95% confidence interval

(x to x) and P-value. P values of o 0.05 were considered

statistically significant.

Results

Susceptibility profiles of C. albicans isolates

The MICs for fluconazole of all the 19 C. albicans isolates

ranged from 1 to 128 mg L�1, with a geometric mean of

33 mg L�1 (1). The geometric mean of the three

susceptible isolates with fluconazole MICs � 8 mg L�1 was

3.63 mg L�1 (range 1–8). All susceptible isolates were

isolated from different patients (Patients 2, 4 and 5). The

geometric mean of the 11 susceptible DD isolates

with fluconazole MICs 16 � MIC � 64 mg L�1 was 37.9

(range 24–48). The geometric mean of the five resistant

with fluconazole MICsZ64 mg L�1 isolates was 91.6

(range 64–128).

CDR1/2 and ADH1mRNA expression

As predicted, the relative mRNA expression of CDR1

correlated with the relative mRNA expression of CDR2

[rS = 0.8810 (0.6862–0.9579)]. Isolate 1995 from Pat2 with

the lowest MIC for fluconazole (1 mg L�1) showed the low-

est relative expression of CDR1 (0.05-fold). Isolate 2005

from Pat6 showed a low relative expression of both CDR1

(0.06-fold) and CDR2 (0.01-fold) despite an increased

fluconazole MIC of 32 mg L�1. This isolate, however, has

been found to carry a mutation S405F in the ERG11 gene

contributing to the increased MIC (Siikala et al., 2010). The

relative expression of CDR2 was found to be low in several

isolates with increased MICs (Table 1).

There was a highly significant negative correlation

between the relative expressions of ADH1 and both CDR1

[rS =� 0.8540 (� 0.9478 to � 0.6238), Po 0.0001] and

CDR2 [rS =� 0.7571 (� 0.9102 to � 0.4218), P = 0.0004]

(Fig. 2a and b). No correlation was found to exist between

the expression level of ADH1 and the MICs of the isolates to

fluconazole [rS =� 0.1671 (� 0.5872 to 0.3238)].

25

30

10

15

20

ADH1

0

5

2001

2004

2004

2001

2006

2007

2006

1996

2001

2004

1995

2001

2004

2004

2005

1995

2001

2004

2006

Rel

ativ

e ex

pres

sion

(fo

ld)

Rel

ativ

e ex

pres

sion

(fo

ld) 1.0

1.2

Pat1 Pat2 Pat3 Pat4 Pat5 Pat6 Pat7 Pat8 Pat9

0.6

0.4

0.8

CDR1

CDR2

0.0

0.2

001

004

004

0 01

006

007

0 06

996

001

004

9 95

001

004

004

005

995

001

0 04

006

2 0 20 20 20 20 20 20 19 20 20 19 20 20 20 2 0 19 20 20 20

Pat1 Pat2 Pat3 Pat4 Pat5 Pat6 Pat7 Pat8 Pat9

Fig. 1. Expression levels of ADH1, CDR1 and

CDR2. Isolate with the lowest ADH1 expression

(Patient 1, 2004) was used as the baseline control

(marked with an arrow).

FEMS Yeast Res 11 (2011) 494–498c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

496 E. Siikala et al.

Discussion

We have shown previously that C. albicans isolates from

APECED patients are capable of producing high levels of

carcinogenic acetaldehyde in vitro (Uittamo et al., 2009). In

C. albicans, alcohol dehydrogenase, encoded by ADH1,

catalyses the oxidation of ethanol to acetaldehyde. ADH1

expression is a prerequisite to the production of ADH and

acetaldehyde from ethanol. According to the results pre-

sented here, ADH1 is highly upregulated in isolates with low

CDR1 and CDR2 expression, also in those resistant to azoles.

The mechanisms underlying resistance in the isolates used in

this study have been published previously (Siikala et al.,

2010). The azole-resistant isolates with low CDR1/2 expres-

sion have mutations in the ERG11 leading to increased

fluconazole MICs. No correlations between the ERG11

mutations and the data presented in this study were found.

We also showed that the isolates are not phylogenetically

related to each other and appear to be randomly distributed

throughout the taxon.

ADH1 is expressed at low levels in fluconazole-resistant

isolates where CDR1 and CDR2 are highly expressed. A

highly significant negative correlation was seen between the

expression of ADH1 and CDR1 and CDR2 (Po 0.0001 for

CDR1 and P = 0.0004 for CDR2) in the C. albicans strains

isolated from APECED patients. This is a novel link between

candidal two-carbon metabolism genes and CDR1 and

CDR2, genes conferring azole resistance.

The correlation seen may be due to metabolic changes in

the isolates. For example, catabolite repression leading to

ADH repression could induce CDRs. According to previous

studies, the expression of ADH1 in C. albicans is regulated in

response to the growth phase and the carbon source

(Bertram et al., 1996). ADH1 mRNA appears to be less

abundant during growth in glucose or ethanol than during

growth in other carbon sources. Elevated ADH1 expression

is an indicator of the candidal metabolic flux via the

pyruvate bypass route. In the present study, before extrac-

tion of RNA, C. albicans isolates were grown aerobically in

YEPD with 2% glucose. Despite the fact that this is clearly a

concentration higher than that generally found in the

human host and that alcohol fermentation occurs in Candida

under hypoxic conditions, the expression level of ADH1 was

remarkably high in many cases. Our experiments were

performed in the absence of either azoles or ethanol; thus,

the results cannot be explained by ethanol induction of

ADH1 expression or by fluconazole induction of CDR1 and

Table 1. Relative expression levels of ADH1, CDR1 and CDR2

Strains

Fluconazole

MIC (mg L�1) DST

Ratio

ADH CDR1 CDR2

Pat1/2001 24 1152 2.80 0.73 0.60

Pat1/2004� 64 1156 1.0

(851 377)

1.0

(1 703 487)

1.0

(5 057 978)

Pat2/1995 1 360 20.25 0.05 0.02

Pat2/2001 64 360 16.39 0.20 0.17

Pat3/2006 48 1157 2.59 0.45 0.24

Pat4/2001 48 1151 4.91 0.70 0.21

Pat4/2006 6 1493 15.61 0.22 0.23

Pat4/2007 32 1493 2.76 0.95 0.60

Pat5/1996 8 1489 3.26 0.51 0.44

Pat5/2001 48 1489 8.60 0.23 0.20

Pat5/2004 32 1318 5.13 0.42 0.36

Pat6/2004 128 1494 11.17 0.34 0.12

Pat6/2005 32 1494 22.28 0.06 0.01

Pat7/1995 48 1490 22.53 0.18 0.23

Pat7/2001 48 1158 6.44 0.11 0.02

Pat7/2004 128 1490 3.77 0.78 0.35

Pat8/2004 48 1154 11.09 0.20 0.01

Pat8/2006 24 1492 25.40 0.10 0.01

Pat9/2004 96 203 6.85 0.71 0.41

The expression levels of ADH1, CDR1 and CDR2 were normalized against

the expression of ACT1 and the relative expression levels were quantified

relative to the control strain with the lowest ADH1 expression (Pat1/2004

marked with�). The absolute expression levels before normalization

correspond to the relative fluorescence units and are given in parenth-

eses for this isolate.

rS= −0.8539; P<0.0001

0 10 20 300.0

0.5

1.0

1.5

Relative expression of ADH1

Rel

ativ

eex

pres

sion

ofC

DR

1

rS = − 0.7673; P=0.0001

0 10 20 300.0

0.5

1.0

1.5

Relative expression of ADH1R

elat

ive

expr

essi

onof

CD

R2

(a)

(b)

Fig. 2. Correlations (Spearman’s rho) and polynomial regression

between the relative expression levels of ADH1 and CDR1 [rS = �0.8540

(� 0.9478 to �0.6238), Po 0.0001] (a) and ADH1 and CDR2

[rS = � 0.7571 (�0.9102 to � 0.4218), P = 0.0004] (b). Susceptible

isolates are marked in white, susceptible DD isolates in grey and resistant

isolates in black.

FEMS Yeast Res 11 (2011) 494–498 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

497ADH1 and CDR correlate in azole-resistant Candida

CDR2 expression. Interestingly, progesterone exposure has

been shown to upregulate CDR1 and CDR2 expression and

to a lesser extent ADH1 expression in C. albicans (Banerjee

et al., 2007).

ADH1 has been shown to be downregulated in Candida

biofilms and it has been suggested to play a role in quorum

sensing during their formation (Chandra et al., 2001;

Mukherjee et al., 2006; De Monte et al., 2007). Biofilm

formation contributes to antifungal resistance in Candida

(Chandra et al., 2001). In our study, however, experiments

were performed with planktonic cells and low ADH1

expression by some isolates cannot be explained by biofilm

formation. It is possible that the expression of both ADH1

and CDR1 is affected by the same regulator. Regardless of

the mechanism, azole exposure resulting in CDR upregula-

tion could lead to ADH1 downregulation and decreased

exposure carcinogenic acetaldehyde. The mechanisms

behind this phenomenon will require further study.

Acknowledgements

We thank Francoise Ischer for her technical help and

expertise and Professor Seppo Sarna for statistical assistance.

The study was supported by grants from Helsinki University

Central Hospital, the Finnish Dental Association Apollonia,

the Yrjo Jahnsson Foundation, the Daisy and Yrjo Eskola

trust fund of the Paulo Foundation and the University of

Helsinki.

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