International Journal of Antimicrobial Agents 33 (2009) 574–578

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Mutations in the CgPDR1 and CgERG11 genes in azole-resistant Candida glabrata
clinical isolates from Slovakia

Norbert Berila, Silvia Borecka, Vladimira Dzugasova, Jaroslav Bojnansky, Julius Subik

∗

Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynska dolina B-2, 842 15 Bratislava 4, Slovak Republic

a r t i c l e i n f o

Article history:
Received 14 August 2008
Accepted 24 November 2008

Keywords:
Azole resistance
Candida glabrata
CDR1
ERG11
PDR1

a b s t r a c t

Candida glabrata is an important human pathogen that is naturally less susceptible to antimycotics com-
pared with Candida albicans. Ten unmatched C. glabrata clinical isolates were selected from a collection
of isolates exhibiting decreased susceptibilities to azole antifungals. Overexpression of the CgPDR1 gene,
encoding the main multidrug resistance transcription factor, and its target genes CgCDR1 and CgCDR2,
coding for drug efflux transporters, was observed in six fluconazole-resistant isolates. Sequence analy-
sis of the polymerase chain reaction (PCR)-amplified DNA fragments of each isolate’s CgPDR1 gene was
used to identify two novel L347F and H576Y mutations in CgPdr1p. These proved to be responsible for
fluconazole resistance in transformants of the C. glabrata pdr1

 mutant strain. Five isolates harbour-

ing the H576Y mutation also contained the mutation E502V in CgErg11p 14C-lanosterol-demethylase.
Heterologous expression of the CgERG11 mutant allele did not provide evidence for its involvement in
azole resistance. In four fluconazole-sensitive isolates that were itraconazole-resistant, slightly enhanced
CgCDR2 expression was observed. No upregulation of the CgERG11 gene was observed in any of the ten
isolates. The results demonstrate that decreased susceptibilities of C. glabrata clinical isolates to azole anti-
fungals mainly results from gain-of-function mutations in the gene encoding the CgPdr1p transcription
factor.

© 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

1. Introduction

Candida glabrata is the second most important human pathogen

responsible for candidaemia

[1]

. This species is evolutionarily more

related to Saccharomyces cerevisiae than to Candida albicans and,
in contrast to the latter, is haploid, having survived deletions or
a complete loss of its mitochondrial genome

[2]

. It is inherently

less susceptible to azole antifungals that selectively inhibit 14-

␣-

demethylase of lanosterol encoded by the ERG11 gene

[2]

.

Compared with C. albicans, there have been fewer studies on C.

glabrata dealing with the molecular mechanisms of drug resistance

[2]

. In azole-resistant clinical isolates of C. glabrata, upregulation of

the ABC transporter genes CgCDR1, CgCDR2

[3–8]

and even CgSNQ2

[6]

was the major cause of drug resistance. Upregulation of ABC

transporter genes resulted from mutations in the CgPDR1 gene

[7,8]

,

a single orthologue of the PDR1 and PDR3 genes encoding transcrip-
tional activators of multidrug resistance in S. cerevisiae

[2]

.

The aim of this study was to investigate the molecular mecha-

nisms involved in the decreased susceptibility to azole antifungals
in unmatched C. glabrata clinical isolates recovered from different
patients treated in two university hospitals in Slovakia.

∗ Corresponding author. Tel.: [phone]; fax: [phone].

E-mail address:

[e-mail]

(J. Subik).

2. Materials and methods

2.1. Microorganisms, media and drugs

The C. glabrata clinical isolates used in this study are listed

in

Table 1

. They were recovered from patients treated at Uni-

versity Hospital in Nitra or collected from vaginal samples of
patients in University Hospital in Bratislava, Slovakia

[9]

. Can-

dida glabrata ATCC 2001, two well-characterised C. glabrata
isolates including a fluconazole-susceptible isolate DSY562

[3]

and a fluconazole-resistant isolate DSY565

[3]

, as well as the C.

glabrata 84u (ura3) wild-type and its C. glabrata B4u pdr1

ura3

mutant strain

[7]

were used as controls. Saccharomyces cerevisiae

Y26604 (MATa/MAT

˛ his31/his31 leu20/leu20 lys20/LYS2

MET15/met15

0 ura30/ura30 erg11::kanMX4/ERG11) diploid

mutant strain from EUROSCARF (Frankfurt, Germany) was used to
assess the contribution of mutation in CgErg11p to azole resistance
in C. glabrata. The strains were grown at 30

◦

C or 37

◦

C in either

complete YEPD medium (2% Bacto-Peptone, 1% yeast extract and 2%
glucose), in RPMI 1640 medium with

l-glutamate [without sodium

bicarbonate supplemented with 2% glucose and buffered to pH 7.0
with 0.165 M morpholinepropanesulfonic acid (MOPS)] or in mini-
mal YNB medium (0.67% Yeast Nitrogen Base without amino acids,
2% glucose). When grown on solid media, 2% agar was added to the
media. Isolates were identified and stored as described previously

0924-8579/$ – see front matter © 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
doi:

10.1016/j.ijantimicag.2008.11.011

N. Berila et al. / International Journal of Antimicrobial Agents 33 (2009) 574–578

575

Table 1
In vitro drug susceptibilities of Candida glabrata clinical isolates.

Isolate

Site of isolation

MIC

80

(

␮g/mL)

a

Fluconazole

Itraconazole

Voriconazole

1

Endotracheal sputum

128

8

32

2

Urine

1

0.25

0.75

3

Tonsil

128

2

>32

4

Urine

2

0.125

1

5

Endotracheal sputum

2

0.25

0.75

6

Tissue

4

0.25

1.5

7

Endotracheal sputum

128

4

32

8

Endotracheal sputum

2

0.5

1

9

Tongue

4

0.125

0.38

10

Oral cavity

2

0.25

0.5

11

Trachea

32

0.125

0.5

12

Vagina

8

0.5

0.5

13

Endotracheal sputum

16

0.25

>32

14

Abscess

32

8

>32

15

Endotracheal sputum

8

2

>32

16

Tracheal cannula

64

8

>32

17

Endotracheal sputum

64

8

8

18

Tonsil

32

8

>32

19

Tonsil

16

8

>32

20

Urine

128

0.5

0.75

21

Endotracheal sputum

128

>8

>32

22

Endotracheal sputum

128

>8

>32

23

Blood

128

>8

>32

24

Endotracheal sputum

8

1

0.75

25

Trachea

128

>8

>32

26

Urine

4

1

1.5

27

Urine

128

>8

>32

28

Vagina

4

8

0.38

29

Vagina

4

4

0.38

30

Vagina

4

4

1

31

Vagina

32

2

1.5

32

Vagina

2

1

4

33

Vagina

16

1

1.5

34

Vagina

4

1

1.5

35

Vagina

32

1

0.75

36

Vagina

16

0.5

0.75

37

Vagina

4

2

0.25

38

Vagina

2

1

0.125

DSY562

4

0.5

1

DSY565

128

4

4

ATCC 2001

2

0.5

N.D.

MIC

80

, minimum inhibitory concentration for that resulted in 80% reduction of fungal growth after 48 h compared with the drug-free control; N.D., not determined; SDD,

susceptible dose-dependent.

a

MIC breakpoints

[10]

: fluconazole, susceptible,

≤8 ␮g/mL; SDD, 16–32 ␮g/mL; resistant ≥64 ␮g/mL; itraconazole, susceptible ≤0.125 ␮g/mL; SDD, 0.25–0.5 ␮g/mL;

resistant

≥1 ␮g/mL; voriconazole, susceptible ≤1 ␮g/mL; resistant ≥4 ␮g/mL.

[9]

. Fluconazole was used as a commercial solution (Pfizer, New

York, NY). Itraconazole (Janssen, Beerse, Belgium) was dissolved in
100% dimethyl sulphoxide (DMSO).

2.2. Drug susceptibility testing

Susceptibilities of the isolates to fluconazole and itraconazole

were assayed by the broth microdilution method in 96-well plates
according to the proposed Clinical and Laboratory Standards Insti-
tute M27-A2 standard guidelines as described previously

[9,10]

.

Etest assays (AB BIODISK, Solna, Sweden) on RPMI medium supple-
mented with 2% glucose and the zone inhibition assays on Antibiotic
Medium 3 were used for determination of susceptibilities of isolates
to voriconazole and polyenes (each at 50

␮g per disk), respectively.

2.3. Plasmids, polymerase chain reaction (PCR) amplification,
DNA sequencing and quantitative real-time reverse transcription
(RT)-PCR

Centromeric plasmids pFL38 and pACU-5

[11]

containing

the URA3 selectable marker were used for cloning DNA frag-
ments of S. cerevisiae and C. glabrata, respectively. Plasmid
pCgPDR1

4672

was used as a source of the CgPDR1 gene

[7]

.

Genomic DNA from isolates was extracted and used as a tem-
plate for amplification of the CgERG11 gene and fragments of
the CgPDR1 gene. PCR was carried out with a high-fidelity
KOD Hot Star DNA Polymerase (Sigma–Aldrich, St Louis, MO)
and Extensor Hi-Fidelity PCR Enzyme Kit (ABgene, Hamburg,
Germany) with the following primer pairs: CgERG11 Prom 5



-

TAATATTGAGCTCCGAAGAGGTACGAAACATCC-3



(forward)

and

CgERG11 End

5



-TATTACTCTGCAGTGGGATCAACCAACTTTGTC-3



(reverse) containing flanking restriction sites for SacI and PstI
(underlined), respectively; CgERG11-forward 5



-GCGATCCCTTCA-

TGTCCATTGTC-3



and CgERG11-reverse 5



-GGCTAATGAATCAGC-

GTATATCCCG-3



; CgPDR1-F2 5



-GTGACTCGGAAGAAAGGGAC-3



and

CgPDR1-REV 5



-CACTGGTAACTATTGTAAGGGCC-3



; and CgPDR1-F5

5



-CAGAGACATCATATGAGGCAATCAG-3



and EcoRICgPDR1-STOP

5



-GATATATGAATTCTCATTCAGAATCGAAGGG-3



. The CgPDR1 DNA

fragments used with pCgPDR1

4672

plasmid in co-transformation

experiments were PCR-amplified using genomic DNA of isolate 3
and paired primers CgPDR1 F 5



-GGTAAATCAAAACCAACAGGGA-3



(forward)

and

CgPDR1-RI

5



-GACAATGGAATCGTAATCGCTC-3



(reverse), or genomic DNA of isolate 1 and paired primers
CgPDR1 F

5



-GGTAAATCAAAACCAACAGGGA-3



(forward)

and

CgPDR1-R 5



-CCGATAAGGGAGATGCAGTT-3



(reverse). Resulting

576

N. Berila et al. / International Journal of Antimicrobial Agents 33 (2009) 574–578

amplicons were purified using a QIAquick PCR Purification Kit
(Qiagen, Hilden, Germany) and the nucleotide sequences for
both strands were determined by primer elongation using an
automated DNA sequencer (ABI Prism 3100; Applied Biosys-
tems, Foster City, CA). DNA sequencing primers were the
same as those used for PCR amplification and supplemented
as

follows:

CgERG11-Srev

5



-AGGCAAGTTAGGGAAGACGA-3



,

CgPDR1-F3 5



-GGTCTTGGTTACTGTGTTCACCT-3



and CgPDR1-F6 5



-

TTTCTGAAGTATGCCCTGACC-3



. Sequence data were compared with

standard gene sequences (

http://cbi.labri.fr/Genolevures/elt/CAGL

)

using the BLAST program. Quantitative real-time RT-PCR was car-
ried out as described previously

[11]

. Target nucleic acids were

quantified using the standard curve method for determining the
ratio between the relative quantity of target gene and the CgACT1
reference gene.

3. Results and discussion

3.1. Susceptibilities of clinical isolates to antimycotics

Thirty-eight C. glabrata clinical isolates, originating from differ-

ent patients hospitalised in intensive care units, oncology wards
or examined at the gynaecology clinic, were screened for antifun-
gal resistance using standard susceptibility testing methods. As
shown in

Table 1

, the isolates exhibited different levels of sus-

ceptibility to fluconazole, itraconazole and voriconazole. Among
the isolates studied, 28.9%, 68.4% and 42.1% were resistant to flu-
conazole, itraconazole and voriconazole, respectively. Considerable
cross-resistance was observed among the antimycotics. With the
exception of isolate 20, all other fluconazole-resistant isolates were
also resistant to itraconazole and voriconazole. All clinical isolates
were susceptible to amphotericin B and nystatin (data not shown)
and grew on complex medium containing glycerol plus ethanol.

3.2. Expression of multidrug resistance-related genes

Six isolates with simultaneous resistance to fluconazole, itra-

conazole and voriconazole (isolates 1, 3, 7, 21, 22 and 27) as well as

four isolates sensitive to fluconazole but resistant to itraconazole
(isolates 28, 29, 30 and 32) were randomly selected to investigate
the molecular mechanisms underlying the development of azole
resistance. Compared with three azole-susceptible control strains
(ATCC 2001, DSY562 and 84u), a simultaneous increased expres-
sion of the CgPDR1, CgCDR1 and CgCDR2 genes was observed both in
the azole-resistant DSY565 control strain and six other fluconazole-
resistant clinical isolates (1, 3, 7, 21, 22 and 27) (

Fig. 1

). The CgPDR1

transcript level increased a maximum of 5.07-fold (isolate 7), whilst
the levels of CgCDR1 expression were 9.46–21.43-fold higher than
that in the control strain ATCC 2001. A slightly increased abundance
of CgCDR2 mRNA, but not of CgPDR1 and CgCDR1, was observed in
four fluconazole-sensitive and itraconazole-resistant clinical iso-
lates (28, 29, 30 and 32). The increased amount of CgPDR1 mRNA
was observed even in the pdr1

 mutant strain but without upreg-

ulation of CgCDR1 and CgCDR2, corroborating the results of Tsai et
al.

[7]

. No upregulation of the CgERG11 gene was observed in any of

the ten isolates analysed.

3.3. Mutations in the CgPDR1 and CgERG11 genes

Two parts of CgPDR1 from six fluconazole-resistant clinical

isolates overexpressing drug efflux transporter genes were PCR-
amplified using genomic DNA and pairs of primers as described in
Section

2.3

. Sequences of resulting amplicons covering the central

regulatory domain (539–2000 bp) and the C-terminal activation
domain (2138–3554 bp) and known to contain gain-of-function
mutations in homologous ScPDR1

[12]

and ScPDR3

[13]

genes were

determined and compared with the published CgPDR1 sequence.
Despite the independent origin of isolates, the CgPDR1 sequences
of five isolates (1, 7, 21, 22 and 27) contained the same nucleotide
variations. One of the nucleotide mutations, C1726T, led to H576Y
amino acid alteration in CgPdr1p (

Table 2

). In isolate 3, along with

known nucleotide variations in the CgPDR1 gene, another point
mutation, C1039T (resulting in the L347F amino acid substitution
in CgPdr1p) was identified. The position of the L347F mutation
exactly corresponds to the I252M gain-of-function mutation found

Fig. 1. Expression of the CgPDR1, CgCDR1, CgCDR2 and CgERG11 genes in Candida glabrata clinical isolates as determined by real-time reverse transcription polymerase chain
reaction (RT-PCR). The results are the mean

± standard deviation for the three independent experiments.

N. Berila et al. / International Journal of Antimicrobial Agents 33 (2009) 574–578

577

Table 2
Nucleotide and amino acid substitutions in the CgPDR1 and CgERG11 genes in Candida glabrata clinical isolates.

Gene

Base substitutions in clinical isolates

Amino acid substitutions

1, 7, 21, 22, 27

3

28

29, 30, 32

CgPDR1

C705T

C705T

–

C765T

C765T

–

–

C837T

–

T871C

T871C

–

–

C1039T

L347F

C1726T

–

H576Y

C1749T

C1749T

–

A2319T

A2319T

–

T2578C

T2578C

–

T2994C

T2994C

–

G3156A

G3156A

–

CgERG11

C588T

–

–

C588T

–

T768C

T768C

T768C

T768C

–

C918T

–

–

C918T

–

–

G927A

G927A

–

–

A1023G

A1023G

A1023G

A1023G

–

A1505T

–

–

–

E502V

T1557A

T1557A

T1557A

T1557A

–

in hyperactive ScPdr3p

[13]

. The H576Y mutation occurs in the

vicinity of a previously described F575L amino acid substitution
in hyperactive CgPdr1p

[7]

.

To prove that these mutations are responsible for azole

resistance, the C. glabrata B4u pdr1

ura3 mutant strain was co-

transformed with a gapped pCgPDR1

4672

plasmid

[7]

(restricted

either by DraIII or DraIII and PacI endonucleases) and the corre-
sponding DNA fragments overlapping these gaps in CgPDR1 that
were amplified by PCR using primer pairs and genomic DNA of iso-
lates 3 or 1, respectively. The resulting transformants containing
plasmid-borne functional CgPDR1 mutant alleles, checked by DNA
sequencing, were found to be resistant to fluconazole [minimum
inhibitory concentration for concentrations that resulted in 80%
reduction of fungal growth after 48 h compared with the drug-free
control; (MIC

80

)

≥128 ␮g/mL]. These results clearly demonstrate

that the identified Leu347Phe and His576Tyr mutations occurring
in the central inhibitory domain of CgPdr1p are responsible for
activation of CgPdr1p and the establishment of azole resistance.

All ten clinical isolates displaying decreased susceptibility either

to fluconazole or itraconazole were also subjected to CgERG11
sequence analysis. With the exception of isolate 3, five fluconazole-
resistant isolates (1, 7, 21, 22 and 27) had overexpressing drug
efflux transporter genes and displayed the same silent nucleotide
variations and A1505T mutation leading to E502V amino acid
substitution in the C-terminal part of CgErg11p (

Table 2

). The

appearance of the same pattern of nucleotide variations in the
CgPDR1 and CgERG11 genes in five fluconazole-resistant isolates
recovered from different patients treated in 2006 and 2007 at Uni-
versity Hospital in Nitra, together with the results of microsatellite
analysis using RPM2, MTI and Cg6 markers

[14,15]

, indicates the

common origin of these isolates. Apparently, the same is also true
for the other three isolates (29, 30 and 32) resistant to itracona-
zole in that they have the same nucleotide variations in CgERG11
as indicated by their display of the same sizes of DNA fragments in
microsatellite analysis using the polymorphic markers mentioned
above (unpublished results).

To our knowledge, E502V is the first amino acid substitu-

tion found in C. glabrata Erg11p. To assess its contribution to
azole resistance in yeast, the CgERG11 gene with its promoter
was amplified by PCR using genomic DNA of isolates 1 or 29
and paired primers CgERG11 Prom and CgERG11 End. Amplicons
were inserted into the pFL38 centromeric vector as SacI–PstI DNA
fragments. The resulting plasmids, containing either CgERG11-

E502V mutant or CgERG11 wild-type alleles, were introduced by
transformation into a S. cerevisiae Y26604 diploid strain contain-
ing one chromosomal ERG11 gene disrupted with a kanamycin
cassette. Transformants were subjected to sporulation and the
resulting haploid kanamycin-resistant Ura

+

spores were anal-

ysed for susceptibility to fluconazole and itraconazole. Since both
the CgERG11-E502V mutant and CgERG11 wild-type alleles in the
genetic background of Scerg11::kanMX conferred the same level
of fluconazole susceptibility (MIC

80

= 8

␮g/mL), it was concluded

that the E502V mutation in CgErg11p does not contribute to azole
resistance in C. glabrata.

Decreased susceptibilities to itraconazole in four vaginal yeast

isolates (28, 29, 30 and 32) were not associated with upregulation of
CgPDR1 and CgCDR1. In these isolates, no mutations altered amino
acids and no upregulation of CgERG11 was observed. Whether
a slightly enhanced expression of CgCDR2 in these isolates con-
tributes to their drug susceptibility pattern is difficult to decide
since a collection of unmatched clinical isolates of different origin
was analysed without knowing the exact levels of gene expression
in corresponding parental sensitive strains. Therefore, one cannot
rule out the participation of other drug transporters or additional
mechanisms in the regulation of itraconazole susceptibility in C.
glabrata.

Taken together, we identified two new mutations in CgPDR1 that

were associated with decreased susceptibilities to azole antifun-
gals in C. glabrata clinical isolates. They resulted in upregulation
of both CgPDR1 and its CgCDR1 and CgCDR2 targets. In selected
unmatched yeast isolates, upregulation of the CgERG11 gene was
not observed. The E502V mutation in the CgERG11 gene, found in
some fluconazole-resistant isolates, apparently did not contribute
to their azole resistance.

Acknowledgments

The authors thank Drs K. Kuchler, D. Sanglard, M. Sojakova and

H.F. Tsai for the strains and plasmids as well as D. Hanson for careful
reading of the manuscript.

Funding: This work was supported by grants from the Slovak

Research and Developmental Agency (APVV-20-00604, LPP-0022-
06, LPP-0011-07 and VVCE-0064-07), the Slovak Grant Agency of
Science (VEGA 1/3250/06) and Comenius University (Grant UK
342/08).

578

N. Berila et al. / International Journal of Antimicrobial Agents 33 (2009) 574–578

Competing interests: None declared.
Ethical approval: Not required.

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