International Journal of Antimicrobial Agents 33 (2009) 574 578
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International Journal of Antimicrobial Agents
journal homepage: http://www.elsevier.com/locate/ijantimicag
Short communication
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 a b s t r a c t
Article history:
Candida glabrata is an important human pathogen that is naturally less susceptible to antimycotics com-
Received 14 August 2008
pared with Candida albicans. Ten unmatched C. glabrata clinical isolates were selected from a collection
Accepted 24 November 2008
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,
Keywords:
coding for drug efflux transporters, was observed in six fluconazole-resistant isolates. Sequence analy-
Azole resistance
sis of the polymerase chain reaction (PCR)-amplified DNA fragments of each isolate s CgPDR1 gene was
Candida glabrata
used to identify two novel L347F and H576Y mutations in CgPdr1p. These proved to be responsible for
CDR1
fluconazole resistance in transformants of the C. glabrata pdr1 mutant strain. Five isolates harbour-
ERG11
ing the H576Y mutation also contained the mutation E502V in CgErg11p 14C-lanosterol-demethylase.
PDR1
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 2. Materials and methods
Candida glabrata is the second most important human pathogen
2.1. Microorganisms, media and drugs
responsible for candidaemia [1]. This species is evolutionarily more
related to Saccharomyces cerevisiae than to Candida albicans and,
The C. glabrata clinical isolates used in this study are listed
in contrast to the latter, is haploid, having survived deletions or
in Table 1. They were recovered from patients treated at Uni-
a complete loss of its mitochondrial genome [2]. It is inherently
versity Hospital in Nitra or collected from vaginal samples of
less susceptible to azole antifungals that selectively inhibit 14- -
patients in University Hospital in Bratislava, Slovakia [9]. Can-
demethylase of lanosterol encoded by the ERG11 gene [2].
dida glabrata ATCC 2001, two well-characterised C. glabrata
Compared with C. albicans, there have been fewer studies on C.
isolates including a fluconazole-susceptible isolate DSY562 [3]
glabrata dealing with the molecular mechanisms of drug resistance
and a fluconazole-resistant isolate DSY565 [3], as well as the C.
[2]. In azole-resistant clinical isolates of C. glabrata, upregulation of
glabrata 84u (ura3) wild-type and its C. glabrata B4u pdr1 ura3
the ABC transporter genes CgCDR1, CgCDR2 [3 8] and even CgSNQ2
mutant strain [7] were used as controls. Saccharomyces cerevisiae
[6] was the major cause of drug resistance. Upregulation of ABC
Y26604 (MATa/MATÛ his3 1/his3 1 leu2 0/leu2 0 lys2 0/LYS2
transporter genes resulted from mutations in the CgPDR1 gene [7,8],
MET15/met15 0 ura3 0/ura3 0 erg11::kanMX4/ERG11) diploid
a single orthologue of the PDR1 and PDR3 genes encoding transcrip-
mutant strain from EUROSCARF (Frankfurt, Germany) was used to
tional activators of multidrug resistance in S. cerevisiae [2].
assess the contribution of mutation in CgErg11p to azole resistance
ć% ć%
The aim of this study was to investigate the molecular mecha-
in C. glabrata. The strains were grown at 30 C or 37 C in either
nisms involved in the decreased susceptibility to azole antifungals
complete YEPD medium (2% Bacto-Peptone, 1% yeast extract and 2%
in unmatched C. glabrata clinical isolates recovered from different
glucose), in RPMI 1640 medium with l-glutamate [without sodium
patients treated in two university hospitals in Slovakia.
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
"
Corresponding author. Tel.: [phone]; fax: [phone].
media. Isolates were identified and stored as described previously
E-mail address: [e-mail] (J. Subik).
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 MIC80 ( 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.
MIC80, 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, d"8 g/mL; SDD, 16 32 g/mL; resistant e"64 g/mL; itraconazole, susceptible d"0.125 g/mL; SDD, 0.25 0.5 g/mL;
resistant e"1 g/mL; voriconazole, susceptible d"1 g/mL; resistant e"4 g/mL.
[9]. Fluconazole was used as a commercial solution (Pfizer, New Genomic DNA from isolates was extracted and used as a tem-
York, NY). Itraconazole (Janssen, Beerse, Belgium) was dissolved in plate for amplification of the CgERG11 gene and fragments of
100% dimethyl sulphoxide (DMSO). the CgPDR1 gene. PCR was carried out with a high-fidelity
KOD Hot Star DNA Polymerase (Sigma Aldrich, St Louis, MO)
2.2. Drug susceptibility testing
and Extensor Hi-Fidelity PCR Enzyme Kit (ABgene, Hamburg,
Germany) with the following primer pairs: CgERG11 Prom 5 -
Susceptibilities of the isolates to fluconazole and itraconazole
TAATATTGAGCTCCGAAGAGGTACGAAACATCC-3 (forward) and
were assayed by the broth microdilution method in 96-well plates
CgERG11 End 5 -TATTACTCTGCAGTGGGATCAACCAACTTTGTC-3
according to the proposed Clinical and Laboratory Standards Insti-
(reverse) containing flanking restriction sites for SacI and PstI
tute M27-A2 standard guidelines as described previously [9,10].
(underlined), respectively; CgERG11-forward 5 -GCGATCCCTTCA-
Etest assays (AB BIODISK, Solna, Sweden) on RPMI medium supple-
TGTCCATTGTC-3 and CgERG11-reverse 5 -GGCTAATGAATCAGC-
mented with 2% glucose and the zone inhibition assays on Antibiotic
GTATATCCCG-3 ; CgPDR1-F2 5 -GTGACTCGGAAGAAAGGGAC-3 and
Medium 3 were used for determination of susceptibilities of isolates
CgPDR1-REV 5 -CACTGGTAACTATTGTAAGGGCC-3 ; and CgPDR1-F5
to voriconazole and polyenes (each at 50 g per disk), respectively.
5 -CAGAGACATCATATGAGGCAATCAG-3 and EcoRICgPDR1-STOP
5 -GATATATGAATTCTCATTCAGAATCGAAGGG-3 . The CgPDR1 DNA
2.3. Plasmids, polymerase chain reaction (PCR) amplification,
fragments used with pCgPDR14672 plasmid in co-transformation
DNA sequencing and quantitative real-time reverse transcription
experiments were PCR-amplified using genomic DNA of isolate 3
(RT)-PCR
and paired primers CgPDR1 F 5 -GGTAAATCAAAACCAACAGGGA-3
(forward) and CgPDR1-RI 5 -GACAATGGAATCGTAATCGCTC-3
Centromeric plasmids pFL38 and pACU-5 [11] containing
(reverse), or genomic DNA of isolate 1 and paired primers
the URA3 selectable marker were used for cloning DNA frag-
CgPDR1 F 5 -GGTAAATCAAAACCAACAGGGA-3 (forward) and
ments of S. cerevisiae and C. glabrata, respectively. Plasmid
CgPDR1-R 5 -CCGATAAGGGAGATGCAGTT-3 (reverse). Resulting
pCgPDR14672 was used as a source of the CgPDR1 gene [7].
576 N. Berila et al. / International Journal of Antimicrobial Agents 33 (2009) 574 578
amplicons were purified using a QIAquick PCR Purification Kit four isolates sensitive to fluconazole but resistant to itraconazole
(Qiagen, Hilden, Germany) and the nucleotide sequences for (isolates 28, 29, 30 and 32) were randomly selected to investigate
both strands were determined by primer elongation using an the molecular mechanisms underlying the development of azole
automated DNA sequencer (ABI Prism 3100; Applied Biosys- resistance. Compared with three azole-susceptible control strains
tems, Foster City, CA). DNA sequencing primers were the (ATCC 2001, DSY562 and 84u), a simultaneous increased expres-
same as those used for PCR amplification and supplemented sion of the CgPDR1, CgCDR1 and CgCDR2 genes was observed both in
as follows: CgERG11-Srev 5 -AGGCAAGTTAGGGAAGACGA-3 , the azole-resistant DSY565 control strain and six other fluconazole-
CgPDR1-F3 5 -GGTCTTGGTTACTGTGTTCACCT-3 and CgPDR1-F6 5 - resistant clinical isolates (1, 3, 7, 21, 22 and 27) (Fig. 1). The CgPDR1
TTTCTGAAGTATGCCCTGACC-3 . Sequence data were compared with transcript level increased a maximum of 5.07-fold (isolate 7), whilst
standard gene sequences (http://cbi.labri.fr/Genolevures/elt/CAGL) the levels of CgCDR1 expression were 9.46 21.43-fold higher than
using the BLAST program. Quantitative real-time RT-PCR was car- that in the control strain ATCC 2001. A slightly increased abundance
ried out as described previously [11]. Target nucleic acids were of CgCDR2 mRNA, but not of CgPDR1 and CgCDR1, was observed in
quantified using the standard curve method for determining the four fluconazole-sensitive and itraconazole-resistant clinical iso-
ratio between the relative quantity of target gene and the CgACT1 lates (28, 29, 30 and 32). The increased amount of CgPDR1 mRNA
reference gene. was observed even in the pdr1 mutant strain but without upreg-
ulation of CgCDR1 and CgCDR2, corroborating the results of Tsai et
3. Results and discussion
al. [7]. No upregulation of the CgERG11 gene was observed in any of
the ten isolates analysed.
3.1. Susceptibilities of clinical isolates to antimycotics
3.3. Mutations in the CgPDR1 and CgERG11 genes
Thirty-eight C. glabrata clinical isolates, originating from differ-
ent patients hospitalised in intensive care units, oncology wards
Two parts of CgPDR1 from six fluconazole-resistant clinical
or examined at the gynaecology clinic, were screened for antifun-
isolates overexpressing drug efflux transporter genes were PCR-
gal resistance using standard susceptibility testing methods. As
amplified using genomic DNA and pairs of primers as described in
shown in Table 1, the isolates exhibited different levels of sus-
Section 2.3. Sequences of resulting amplicons covering the central
ceptibility to fluconazole, itraconazole and voriconazole. Among
regulatory domain (539 2000 bp) and the C-terminal activation
the isolates studied, 28.9%, 68.4% and 42.1% were resistant to flu-
domain (2138 3554 bp) and known to contain gain-of-function
conazole, itraconazole and voriconazole, respectively. Considerable
mutations in homologous ScPDR1 [12] and ScPDR3 [13] genes were
cross-resistance was observed among the antimycotics. With the
determined and compared with the published CgPDR1 sequence.
exception of isolate 20, all other fluconazole-resistant isolates were
Despite the independent origin of isolates, the CgPDR1 sequences
also resistant to itraconazole and voriconazole. All clinical isolates
of five isolates (1, 7, 21, 22 and 27) contained the same nucleotide
were susceptible to amphotericin B and nystatin (data not shown)
variations. One of the nucleotide mutations, C1726T, led to H576Y
and grew on complex medium containing glycerol plus ethanol.
amino acid alteration in CgPdr1p (Table 2). In isolate 3, along with
known nucleotide variations in the CgPDR1 gene, another point
3.2. Expression of multidrug resistance-related genes
mutation, C1039T (resulting in the L347F amino acid substitution
Six isolates with simultaneous resistance to fluconazole, itra- in CgPdr1p) was identified. The position of the L347F mutation
conazole and voriconazole (isolates 1, 3, 7, 21, 22 and 27) as well as 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 Ä… 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 E502V mutant or CgERG11 wild-type alleles, were introduced by
vicinity of a previously described F575L amino acid substitution transformation into a S. cerevisiae Y26604 diploid strain contain-
in hyperactive CgPdr1p [7]. ing one chromosomal ERG11 gene disrupted with a kanamycin
To prove that these mutations are responsible for azole cassette. Transformants were subjected to sporulation and the
resistance, the C. glabrata B4u pdr1 ura3 mutant strain was co- resulting haploid kanamycin-resistant Ura+ spores were anal-
transformed with a gapped pCgPDR14672 plasmid [7] (restricted ysed for susceptibility to fluconazole and itraconazole. Since both
either by DraIII or DraIII and PacI endonucleases) and the corre- the CgERG11-E502V mutant and CgERG11 wild-type alleles in the
sponding DNA fragments overlapping these gaps in CgPDR1 that genetic background of Scerg11::kanMX conferred the same level
were amplified by PCR using primer pairs and genomic DNA of iso- of fluconazole susceptibility (MIC80 =8 g/mL), it was concluded
lates 3 or 1, respectively. The resulting transformants containing that the E502V mutation in CgErg11p does not contribute to azole
plasmid-borne functional CgPDR1 mutant alleles, checked by DNA resistance in C. glabrata.
sequencing, were found to be resistant to fluconazole [minimum Decreased susceptibilities to itraconazole in four vaginal yeast
inhibitory concentration for concentrations that resulted in 80% isolates (28, 29, 30 and 32) were not associated with upregulation of
reduction of fungal growth after 48 h compared with the drug-free CgPDR1 and CgCDR1. In these isolates, no mutations altered amino
control; (MIC80) e"128 g/mL]. These results clearly demonstrate acids and no upregulation of CgERG11 was observed. Whether
that the identified Leu347Phe and His576Tyr mutations occurring a slightly enhanced expression of CgCDR2 in these isolates con-
in the central inhibitory domain of CgPdr1p are responsible for tributes to their drug susceptibility pattern is difficult to decide
activation of CgPdr1p and the establishment of azole resistance. since a collection of unmatched clinical isolates of different origin
All ten clinical isolates displaying decreased susceptibility either was analysed without knowing the exact levels of gene expression
to fluconazole or itraconazole were also subjected to CgERG11 in corresponding parental sensitive strains. Therefore, one cannot
sequence analysis. With the exception of isolate 3, five fluconazole- rule out the participation of other drug transporters or additional
resistant isolates (1, 7, 21, 22 and 27) had overexpressing drug mechanisms in the regulation of itraconazole susceptibility in C.
efflux transporter genes and displayed the same silent nucleotide glabrata.
variations and A1505T mutation leading to E502V amino acid Taken together, we identified two new mutations in CgPDR1 that
substitution in the C-terminal part of CgErg11p (Table 2). The were associated with decreased susceptibilities to azole antifun-
appearance of the same pattern of nucleotide variations in the gals in C. glabrata clinical isolates. They resulted in upregulation
CgPDR1 and CgERG11 genes in five fluconazole-resistant isolates of both CgPDR1 and its CgCDR1 and CgCDR2 targets. In selected
recovered from different patients treated in 2006 and 2007 at Uni- unmatched yeast isolates, upregulation of the CgERG11 gene was
versity Hospital in Nitra, together with the results of microsatellite not observed. The E502V mutation in the CgERG11 gene, found in
analysis using RPM2, MTI and Cg6 markers [14,15], indicates the some fluconazole-resistant isolates, apparently did not contribute
common origin of these isolates. Apparently, the same is also true to their azole resistance.
for the other three isolates (29, 30 and 32) resistant to itracona-
zole in that they have the same nucleotide variations in CgERG11
Acknowledgments
as indicated by their display of the same sizes of DNA fragments in
microsatellite analysis using the polymorphic markers mentioned
above (unpublished results). The authors thank Drs K. Kuchler, D. Sanglard, M. Sojakova and
To our knowledge, E502V is the first amino acid substitu- H.F. Tsai for the strains and plasmids as well as D. Hanson for careful
tion found in C. glabrata Erg11p. To assess its contribution to reading of the manuscript.
azole resistance in yeast, the CgERG11 gene with its promoter Funding: This work was supported by grants from the Slovak
was amplified by PCR using genomic DNA of isolates 1 or 29 Research and Developmental Agency (APVV-20-00604, LPP-0022-
and paired primers CgERG11 Prom and CgERG11 End. Amplicons 06, LPP-0011-07 and VVCE-0064-07), the Slovak Grant Agency of
were inserted into the pFL38 centromeric vector as SacI PstI DNA Science (VEGA 1/3250/06) and Comenius University (Grant UK
fragments. The resulting plasmids, containing either CgERG11- 342/08).
578 N. Berila et al. / International Journal of Antimicrobial Agents 33 (2009) 574 578
Competing interests: None declared. resistance in clinical isolates and petite mutants. Antimicrob Agents Chemother
2006;50:1384 92.
Ethical approval: Not required.
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