Mutations in the CgPDR1 and CgERG11 genes in azole-resistant C.glabrata.pdf

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

article info

abstract

Article history:

Received 14 August 2008

Accepted 24 November 2008

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

Keywords:

Azole resistance

Candida glabrata

CDR1

ERG11

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.

1. Introduction

2. Materials and methods

Candida glabrata is the secondmost important human pathogen

responsible for candidaemia [1] . This species is evolutionarilymore

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-

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

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 frommutations 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.

ura3

mutant strain [7] were used as controls. Saccharomyces cerevisiae

Y26604 ( MATa / MAT

his3

1 / his3

1 leu2

0 / leu2

0lys2

0 / LYS2

0 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 ◦ Cor37 ◦ C in either

complete YEPDmedium (2% Bacto-Peptone, 1% yeast extract and 2%

glucose), in RPMI 1640 mediumwith

0ura3

0 / ura3

-glutamate [without sodium

bicarbonate supplemented with 2% glucose and buffered to pH 7.0

with 0.165Mmorpholinepropanesulfonic 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

∗

Corresponding author. Tel.: +421 2 6029 6631; fax: +421 2 6542 9064.

E-mail address: subik@fns.uniba.sk (J. Subik).

doi: 10.1016/j.ijantimicag.2008.11.011

MET15 / met15

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

g/mL) a

Fluconazole

Itraconazole

Voriconazole

Endotracheal sputum

128

Urine

0.25

0.75

Tonsil

128

>32

Urine

0.125

Endotracheal sputum

0.25

0.75

Tissue

0.25

1.5

Endotracheal sputum

128

Endotracheal sputum

0.5

Tongue

0.125

0.38

Oral cavity

0.25

0.5

Trachea

0.125

0.5

Vagina

0.5

Endotracheal sputum

0.25

>32

Abscess

>32

Endotracheal sputum

>32

Tracheal cannula

>32

Endotracheal sputum

Tonsil

>32

Tonsil

>32

Urine

128

0.5

0.75

Endotracheal sputum

128

>32

Endotracheal sputum

128

>32

Blood

128

>32

Endotracheal sputum

0.75

Trachea

128

>32

Urine

1.5

Urine

128

>32

Vagina

0.38

Vagina

0.38

Vagina

1.5

Vagina

1.5

Vagina

1.5

Vagina

0.75

Vagina

0.5

0.75

Vagina

0.25

Vagina

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,

≤

g/mL; SDD, 16–32

g/mL; resistant

≥

g/mL; itraconazole, susceptible

≤

0.125

g/mL; SDD, 0.25–0.5

g/mL;

resistant

≥

g/mL; voriconazole, susceptible

≤

g/mL; resistant

≥

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).

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 -

TAATATT GAGCTC CGAAGAGGTACGAAACATCC-3 (forward) and

CgERG11 End 5 -TATTACT CTGCAG TGGGATCAACCAACTTTGTC-3

(reverse) containing ﬂanking restriction sites for Sac I and Pst I

(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 F5 -GGTAAATCAAAACCAACAGGGA-3

(forward) and CgPDR1-RI 5 -GACAATGGAATCGTAATCGCTC-3

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

CgPDR1 F5 -GGTAAATCAAAACCAACAGGGA-3

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-

mentedwith2%glucose and the zone inhibitionassays onAntibiotic

Medium3were used for determinationof 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] .

(forward) and

CgPDR1-R 5 -CCGATAAGGGAGATGCAGTT-3

(reverse). Resulting

MIC 80 (

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 datawere comparedwith

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.

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 amaximumof 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

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.

3.3. 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 C gPDR1 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

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

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.

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.

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

29, 30, 32

CgPDR1

C705T

–

C765T

–

C837T

–

T871C

–

C1039T

L347F

C1726T

–

H576Y

C1749T

–

A2319T

–

T2578C

–

T2994C

–

G3156A

–

CgERG11

C588T

–

C588T

–

T768C

–

C918T

–

C918T

–

G927A

–

A1023G

–

A1505T

–

E502V

T1557A

–

ura3 mutant strain was co-

transformed with a gapped pCgPDR1 4672 plasmid [7] (restricted

either by Dra III or Dra III and Pac I 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 )

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 associatedwith 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 newmutations 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.

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 Sac I– Pst IDNA

fragments. The resulting plasmids, containing either CgERG11-

≥

128

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).

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

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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