Microarray and Molecular Analyses of the Azole Resistance Mechanism in C.glabrata.pdf

A NTIMICROBIAL A GENTS AND C HEMOTHERAPY , Aug. 2010, p. 3308–3317

Vol. 54, No. 8

0066-4804/10/$12.00 doi:10.1128/AAC.00535-10

Timothy G. Myers, 2 Kieren A. Marr, 3 and John E. Bennett 1 *

Clinical Mycology Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases,

National Institutes of Health, Bethesda, Maryland 20892 1 ; Genomic Technologies Section, Research Technologies Branch,

National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892 2 ; and

Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 3

Xiaozhen Zhang, 1

Sara D. Sufﬁs, 1

Qin Su, 2

Received 20 April 2010/Returned for modiﬁcation 15 May 2010/Accepted 30 May 2010

DNA microarrays were used to analyze Candida glabrata oropharyngeal isolates from seven hematopoietic

stem cell transplant recipients whose isolates developed azole resistance while the recipients received ﬂucon-

azole prophylaxis. Transcriptional proﬁling of the paired isolates revealed 19 genes upregulated in the majority

of resistant isolates compared to their paired susceptible isolates. All seven resistant isolates had greater than

2-fold upregulation of C. glabrata PDR1 (Cg PDR1 ), a master transcriptional regulator of the pleiotropic drug

resistance (PDR) network, and all seven resistant isolates showed upregulation of known Cg PDR1 target genes.

The altered transcriptome can be explained in part by the observation that all seven resistant isolates had

acquired a single nonsynonymous mutation in their Cg PDR1 open reading frame. Four mutations occurred in

the regulatory domain (L280P, L344S, G348A, and S391L) and one in the activation domain (G943S), while two

mutations (N764I and R772I) occurred in an undeﬁned region. Association of azole resistance and the Cg PDR1

mutations was investigated in the same genetic background by introducing the Cg PDR1 sequences from one

sensitive isolate and ﬁve resistant isolates into a laboratory azole-hypersusceptible strain (Cg pdr1 strain) via

integrative transformation. The Cg pdr1 strain was restored to wild-type ﬂuconazole susceptibility when trans-

formed with Cg PDR1 from the susceptible isolate but became resistant when transformed with Cg PDR1 from

the resistant isolates. However, despite the identical genetic backgrounds, upregulation of Cg PDR1 and

Cg PDR1 target genes varied between the ﬁve transformants, independent of the domain locations in which the

mutations occurred. In summary, gain-of-function mutations in Cg PDR1 contributed to the clinical azole

resistance, but different mutations had various degrees of impact on the Cg PDR1 target genes.

Candida glabrata is a haploid yeast and closely related to

Saccharomyces cerevisiae. To date, second to Candida albicans ,

C. glabrata has emerged as the most common cause of blood-

stream infection (candidemia) in many countries (15, 17). Er-

gosterol is an important component of fungal cell membranes,

and the ergosterol biosynthetic pathway has been a primary

target of antifungal drugs, including azoles (e.g., ﬂuconazole)

and allylamines (terbinaﬁne). ERG11 encodes a cytochrome

P-450-dependent C 14 lanosterol demethylase (Erg11p) and is

essential in ergosterol biosynthesis. Azole antifungals inhibit

Erg11p activity and lead to the depletion of ergosterol. How-

ever, C. glabrata possesses intrinsically low susceptibility to

ﬂuconazole compared to C. albicans and frequently further

develops resistance during prolonged treatment with ﬂucon-

azole (4, 16, 18, 19, 27).

Azole resistance in pathogenic fungi has been reviewed re-

cently (14). Drug efﬂux due to ATP-binding-cassette (ABC)

transporters has been found to be a major contributor to azole

resistance in several species, including C. glabrata . C. glabrata

Pdr1p (CgPdr1p), a master transcriptional regulator of pleio-

tropic drug resistance (PDR), contributes to azole resistance

by regulating gene expression of various transporters and plays

a central role in ﬂuconazole resistance acquired by C. glabrata

(8, 25, 28–30). Gain-of-function (GOF) mutations in the tran-

scriptional regulator, CgPdr1p, have been found in C. glabrata

clinical isolates (8, 28) and in a laboratory strain (30). The

mutations have been accompanied by an increased expression

of drug efﬂux pumps and other target genes involved in the

response to xenobiotics. The resistant isolates have varied in

their regulation of the three ABC transporter genes most im-

portant for azole resistance Cg CDR1 , PDH1 (Cg CDR2 ), and

Cg SNQ2 (8, 26).

The relationship between the CgPdr1p protein domain and

downstream effects of these mutations in C. glabrata appeared

worthy of further analysis. We selected seven pairs of isolates

from patients receiving ﬂuconazole prophylaxis following he-

matopoietic stem cell transplantation. Pairs from the same

patient had the same contour-clamped homogeneous electric

ﬁeld (CHEF) gel patterns but differed in ﬂuconazole suscep-

tibility (4). We identiﬁed the nonsynonymous mutations in

Cg PDR1 of the seven clinical pairs and analyzed the transcrip-

tome of each clinical pair by DNA microarray analysis. To

eliminate the possibility that differences within the clinical

pairs were due to mutations other than those in Cg PDR1 ,we

expressed the Cg PDR1 gene from one susceptible isolate and

ﬁve resistant isolates in the same Cg pdr1 host. The impact of

Cg PDR1 GOF mutations on the transcription of Cg PDR1 and

four of the Cg PDR1 target genes was determined by quantita-

tive real-time PCR (qRT-PCR). Despite expression in the

* Corresponding author. Mailing address: NIH, 10 Center Drive,

Building 10, Room 12C103, Bethesda, MD 20892. Phone: (301) 496-

3461. Fax: (301) 480-0050. E-mail: Jbennett@niaid.nih.gov.

Published ahead of print on 14 June 2010.

3308

Lindsay R. Sammons, 1

Microarray and Molecular Analyses of the Azole Resistance Mechanism

in Candida glabrata Oropharyngeal Isolates

Huei-Fung Tsai, 1

V OL . 54, 2010

AZOLE RESISTANCE OF C. GLABRATA 3309

TABLE 1. Candida glabrata strains used in this study

Strain

Parental

strain

Genotype or description

Reference(s) or source

NCCLS84

Wild-type strain 84

ATCC 90030 a

84u

NCCLS84

ura3

CgB4

84u

ura3 cg pdr1 ::Tn5

Cm URA3

Cg1S

Clinical susceptible isolate, Cg12581, pair 1 b

4, 28

Cg2R

Clinical resistant isolate, Cg13928, pair 1 b

4, 28

Cg3S

Clinical susceptible isolate, pair 2 b

Cg4R

Clinical resistant isolate, pair 2 b

Cg5S

Clinical susceptible isolate, pair 3 b

Cg6R

Clinical resistant isolate, pair 3 b

Cg7S

Clinical susceptible isolate, Cg1660, pair 4 b

4, 28

Cg8R

Clinical resistant isolate, Cg4672, pair 4 b

4, 28

Cg11S

Clinical susceptible isolate, pair 6 b

Cg12R

Clinical resistant isolate, pair 6 b

Cg13S

Clinical susceptible isolate, pair 7 b

Cg14R

Clinical resistant isolate, pair 7 b

Cg15S

Clinical susceptible isolate, pair 8 b

Cg16R

Clinical resistant isolate, pair 8 b

Cg17S

Clinical susceptible isolate, pair 9 b

Cg18R

Clinical resistant isolate, pair 9 b

Cg21S

Clinical susceptible isolate, pair 11 b

Cg22R

Clinical resistant isolate, pair 11 b

C1Sa c

CgB4

ura3 Cg PDR1 -Cg1S

This study

C1Sb c

CgB4

ura3 Cg PDR1 -Cg1S

This study

C4Ra c

CgB4

ura3 Cg PDR1 -Cg4R

This study

C4Rb c

CgB4

ura3 Cg PDR1 -Cg4R

This study

C6Ra c

CgB4

ura3 Cg PDR1 -Cg6R

This study

C6Rb c

CgB4

ura3 Cg PDR1 -Cg6R

This study

C14Ra c

CgB4

ura3 Cg PDR1 -Cg14R

This study

C14Rb c

CgB4

ura3 Cg PDR1 -Cg14R

This study

C16Ra c

CgB4

ura3 Cg PDR1 -Cg16R

This study

C16Rb c

CgB4

ura3 Cg PDR1 -Cg16R

This study

C18Ra c

CgB4

ura3 Cg PDR1 -Cg18R

This study

C18Rb c

CgB4

ura3 Cg PDR1 -Cg18R

This study

a American Type Culture Collection, Manassas, VA.

b Comparsion of the more susceptible and more resistant isolates within each paired clinical isolate.

c Complementation by integrative transformation. Two independent complemented transformants were selected for each Cg PDR1 GOF mutation, labeled “a” and “b.”

same host, the GOF mutations differed in the upregulation

of Cg PDR1 and in the upregulation of its four target genes.

Microarray analysis. DNA microarray analysis was used to identify genes with

altered expression in the resistant clinical isolates and the Cg PDR1 -comple-

mented strains. Total RNA was isolated from the log phase culture of C. glabrata

grown in YPD by using Trizol (Invitrogen) and the RNeasy MiniElute cleanup

kit (Qiagen, Valencia, CA). Pin-spotted 70-mer oligonucleotide in-house arrays

fabricated at the NIAID were used for analysis of clinical pairs initially, but later,

Agilent custom arrays (Agilent Technologies, Santa Clara, CA) were used for

analysis of the Cg PDR1 -complemented strains, as the in-house printing of arrays

was discontinued.

For the in-house microarrays, a total of 5,908 70-mer oligonucleotides were

purchased from Institut Pasteur (Paris, France) and were used for microarray

printing at the NIAID Microarray Research Facility. Expression of each open

reading frame (ORF) is measured by hybridization to a speciﬁc 70-mer oligo-

nucleotide (7, 12). Thirty micrograms of total RNA from sensitive isolates and

resistant isolates was reversed transcribed to cDNA to incorporate the ﬂuores-

cent Cy3-dUTP and Cy5-dUTP (GE Health Care, Piscataway, NJ), respectively.

The labeled cDNA of paired sensitive/resistant isolates was combined and used

for microarray hybridization. Each group consisted of a sensitive/resistant pair

with ﬁve microarrays, including one or two with reciprocal labeling. The microar-

rays were prehybridized at 42°C in prehybridization buffer (5 SSC [1 SSC is

0.15 M NaCl plus 0.015 M sodium citrate], 1% bovine serum albumin [BSA],

0.1% SDS) for 30 to 60 min and then hybridized to the labeled cDNA in 50 l

of hybridization buffer (25% formamide, 5 SSC, 0.2% SDS, 20

MATERIALS AND METHODS

Strains and culture conditions. Plasmids were maintained in Escherichia coli

XL1-Blue (Stratagene, La Jolla, CA), TOP10 (Invitrogen, Carlsbad, CA), or

TOP10F

(Invitrogen) host cells grown in LB with 50

g/ml ampicillin, 50

g/ml

g/ml chloramphenicol, depending on the plasmids.

Candida glabrata strains, including four strains from a previous study (Table

1), were cultured on either yeast extract-peptone-dextrose (YPD) containing 1%

Bacto yeast extract (Difco Laboratories, Detroit, MI), 2% Bacto peptone (Difco

Laboratories), and 2% glucose (Sigma, St. Louis, MO) or minimum medium

(MIN) containing 0.67% yeast nitrogen base without amino acids (Difco Labo-

ratories) plus 2% glucose.

The seven pairs of oropharyngeal sequential isolates were chosen for study

because each pair came from an individual hematopoietic stem cell transplant

recipient receiving ﬂuconazole (FHCRC protocol number 954). The more resis-

tant isolate of each pair acquired increased ﬂuconazole resistance during ther-

apy, while the karyotype remained unchanged from its paired more-susceptible

isolate (4).

Drug sensitivity assay. MIC of ﬂuconazole (courtesy of Pﬁzer, Sandwich,

United Kingdom) was determined with the CSLI (formerly NCCLS) microtiter

test by using the MIC producing 80% growth reduction (MIC 80 ) as the MIC; the

test was modiﬁed by addition of 2% glucose to the buffered RPMI medium

(Sigma) and incubation at 37°C for 48 h with 250 cells of inoculum per well. In

the case of the ura3 mutant, the RPMI medium was supplemented with 20

g/ml

g/ml yeast tRNA) over-

night at 42°C. The microarrays were washed three times in wash buffer A (1

g/ml Cot-1 DNA [Invitrogen], 80

SSC, 0.05% SDS) and washing buffer B (0.1 SSC). The in-house arrays were

scanned with a GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA). All

microarray data archive and analyses were done in the Web-based mAdb system

g/ml

of uracil (Sigma).

kanamycin, or 12.5

poly[dA] 40–60 , 200

3310

TSAI ET AL.

A NTIMICROB .A GENTS C HEMOTHER .

TABLE 2. Primers and TaqMan probes used in this study

Primer or probe

Sequence (5 –3 )

DNA sequence analysis of Cg PDR1 . Genomic DNA from the seven pairs of

oropharyngeal isolates was used as the templates for PCR to amplify the

Cg PDR1 ORF as well as its 2.5-kb promoter region. PfuUltra High-Fidelity DNA

polymerase (Stratagene) was used for PCR ampliﬁcation for reducing the gen-

eration of mutations during PCR ampliﬁcation. Two independent PCR ampliﬁ-

cations were performed for each isolate to obtain the DNA for sequencing.

Primer set PDR8S and PDR5AS (Table 2) was used for amplifying the ORF

region with the following parameters: 95°C for 2 min; 30 cycles of 95°C for 30 s,

53°C for 30 s, 72°C for 5 min, with an extension on the last cycle at 72°C for 10

min. Primer set PDR9S and PDR17AS was used for amplifying the promoter

region with the same parameters as described above. The PCR products were

then sequenced and analyzed.

Plasmid construction. The plasmid pCgACU-P2F5 carrying the Cg PDR1 gene

on a 8-kb KpnI DNA fragment from the clinical isolate Cg8R (Cg4672) was used

as the backbone vector (11). For the cloning of CgPDR1 from several clinical

isolates, the Cg PDR1 ORFs from one susceptible isolate (Cg1S) and two resis-

tant isolates (Cg4R and Cg18R) with mutations in the regulatory domain (RD)

were obtained by PCR using the primers PDR8S and PDR5AS as described

above. The PCR products were digested with DraIII, and the 0.9-kb DraIII DNA

fragments were then cloned into the DraIII site of pCgACU-P2F5 to give

pCgACU-Cg1S, pCgACU-Cg4R, and pCgACU-Cg18R, respectively. The

Cg PDR1 ORFs from the resistant isolates with mutations in the activation do-

main (Cg6R) or undeﬁned region (Cg14R and Cg16R) were obtained by PCR

using the primer set PDR8S and PDR5AS and the parameters described above.

The PCR products were digested with HpaI and PacI. The 1.2-kb HpaI-PacI

DNA fragment containing the activation domain and the undeﬁned region was

then cloned into the HpaI-PacI site of pCgACU-1S to give the plasmids

pCgACU-Cg14R, pCgACU-Cg16R, and pCgACU-Cg6R, respectively.

Cg PDR1 complementation. The Cg PDR1 of clinical isolates was introduced

into a laboratory cg pdr1 mutant (CgB4) (28), which allowed us to determine the

impact of the Cg PDR1 mutations on ﬂuconazole resistance in the same genetic

background. To introduce the Cg PDR1 into the Tn

PCR and sequencing

primers

CgPDR1AS ..............................GGACAGAAATTGGAACATCG

CgPDR2S .................................TATCCTAAGTATGGACAACG

CgPDR4AS ..............................GATTCCTTAAGCCCGATAAG

CgPDR5AS ..............................GGTTACACCACT ACTAGT TG a

CgPDR8S .................................GGTG GAGCTC TTTAGCTACGTTATT

GAG a

CgPDR9S .................................TGAGATGAAAGCAATAACTG

CgPDR10S ...............................TCAGTACTACACCTGAGTTG

CgPDR15AS ............................AATCGTTGTCCATACTTAGG

CgPDR16AS ............................ACACTCTCAATAAACGGTTG

CgPDR17AS ............................GTCAATGGATGATTTTATCG

CgPDR18AS ............................ACAAGGTTTTAGCCCATTAC

CgPDR19AS ............................TAATACCTAGTTTTACCCAC

CgPDR21AS ............................AGTATTCCCAACAGTATGAG

CgPDR22AS ............................ATGCTTAGTCTCTGCTCAC

CgPDR24S ...............................ATGTCCTTATCACTAGGTC

qRT-PCR probes

CgACT1P .................................CCACGTTGTTCCAATTTACGCCGG

CgPDR1P .................................TCGAATATTATGCACCATCATGTCTGTG

TTTAGCT

CgCDR1P ................................TTATCTGCTGCGATGGTTCCTGCTTCC

PDH1P .....................................CAGGCTCACATGCAAACCAAGACTA

CCAT

CgSNQ2P .................................CCGATGGTGACGATGCGCACAG

CgYOR1P ................................CTCGCCGGTGCAGGATTACGATCTAGA

qRT-PCR primers

CgACT1F .................................TTGGACTCTGGTGACGGTGTTA

CgACT1R ................................AAAATAGCGTGTGGCAAAGAGAA

CgPDR1F.................................AACGATTATTCAATTGCAACAACG

CgPDR1R ................................CCTCACAATAAGGAAAGTCTGCG

CgCDR1F ................................AGATGTGTTGGTTCTGTCTCAAAGAC

CgCDR1R................................CCGGAATACATTGACAAACCAAG

PDH1F .....................................AATGGATGTTAGAAGTAGTTGGAGCAG

PDH1R.....................................TGTTCGGAATTTCTCCACACCT

CgSNQ2F .................................GCGGAAGATCGCACGAAG

CgSNQ2R ................................GGCGCGAGCGGGATA

CgYOR1F ................................CGCTGGGAAGGCCAAGA

CgYOR1R................................CTCCCCGGACGTCAGAATAG

a Underlined bases are the restriction sites.

-disrupted

cg pdr1 locus in CgB4, the constructs containing Cg PDR1 from the susceptible

isolate (pCgACU-Cg1S) and ﬁve resistant clinical isolates (pCgACU-Cg4R, pC-

gACU-Cg6R, pCgACU-Cg14R, pCgACU-Cg16R, and pCgACU-Cg18R) were

digested with HindIII, and the 2.9-kb HindIII DNA fragments containing the

partial Cg PDR1 ORFs were used to transform the cg pdr1 mutant CgB4, which is

highly susceptible to ﬂuconazole. Putative transformants were obtained based on

the restoration of wild-type ﬂuconazole susceptibility at 50

Cm URA3

g/ml and resistance

to ﬂuoroorotic acid (FOA) (28). To screen for the replacement of Tn

by Cg PDR1 , FOA-resistant transformants were obtained and analyzed

by PCR using the primer set CgPDR2S and CgPDR4AS with the following

parameters: 95°C for 2 min; 35 cycles of 95°C for 30 s, 53°C for 30 s, and 72°C for

2 min; with extension on the last cycle at 72°C for 10 min. Southern hybridization

and DNA sequence analysis were done to conﬁrm the Cg PDR1 gene replace-

ment (data not shown).

qRT-PCR. Total RNA was isolated from C. glabrata log phase cultures grown

in MIN rather than those grown in YPD to increase RNA purity. The total RNA

was treated with DNase to remove the minute contamination of genomic DNA

prior the reverse transcription with a high-capacity cDNA archive kit (Applied

Biosystems, Foster City, CA). The parallel ampliﬁcation between Cg ACT1 and

the gene of interest was conﬁrmed for each with probe-primer sets. Quantitative

real-time PCR (qRT-PCR) was used to determine the expression level of

Cg ACT1 ,Cg PDR1 ,Cg CDR1 , PDH1 ,Cg SNQ2 , and Cg YOR1 in C. glabrata . The

sequences of TaqMan probes and forward and reverse primers are listed in Table

2. Cg ACT1 was used as an internal control for normalization. The threshold cycle

(2 CT ) method was used for calculating the differences in gene expression.

Techniques and reagents. C. glabrata genomic DNA was isolated from over-

night cultures grown in YPD by using the MasterPure yeast puriﬁcation kit

(Epicentre, Madison, WI). Puriﬁed DNA fragments were recovered using the

Strataprep gel DNA extraction kit (Stratagene). Hybond-N nylon membranes

(Amersham, Arlington Heights, IL) were used for Southern hybridization anal-

yses. DNA probes were labeled with [ - 32 P]dCTP or [ - 32 P]dATP (MP Biomed-

ical, Solon, OH) by using the Prime-It II kit (Stratagene). DNA cloning and

hybridization analyses were done according to the standard protocol (20). DNA

sequencing was done using the DNA sequencing kit with a dRhodamine dye

terminator (Applied Biosystems) and an ABI automatic DNA sequencing system

(Perkin-Elmer, Foster City, CA). For sequencing of PCR products, PfuUltra

DNA polymerase (Stratagene) was used for PCR ampliﬁcation to minimize the

rate of PCR-introduced mutations. The PCR products were cleaned with the

Strataprep PCR puriﬁcation kit (Stratagene) and used as the templates for DNA

sequencing.

provided by the Bioinformatics and Molecular Analysis group (BIMAS) at the

Center for Information Technology (CIT), NIH. The data were ﬁltered with the

parameters that included genes present in three or more arrays per group and

each array with 80% or more genes present. The data set of each of the paired

isolates was then analyzed by Student’s t test. The genes with P values less than

0.001 and with at least 2-fold altered gene expression were then selected. The

ﬁnal data set included all the genes with altered expression in at least one clinical

pair.

For Agilent custom microarrays, the array probes were designed against all

NCBI Reference Sequence (RefSeq) mRNA sequences available for C. glabrata

CBS138 as of September 2008. Sixty-base DNA sequences were selected using

the e-Array software (Agilent Technologies), specifying one “best probe” per

transcript, “base composition method,” and “3-prime bias.” Custom microarrays

with 5,125 unique probes (one for each target transcript), replicated to eight spot

features per probe, were manufactured by Agilent in the 4

blocking

reagent at 65°C for 17 h and washed with Agilent gene expression wash buffer 1

at room temperature and gene expression wash buffer 2 at 37°C. Then slides were

dried under nitrogen gas for 3 min at 30°C. The slides were imaged using Agilent

high-resolution DNA microarray scanner G2505C at 5-

gene expression hybridization HI-RPM buffer and 10

m resolution with both

100% and 10% photomultiplier tubes. Agilent Feature Extraction software was

used for image analysis. Statistical calculations were performed on the “pro-

cessed signal” data by using the mAdb analysis system provided by the BIMAS

group at the CIT, NIH. Data were ﬁltered with the parameters that included

genes present in three or more arrays per group and each array with 80% or

more genes present.

URA3

44K format. Ten

micrograms of total RNA was used for each ﬂuorescent labeling. Each group

constituted of four microarrays, including one with reciprocal labeling. Microar-

rays were hybridized using the Tecan HS Pro 4800 hybridization station with

Agilent 2

V OL . 54, 2010

AZOLE RESISTANCE OF C. GLABRATA 3311

TABLE 3. Cg PDR1 mutations and ﬂuconazole susceptibilities of clinical isolates

Domain/region

Isolate

Fluconazole

susceptibility a

MIC 80

( g/ml)

Codon

Amino acid

substitution

Amino acid property

Regulatory

Cg21S

Susceptible

TTG

Nonpolar, aliphatic

Cg22R

Resistant

256

TT T

L280F

Nonpolar, aromatic

Cg7S b

Susceptible

32–64

TGG

Nonpolar, aromatic

Cg8R b

Resistant

512

T C G

W297S

Polar-neutral

Cg17S

Susceptible

TTG

Nonpolar, aliphatic

Cg18R

Resistant

256

T C G

L344S

Polar-neutral

Cg11S

Susceptible

GGT

Nonpolar, aliphatic

Cg12R

Resistant

256

G C T

G348A

Nonpolar, aliphatic

Cg3S

Susceptible

TCG

Polar-neutral

Cg4R

Resistant

256

T T G

S391L

Nonpolar, aliphatic

FSTF c

Cg1S b

Susceptible

TTC

Nonpolar, aromatic

Cg2R b

Resistant

128

C TC

F575L

Nonpolar, aliphatic

Undeﬁned

Cg13S

Susceptible

AAT

Polar-neutral

Cg14R

Resistant

256

A T T

N764I

Nonpolar, aliphatic

Cg15S

Susceptible

AGA

Polar-basic

Cg16R

Resistant

128

A T A

R772I

Nonpolar, aliphatic

Activation

Cg5S

Susceptible

GGT

Nonpolar, aliphatic

Cg6R

Resistant

256

A GT

G943S

Polar-neutral

a Comparison of the more susceptible and more resistant isolates within each paired clinical isolate.

b Reported previously.

c FSTF, fungus-speciﬁc transcriptional factor.

Nucleotide sequence accession numbers. The GenBank accession numbers for

the Cg PDR1 DNA sequences are HM17911 to HM17924. The array layout and

probe sequences have been uploaded to the NCBI GEO microarray repository.

The GEO accession number for the Agilent Cgda array is GPL10325. The

accession number for the in-house array Cgaa is GPL8174. The GEO accession

number for the in-house Cgaa array data is GSE21352, and the GEO accession

number for the Agilent Cgda array data is GSE21355.

there were two amino acid substitutions in an undeﬁned region

(Cg14R, N764; Cg16R, R772I), which is in the vicinity of a

putative nuclear localization signal (NLS; amino acids 793 to

836) based on its similarity to the NLS of Pdr1p (amino acids

725 to 769) reported in S. cerevisiae (5). Together with the two

mutations we identiﬁed previously in the regulatory and fun-

gus-speciﬁc transcription factor domains (Cg8R, 297S; Cg2R,

F575L) (28), a total of four domains/regions in CgPdr1p were

identiﬁed as potentially being involved in the clinical azole

resistance associated with the PDR network, with the regula-

tory domain being the predominant region for the mutations.

Clinical pairs with the Cg PDR1 mutations in the same do-

main exhibited different transcriptional proﬁles. DNA mi-

croarray analysis was performed to determine the potential

impact of different nonsynonymous Cg PDR1 mutations on the

transcriptional proﬁles of the clinical resistant isolates. Total

RNA of the clinical pairs was reverse transcribed to incorpo-

rate Cy3-dUTP or Cy5-dUTP, which were combined and hy-

bridized to the C. glabrata 70-mer oligonucleotide in-house

microarrays. Figure 1A provides the heat map of 45 genes with

signiﬁcant altered expression in at least one clinical pair. Five

arrays for each of the seven clinical pairs and the expression

ratios are shown in a log 2 scale as either upregulation (in red)

or downregulation (in green) of genes in the resistant isolates

compared to its paired sensitive isolates.

The hierarchical cluster I contained genes upregulated in a

majority of the seven clinical resistant isolates (Fig. 1A, panel

I). Differences in the transcriptional proﬁles among seven clin-

ical pairs were also evidenced in the data set. Cluster II in-

cluded many genes upregulated only in three groups, 22R/21S,

12R/11S, and 4R/3S (Fig. 1A, panel II). It was particularly

striking for the pair 16R/15S, which exhibited many downregu-

lated genes (Fig. 1A, panel II). This was not observed in the

RESULTS

All seven clinical azole-resistant isolates had single nonsyn-

onymous mutations in Cg PDR1. The ﬂuconazole MIC 80 of the

more susceptible clinical isolates ranged from 16 to 64

g/ml (Table 3). To investigate whether Cg PDR1 muta-

tions contributed to the azole resistance in the oropharyngeal

isolates of C. glabrata , the Cg PDR1 ORFs (3.3 kb) of seven

clinical azole-susceptible/azole-resistant pairs were sequenced

along with their promoter regions (1.4 kb). DNA sequence

analysis revealed that each of the seven clinical resistant iso-

lates harbored a single nonsynonymous mutation at various

regions of the Cg PDR1 ORF compared to its paired azole-

sensitive isolates (Table 3). No differences in the promoter

sequences were found. All of the point mutations resulted in

single amino acid substitutions. The majority of the amino acid

substitutions resulted in changes in amino acid properties with

the exception of the pair Cg12R/Cg11S, which retained a non-

polar aliphatic amino acid. Four putative functional domains

(DNA binding, regulatory, fungus-speciﬁc transcriptional

factor, and activation) were proposed in the CgPdr1p based

on its similarity to S. cerevisiae Pdr1p. The majority of amino

acid substitutions, four out of seven, were located in the

regulatory domain (RD) (Cg22R, L280F; Cg18R, L344S;

Cg12R, G348A; and Cg4R, S391L). While only one amino acid

substitution occurred in the activation domain (Cg6R, G943S),

g/ml,

while their paired more-resistant isolates ranged from 128 to

512

3312

TSAI ET AL.

A NTIMICROB .A GENTS C HEMOTHER .

FIG. 1. Microarray analysis of clinical sensitive/resistant paired isolates. (A) Heat map of hierarchical gene clustering. For the seven pairs of

azole-susceptible and -resistant isolates, ﬁve arrays for each pair and the expression ratio of resistant isolate over sensitive isolate are shown in log 2

scale to show upregulation (in red) or downregulation (in green) of the genes in the resistant isolates. RD, regulatory domain; UD, undeﬁned

domain; AD, activation domain. Panel I, genes upregulated in the majority of seven groups; II, genes downregulated in 16R/15S. (B) Heat map

of the genes with altered expression in 16R/15S compared to that of the complemented strains matched to the host wild-type strain 84. C1S, Cg pdr1

mutant complemented by Cg PDR1 from Cg1S; C16R, Cg pdr1 mutant complemented by Cg PDR1 from Cg16R. (C) Heat map and annotation of

the 19 genes upregulated in a majority of the clinical resistant isolates (A, panel I). The C. glabrata locus tags of each ORF represented by the

oligonucleotide as well as its S. cerevisiae homologs are listed on the right. ND, not determined. (D) Putative PDRE motif logo of Cg PDR1 . The

1-kb upstream sequences of 18 upregulated genes were used for the motif search with MEME version 4.3.0. The major motif was obtained with

an E-value of 1.4 10 28 .

other clinical pairs analyzed. Both 14R/13S and 16R/15S had

the mutations in the same region, but the transcriptional pro-

ﬁle of 14R/13S in cluster II appeared more similar to that of

6R/5S than to that of 16R/15S. Similarly, the proﬁle of 18R/17S

was different from those of the other three RD pairs (22R/21S,

12R/11S, and 4R/3S), all of which had the mutations in the

same domain as 18R/17S. In conclusion, the domain/region

locations of Cg PDR1 mutations did not show a direct correla-

tion with the degree of similarity in their transcription proﬁles

based on the microarray analysis.

Downregulation of genes in the clinical isolate Cg16R was

unrelated to the GOF mutation of Cg PDR1. Because the clin-

ical pair Cg16R/Cg15S shared a different gene expression pat-

tern from the other pairs (Fig. 1A, panel II), we wished to

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