pdr1 regulates multidrug resistance in candida …pdr1 regulates multidrug resistance in candida...

Pdr1 regulates multidrug resistance in Candidaglabrata: gene disruption and genome-wide expressionstudies

John-Paul Vermitsky,1 Kelly D. Earhart,2

W. Lamar Smith,1 Ramin Homayouni,3

Thomas D. Edlind1* and P. David Rogers2

1Department of Microbiology and Immunology, DrexelUniversity College of Medicine, Philadelphia, PA, USA.2Department of Pharmacy and PharmaceuticalSciences, College of Pharmacy, and Department ofPediatrics, College of Medicine, University of TennesseeHealth Science Center, Children’s Foundation ResearchCenter at Le Bonheur Children’s Medical Center,Memphis, TN, USA.3Department of Neurology, College of Medicine andCenter for Genomics and Bioinformatics, University ofTennessee Health Science Center, Memphis, TN, USA.

Summary

Candida glabrata emerged in the last decade as acommon cause of mucosal and invasive fungal infec-tion, in large part due to its intrinsic or acquired resis-tance to azole antifungals such as fluconazole. InC. glabrata clinical isolates, the predominant mecha-nism behind azole resistance is upregulated expres-sion of multidrug transporter genes CDR1 and PDH1.We previously reported that azole-resistant mutants(MIC � 64 mg ml-1) of strain 66032 (MIC = 16 mg ml-1)similarly show coordinate CDR1-PDH1 upregulation,and in one of these (F15) a putative gain-of-functionmutation was identified in the single homologueof Saccharomyces cerevisiae transcription factorsPdr1–Pdr3. Here we show that disruption ofC. glabrata PDR1 conferred equivalent fluconazolehypersensitivity (MIC = 2 mg ml-1) to both F15 and66032 and eliminated both constitutive andfluconazole-induced CDR1-PDH1 expression. Rein-troduction of wild-type or F15 PDR1 fully reversedthese effects; together these results demonstrate arole for this gene in both acquired and intrinsic azoleresistance. CDR1 disruption had a partial effect,reducing fluconazole trailing in both strains whilerestoring wild-type susceptibility (MIC = 16 mg ml-1) to

F15. In an azole-resistant clinical isolate, PDR1 dis-ruption reduced azole MICs eight- to 64-fold with noeffect on sensitivity to other antifungals. To extendthis analysis, C. glabrata microarrays were generatedand used to analyse genome-wide expression in F15relative to its parent. Homologues of 10 S. cerevisiaegenes previously shown to be Pdr1–Pdr3 targets wereupregulated (YOR1, RTA1, RSB1, RPN4, YLR346c andYMR102c along with CDR1, PDH1 and PDR1 itself)or downregulated (PDR12); roles for these genesinclude small molecule transport and transcriptionalregulation. However, expression of 99 additionalgenes was specifically altered in C. glabrata F15; theirroles include transport (e.g. QDR2, YBT1), lipidmetabolism (ATF2, ARE1), cell stress (HSP12, CTA1),DNA repair (YIM1, MEC3) and cell wall function(MKC7, MNT3). These azole resistance-associatedchanges could affect C. glabrata tissue-specific viru-lence; in support of this, we detected differences inF15 oxidant, alcohol and weak acid sensitivities.C. glabrata provides a promising model for studyingthe genetic basis of multidrug resistance and itsimpact on virulence.

Introduction

Increasing numbers of individuals are immunocompro-mised in association with AIDS, organ and tissue trans-plantation, aggressive treatments for cancer and immune-related diseases, diabetes, premature birth and advancedage. These individuals are at high risk for opportunisticfungal infection, in particular mucosal or systemic candidi-asis. In the previous decade, Candida glabrata emerged asa common cause of these infections (10–30% of yeastisolates), trailing only Candida albicans (Pfaller et al.,1999; Safdar et al., 2001; Ostrosky-Zeichner et al., 2003;Richter et al., 2005). In some populations such as diabeticsand the elderly, C. glabrata may be the dominant pathogen(Diekema et al., 2002; Kontoyiannis et al., 2002; Grimoudet al., 2005; Goswami et al., 2006). In C. glabrata can-didaemia, mortality rates of 38–53% have been reported(Viscoli et al., 1999; Safdar andArmstrong, 2002; Klingsporet al., 2004). Nevertheless, the basis for C. glabrata patho-genicity is not yet clear, because it is deficient in the

Accepted 10 May, 2006. *For correspondence. E-mail [email protected]; Tel. (+1) 215 9918377; Fax (+1) 215 8482271.

Molecular Microbiology (2006) 61(3), 704–722 doi:10.1111/j.1365-2958.2006.05235.xFirst published online 27 June 2006

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd

virulence factors implicated in C. albicans infection: dimor-phism, strong adhesion, secreted hydrolases and biofilmformation (Douglas, 2003; Nikawa et al., 2003; Kaur et al.,2005; Schaller et al., 2005). On the other hand, C. glabratademonstrates relative resistance to azoles, the mostwidely used antifungal group which includes topical imida-zoles such as miconazole and oral/parenteral triazolessuch as fluconazole. Specifically, the fluconazole MICinhibiting 50% of clinical isolates is 8 mg ml-1, comparedwith 0.25 mg ml-1 for C. albicans (Ostrosky-Zeichner et al.,2003; Pfaller et al., 2004). Azoles inhibit lanosterol dem-ethylase, product of the ERG11 gene (CYP51 in moulds),which results in depletion of the membrane componentergosterol and accumulation of toxic sterol products (forreview, see Akins, 2005). The emergence of C. glabrata(from � 5% of yeast isolates in the 1980s) parallels theintroduction in the early 1990s of fluconazole and over-the-counter imidazoles, along with widespread application ofagricultural azole fungicides. Indeed, its intrinsic low-levelazole resistance, the molecular basis for which remainsundefined, may represent a C. glabrata ‘virulence factor’.

Candida glabrata also demonstrates a high capacity foracquired high-level azole resistance, with 8–27% of iso-lates demonstrating a fluconazole MIC � 64 mg ml-1

(Safdar et al., 2002; Ostrosky-Zeichner et al., 2003;Pfaller et al., 2004). RNA analysis of these clinical isolatessuggests that the predominant basis for acquired azoleresistance is the constitutively upregulated expression ofmultidrug transporter genes CDR1 and, to a lesser extent,PDH1 (Miyazaki et al., 1998; Sanglard et al., 1999; 2001;Redding et al., 2003; Bennett et al., 2004; Vermitsky andEdlind, 2004; Sanguinetti et al., 2005). In support of this,CDR1 or CDR1-PDH1 disruption was shown to conferazole hypersensitivity (Sanglard et al., 2001; Izumikawaet al., 2003). In this respect, C. glabrata resemblesC. albicans and other fungi in which azole resistance hasbeen attributed to upregulated expression of multidrugtransporters (Akins, 2005). Initial laboratory studies ofC. glabrata acquired azole resistance using standardglucose-supplemented medium yielded avirulentrespiratory-deficient mitochondrial mutants (Sanglardet al., 2001; Brun et al., 2005). Using glycerol-supplemented medium, we isolated respiratory-competent mutants with coordinately upregulated CDR1-PDH1 analogous to that observed in azole-resistantclinical isolates (Vermitsky and Edlind, 2004). Coordinateupregulation of these genes was also observed followingbrief exposure of susceptible cells to azoles, representinga potential basis for intrinsic low-level resistance.

Coordinate CDR1-PDH1 upregulation implies acommon transcription factor. Although very distinct interms of niche and human pathogenicity, C. glabrata is anevolutionary close relative of Saccharomyces cerevisiae(Barns et al., 1991; Dujon et al., 2004). In the latter, the

coordinate upregulation of multidrug transporter genesPDR5 and SNQ2 is mediated by the paralogous Pdr1 andPdr3 zinc cluster transcription factors (Kolaczkowska andGoffeau, 1999). Many gain-of-function mutations withinPdr1–Pdr3 have been identified that result in constitutiveupregulation of PDR5-SNQ2 along with a diverse group ofadditional genes (Carvajal et al., 1997; DeRisi et al., 2000;Devaux et al., 2001). Our analysis of the recently releasedC. glabrata genome sequence (Dujon et al., 2004)revealed a single PDR1–PDR3 homologue, and a puta-tive gain-of-function mutation in this gene was identified inazole-resistant laboratory mutant F15 (Vermitsky andEdlind, 2004). Here we demonstrate the central role ofC. glabrata PDR1 in acquired azole resistance, and iden-tify a likely role in intrinsic resistance, by characterizingDpdr1 derivatives of laboratory strains and clinicalisolates. Furthermore, we report the first application ofmicroarrays to this organism, which identified multiplegenes coregulated with CDR1-PDH1 that are likely toimpact C. glabrata virulence.

Results and discussion

PDR1 disruption in F15 and parent

The laboratory selection of spontaneous fluconazole-resistant mutants of C. glabrata ATCC strain 66032 waspreviously described (Vermitsky and Edlind, 2004). Oneof these mutants, F15, exhibited strong upregulation ofCDR1 and PDH1, modest upregulation of PDR1, and asingle base change predicted to alter the Pdr1 amino acidsequence. We reasoned that disruption of PDR1 in F15and parent 66032 would provide an initial test of thehypothesis that this single base change is responsible forthe fluconazole resistance. To accomplish this, ura3derivatives of F15 and 66032 were isolated by selectionon 5-fluoroorotic acid (5FOA) and screening for comple-mentation by a URA3-encoding plasmid. Homologousrecombination is relatively non-specific in C. glabrata,especially with short homology regions, but can beenhanced by promoter-dependent disruption of genes(PRODIGE) as previously described (Edlind et al., 2005).This method was used to disrupt PDR1 (Fig. 1A). Trans-formants were screened by polymerase chain reaction(PCR); loss of the PDR1uF-PDR1iR product and genera-tion of the PDR1uF-URA3iR product confirmed PDR1disruption (Fig. 1B).

Broth microdilution assays were used to examine flu-conazole susceptibility of F15Dpdr1, 66032Dpdr1 andtheir parents (Fig. 1C). Similar to previous results withtheir parents (Vermitsky and Edlind, 2004), the 66032 andF15 ura3 derivatives generated 24 h fluconazole MICs of8–16 and � 64 mg ml-1 respectively. In contrast, theirDpdr1 derivatives were fluconazole hypersusceptible, with

Pdr1 regulates multidrug resistance in Candida glabrata 705

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 61, 704–722

equivalent MICs of 2 mg ml-1. Although susceptible, 66032exhibited trailing growth typical of many Candida species(Rex et al., 1998), and by 48 h was fully grown at allfluconazole concentrations tested (Fig. 1C). Trailinggrowth was absent in the PDR1 disruptants. These resultssupport the role of Pdr1 in F15 fluconazole resistance.Furthermore, the reduced MIC and trailing growth asso-ciated with PDR1 disruption in 66032 suggests that Pdr1is an important contributor to the intrinsic low-level resis-tance that is characteristic of this species.

As hypothesized, RNA analysis showed that PDR1 dis-ruption reversed the constitutive upregulation of CDR1and PDH1 in untreated mutant F15 (Fig. 2). Moreover,expression of these genes was reduced relative to theirexpression in untreated parent 66032. This can explainthe greater susceptibility of the Dpdr1 derivatives relativeto 66032. As previously described (Vermitsky and Edlind,2004), fluconazole treatment induced the expression ofCDR1 and PDH1, most clearly in strains 66032 andF15 respectively (Fig. 2A). PDR1 disruption completely

Fig. 1. Disruption of PDR1 and effects onazole sensitivity.A. Diagram illustrating PRODIGE primerdesign for disruption of PDR1 with URA3coding sequence amplified from pRS416. Alsoshown are the upstream forward and twointernal reverse primers used to screentransformants.B. PCR screen of representative Dpdr1transformants selected on DOB-URA and theirparents 66032 and F15. DNA was purifiedfrom isolated colonies, amplified with theindicated primers pairs, and analysed by gelelectrophoresis; loss of the PDR1uF-PDR1iRproduct and formation of thePDR1uF-URA3iR product identified Dpdr1clones.C. Broth microdilution assays examiningfluconazole sensitivities of parent 66032,azole-resistant mutant F15, and theirrespective Dpdr1 disruptants. Absorbance at630 nm was recorded after 24 or 48 hincubation as indicated; growth was plotted aspercentage of drug-free control.

A

B

C

24 48

706 J.-P. Vermitsky et al.


blocked this treatment-dependent upregulation. Finally,we note that PDR1 itself, which is upregulated in F15(Vermitsky and Edlind, 2004), is also induced by flucona-zole treatment in 66032 and F15 (Fig. 2A).

CDR1 disruption

To more directly assess the role in acquired or intrinsicazole resistance of multidrug transporter gene CDR1, itwas similarly disrupted in the ura3 derivatives of 66032and F15 (Fig. 3A). This reversed the fluconazole resis-tance of F15 (Fig. 3B), although the MIC (16 mg ml-1)remained eightfold above that observed with PDR1 dis-ruption (Fig. 1C). With respect to 66032, CDR1 disruptionhad minimal effect on fluconazole MIC at 24 h, but trailinggrowth most apparent at 48 h was eliminated as it waswith PDR1 disruption. These results are consistent withCDR1 being a major but not exclusive contributor to F15azole resistance.

PDR1 replacement

To rigorously test the role of F15 PDR1 in azole resis-tance, we employed gene replacement. The 66032Dpdr1strain (see above) was transformed with PCR productsrepresenting wild-type and F15 PDR1, including 5′ and 3′flanking sequences which should direct PDR1 to its nativelocus (Fig. 4A). We initially attempted to select homolo-

gous recombinants on fluconazole-containing medium.However, this was precluded by a background of sponta-neous fluconazole-resistant mutants in control (no addedDNA) transformations (see below for further characteriza-tion of these mutants). As an alternative, the protein syn-thesis inhibitor cycloheximide is a known substrate forCdr1-like multidrug transporters, and indeed C. glabrataDpdr1 strains are cycloheximide-hypersensitive (Edlindet al., 2005). In contrast to fluconazole, cycloheximide-containing plates yielded no spontaneous mutants whilefive or six transformants were obtained with addition ofwild-type or F15 PDR1 respectively. PCR screening ofthese transformants confirmed homologous recombina-tion into the native locus (Fig. 4B). All F15 PDR1 replace-ments demonstrated fluconazole resistance comparableto F15 itself, while all but one of the wild-type PDR1replacements demonstrated wild-type sensitivity (Fig.4C). Sequencing of a representative F15 PDR1 replace-ment confirmed there were no mutations other than thepreviously described P927L (Vermitsky and Edlind, 2004).

Characterization of Pdr1-independent azole resistance

As noted above, a background of resistant mutants aroseon fluconazole-containing YP-glycerol medium in controltransformations of strain 66032Dpdr1, which involvedplating c. 2 ¥ 107 cells. To more rigorously examine thisPdr1-independent resistance, equivalent numbers

Fig. 2. Expression of multidrug transportergenes CDR1 and PDH1 and transcriptionalactivator gene PDR1 in parent 66032, mutantF15 and their respective Dpdr1 disruptants.A. RNA was isolated from log phase cultures,slot-blotted to membranes, and hybridized tothe indicated gene probes; ACT1 served asloading control. U, untreated cultures; T,treated with 256 mg ml-1 fluconazole for 2.5 h.B. Quantitative real-time RT-PCR analysis ofrelative CDR1 and PDH1 expression in F15versus 66032, F15Dpdr1 versus 66032, and66032Dpdr1 versus 66032. Data are shownas mean ± SD.

A

B



(3 ¥ 105) of 66032 and 66032Dpdr1 cells were plated onYP-glycerol medium with fluconazole ranging from 0 to256 mg ml-1 (Vermitsky and Edlind, 2004). After 4 daysincubation, the MIC was 32 mg ml-1 for 66032, and about30 mutant colonies (frequency = 1 ¥ 10-4) were observedon each of the four plates at or above this concentration.With 66032Dpdr1, the MIC was 4 mg ml-1, one or twocolonies were observed at 4 and 8 mg ml-1, and no coloniesat 16–256 mg ml-1 (frequency � 3 ¥ 10-6). Thus, Pdr1-independent azole resistance occurs at significantlyreduced frequency.

PDR1 disruption in azole-resistant clinical isolates

Strain BG14, a model for C. glabrata pathogenesis (e.g.Domergue et al., 2005), is a ura3 derivative of a clinicalisolate from a patient who failed fluconazole therapy(Cormack and Falkow, 1999). Consistent with this, BG14is fluconazole-resistant (MIC = 256 mg ml-1), the molecu-

lar basis for which is unknown. PDR1 disruption in BG14,conferring cycloheximide hypersensitivity, was previouslyreported (Edlind et al., 2005). Here we show that thisdisruption also largely reversed BG14 azole resistance.The fluconazole MIC decreased 16-fold to 16 mg ml-1

(Fig. 5A); i.e. comparable to the typical clinical isolatebut eightfold above that observed for 66032Dpdr1(above). Ketoconazole, itraconazole and miconazoleMICs were similarly reduced in BG14Dpdr1, but suscep-tibilities to unrelated antifungals terbinafine, caspofunginand amphotericin B were unchanged. Expression ofCDR1 and ERG11 was examined by RNA hybridization(Fig. 5B). In BG14, constitutive expression of CDR1appeared to be modestly upregulated, but remainedresponsive to fluconazole-dependent upregulation. Bothof these were strongly reduced in the Dpdr1 derivative,while no effects on ERG11 expression were observed.

Strain 8512 represents a second azole-resistant clinicalisolate with high constitutive CDR1-PDH1 expression (Ver-

A

B

24 48

Fig. 3. Disruption of CDR1 and effects on azole sensitivity.A. PCR screen of representative Dcdr1 transformants selected on DOB-URA and their parents 66032 and F15. DNA was purified from isolatedcolonies, amplified with the indicated primers pairs, and analysed by gel electrophoresis; loss of the CDR1uF-CDR1iR product and formationof the CDR1uF-URA3iR product identified Dcdr1 clones.B. Broth microdilution assays examining fluconazole sensitivities of parent 66032, azole-resistant mutant F15, and their respective Dcdr1disruptants. Absorbance at 630 nm was recorded after 24 or 48 h incubation as indicated; growth was plotted as percentage of drug-freecontrol.



mitsky and Edlind, 2004). Following 5FOA-mediated con-version to ura3, PDR1 was disrupted in strain 8512 (notshown). Broth microdilution assays demonstrated reduc-tion of fluconazole MIC from � 256 to 32 mg ml-1. Takentogether, these data suggest that PDR1 is a major deter-minant of azole sensitivity in C. glabrata, although addi-tional gene mutations may contribute to clinical resistance.

Microarray analysis: upregulated genes

In light of the major role played by transcription activatorgene PDR1 in C. glabrata azole sensitivity, an examina-tion of genome-wide changes in gene expression inmutant F15 was warranted. We first attempted this withS. cerevisiae microarrays, because these two yeast are

A

B

C

Fig. 4. PDR1 replacement confirms role in azole resistance.A. Diagram illustrating replacement and PCR screening strategies.B. PCR screen of representative PDR1 replacements 66032R and F15R (wild-type and F15-derived PDR1, respectively) selected oncycoheximide-containing plates, and their parent 66032Dpdr1; strains 66032 and F15 were included as positive controls. DNA was purifiedfrom isolated colonies, amplified with the PDR1uF2-PDR1iR primer pair, and analysed by gel electrophoresis; formation of product confirmedreplacement of PDR1 into its native locus in 66032Dpdr1.C. Broth microdilution assay showing that replacement into 66032Dpdr1 of 66032-derived (66032R) or F15-derived (F15R) PDR1 conferredthe expected low or high-level fluconazole resistance associated with 66032 and F15. Absorbance at 630 nm was recorded after 24 hincubation; growth was plotted as percentage of drug-free control.



closely related. However, the only confirmable changewas upregulation of the PDR5 (18-fold) and PDR15(ninefold) homologues (data not shown); both of thesegenes share 73% nucleotide identity with CDR1.

Therefore, C. glabrata microarrays were developed forthe Affymetrix platform (see Experimental procedures)and used to examine changes in F15 relative to 66032. InF15, 78 genes were upregulated � twofold relative to66032. These genes are listed in Table 1 , grouped byprobable function and ordered by expression level.Among the upregulated are homologues of nine genespreviously identified in microarray studies of S. cerevisiaePdr1–Pdr3 gain-of-function mutants (DeRisi et al., 2000;Devaux et al., 2001). Five of these nine genes encodeputative membrane proteins with roles in small moleculetransport or lipid metabolism. These include, in addition toCDR1 and PDH1, the upregulated genes YOR1 involvedin oligomycin efflux, RSB1 involved in sphingoid base-resistance, and RTA1 involved in 7-aminocholesterolresistance (see SGD website (http://www.yeastgenome.org) for further information on these genes andreferences).

The four remaining genes upregulated in bothS. cerevisiae and C. glabrata gain-of-function mutants

include PDR1 itself (as previously reported; Vermitskyand Edlind, 2004), the stress-induced RPN4 encoding aproteasome gene transcription factor, and the uncharac-terized open reading frames (ORFs) YLR346C andYMR102C. The latter encodes a relatively large andevolutionarily conserved protein with a WD40 domaincommonly found in signalling proteins, and its disruptionhas been associated with fluconazole resistance inS. cerevisiae (Anderson et al., 2003). The YLR346Cproduct, in contrast, is not conserved; indeed, theC. glabrata and S. cerevisiae genes are not detectablyhomologous in terms of sequence but rather in terms ofchromosomal synteny, flanked in both yeast by unambigu-ous YLR345W and YLR347C homologues. Also, bothYLR346C products are short (101 and 112 amino acids)and highly charged in their C-terminal regions. InS. cerevisiae, Ylr346c is mitochondria-localized and formsa two-hybrid interaction with MAP kinase Slt2, suggestinga possible role in mitochondria-nucleus retrogradesignalling.

Among the 69 genes whose upregulation appears to beC. glabrata F15-specific (i.e. not similarly upregulated inS. cerevisiae) are three additional homologues encodingsmall molecule transporters including quinidine and bile

Fig. 5. Antifungal sensitivities andCDR1-ERG11 expression in a Dpdr1derivative of azole-resistant clinical isolateBG14.A. MIC values (at 24 h) determined by brothmicrodilution for BG14 and BG14Dpdr1. A logscale was used to facilitate comparison ofMICs over a wide range. Numbers above theBG14Dpdr1 bars indicate the fold-changerelative to BG14. FLU, fluconazole; ITR,itraconazole; KET, ketoconazole; MIC,miconazole; TER, terbinafine; AMB,amphotericin B; and CAS, caspofungin.B. RNA was isolated from log-phase BG14and BG14Dpdr1 cultures exposed to256 mg ml-1 fluconazole for 0–2.5 h,slot-blotted to membranes, and hybridized tothe indicated gene probes.

A

B



http://www.yeastgenome.org


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394,

552

IPF

5932

SU

T1

CA

GL0

I042

46g

Tran

scrip

tion

fact

orin

volv

edin

ster

olup

take

152.

4–

IPF

3325

d PD

R1

CA

GL0

A00

451g

Tran

scrip

tion

fact

orin

volv

edin

mul

tidru

gre

sist

ance

9.6

2.3

557,

701

IPF

7202

TAF

9C

AG

L0M

0500

5gS

ubun

itof

TF

IIDan

dS

AG

Aco

mpl

exes

1.0

6.3

–IP

F63

66Y

PR

013C

CA

GL0

M01

870g

Unc

hara

cter

ized

;po

tent

ialz

inc

finge

r0.

82.

9–

IPF

2113

SIP

3C

AG

L0I0

1980

gA

ctiv

ates

tran

scrip

tion

thro

ugh

DN

A-b

ound

Snf

10.

42.

2–

IPF

118

HO

T1

CA

GL0

H08

866g

Tran

scrip

tion

fact

orin

volv

edin

osm

ostr

ess

resp

onse

0.1

4.9

228

DN

Are

plic

atio

nan

dda

mag

ere

spon

se

IPF

9036

IPF

9035

IPF

2521

YIM

1M

EC

3D

BF

4

CA

GL0

M09

713g

CA

GL0

M09

735g

CA

GL0

E04

576g

Impl

icat

edin

DN

Ada

mag

ere

spon

seD

NA

dam

age

chec

kpoi

ntR

egul

ator

ysu

buni

tof

Cdc

7p-D

bf4p

kina

seco

mpl

ex

40 1.6

0.7

12 15 2.4

127,

179

110,

162

–IP

F78

5D

PB

3C

AG

L0B

0335

5gD

NA

poly

mer

ase

IIsu

buni

t0.

22.

7–

Pro

tein

synt

hesi

s,m

odifi

catio

n,or

degr

adat

ion

IPF

3014

IPF

6742

IPF

3846

OC

H1

UF

D1

NC

E3

CA

GL0

A01

738g

CA

GL0

J080

96g

CA

GL0

G01

540g

Man

nosy

ltran

sfer

ase

ofci

s-G

olgi

appa

ratu

sR

ecog

nitio

nof

poly

ubiq

uitin

ated

prot

eins

Car

boni

can

hydr

ase-

like;

non-

clas

sica

lpro

tein

expo

rt

5.2

4.4

3.1

2.5

2.2

2.9

– – –IP

F30

72R

PN

8C

AG

L0K

0886

6gN

on-A

TP

ase

regu

lato

rysu

buni

tof

26S

prot

easo

me

2.9

2.9

–IP

F84

84P

CI8

CA

GL0

M12

749g

Pos

sibl

esh

ared

subu

nit

ofC

op9

sign

alos

ome

and

eIF

30.

22.

4–

Ves

icul

aran

dpr

otei

ntr

ansp

ort

IPF

7414

IPF

8257

GS

F2

YP

T52

CA

GL0

L014

85g

CA

GL0

G07

689g

ER

mem

bran

e,he

xose

tran

spor

ter

secr

etio

nG

TP

ase

requ

ired

for

vacu

olar

prot

ein

sort

ing

10 1.6

2.4

2.8

208

–IP

F84

39M

EH

1C

AG

L0L0

2211

gC

ompo

nent

ofth

eE

GO

com

plex

;m

icro

auto

phag

y0.

55.

7–

IPF

4445

VP

S28

CA

GL0

H05

181g

Com

pone

ntof

ES

CR

T-I

com

plex

;pr

otei

ntr

affic

king

0.5

2.2

–IP

F41

73V

TI1

CA

GL0

L106

04g

Invo

lved

inci

s-G

olgi

mem

bran

etr

affic

0.3

6.7

741

IPF

3260

GY

L1C

AG

L0K

1093

4gpu

tativ

egA

Pfo

rY

pt1

invo

lved

inpo

lariz

edex

ocys

tosi

s0.

34.

0–

IPF

271

VP

S51

CA

GL0

H06

809g

Gol

gi-a

ssoc

iate

dre

trog

rade

prot

ein

com

plex

0.2

6.1

–



Tab

le1.

cont

.

Gro

upS

yste

mat

icna

me

S.c

erev

isia

eho

mol

ogue

aC

.gla

brat

ade

sign

atio

nD

escr

iptio

n

Exp

ress

ionb

PD

RE

cF

15F

15/6

6032

Sig

nal

tran

sduc

tion

IPF

1489

IPF

8227

BA

G7

CD

C25

CA

GL0

I072

49g

CA

GL0

D06

512g

GA

Pfo

rR

ho1;

cell

wal

land

cyto

skel

eton

hom

eost

asis

Mem

bran

ebo

und

GE

Ffo

rR

as1-

Ras

21.

61.

22.

95.

8– –

IPF

351

GA

C1

CA

GL0

F04

917g

Reg

ulat

ory

subu

nit

forg

lc7

prot

ein

phos

phat

ase

0.9

4.7

–IP

F23

82Y

NL2

34W

CA

GL0

J075

02g

Sim

ilar

togl

obin

sw

ithha

em-b

indi

ngdo

mai

n0.

53.

1–

IPF

512

GP

G1

CA

GL0

F07

117g

Sub

unit

ofhe

tero

trim

eric

gpr

otei

n,in

tera

cts

with

grp1

0.4

2.6

–IP

F59

14K

IN3

CA

GL0

I044

22g

Pro

tein

kina

se0.

25.

6–

Mito

chon

dria

lIP

F21

22F

MP

48C

AG

L0K

0430

1gS

er/T

hrpr

otei

nki

nase

;m

itoch

ondr

ial

112.

8–

IPF

7121

YG

R04

6WC

AG

L0G

0386

1gE

ssen

tialp

rote

inin

volv

edin

mito

chon

dria

tran

spor

t0.

44.

4–

Cel

lwal

lIP

F95

49F

LO1

CA

GL0

E00

209g

Flo

1-lik

efa

mily

ofce

llw

allp

rote

ins

2.6

3.0

284,

419

Am

ino

acid

and

carb

ohyd

rate

met

abol

ism

IPF

496

IPF

4499

IPF

5315

PY

C1

ST

R3

ME

T8

CA

GL0

F06

941g

CA

GL0

L060

94g

CA

GL0

K06

677g

Pyr

uvat

eca

rbox

ylas

eis

ofor

mC

ysta

thio

nine

beta

-lyas

eB

ifunc

tiona

ldeh

ydro

gena

sean

dfe

rroc

hela

tase

4.6

0.7

0.2

2.3

5.0

7.8

– – –C

hrom

atin

/ch

rom

osom

est

ruct

ure

IPF

8319

IPF

390

IPF

8077

SM

D3

SP

C19

SP

C97

CA

GL0

M04

631g

CA

GL0

F05

467g

CA

GL0

I024

64g

Cor

eS

msp

liceo

som

epr

otei

nS

mD

3C

ompo

nent

ofD

am1

spin

dle

pole

com

plex

Com

pone

ntof

mic

rotu

bule

-nuc

leat

ing

Tub4

com

plex

0.9

0.5

0.5

3.7

13.1 2.8

– – –IP

F27

30S

PC

34C

AG

L0L0

3223

gC

ompo

nent

ofD

am1

spin

dle

pole

com

plex

0.4

2.5

–O

ther

met

abol

ism

IPF

6032

CA

GL0

M14

091g

Put

ativ

equ

inon

ere

duct

ase/

NA

DP

Hde

hydr

ogen

ase

3.8

9.8

244,

532

IPF

6034

AD

H6

CA

GL0

M14

047g

NA

DP

H-d

epen

dent

cinn

amyl

alco

hold

ehyd

roge

nase

2.3

2.7

–IP

F12

79Y

PR

1C

AG

L0A

0281

6g2-

met

hylb

utyr

alde

hyde

redu

ctas

e0.

65.

6–

IPF

4182

INP

1C

AG

L0L1

0736

gP

erip

hera

lmem

bran

epr

otei

nof

pero

xiso

mes

0.2

5.5

–U

ncha

ract

eriz

edIP

F61

16Y

IL07

7CC

AG

L0M

1294

7gU

ncha

ract

eriz

ed20

3847

2,50

2IP

F80

09Y

JL16

3CC

AG

L0M

0842

6gU

ncha

ract

eriz

ed;A

RS

inpr

omot

er9.

110

450

IPF

2520

CA

GL0

E04

554g

Unc

hara

cter

ized

;no

sim

ilarit

ies

8.8

6.4

–IP

F21

96d Y

MR

102C

CA

GL0

K03

377g

Tran

scrib

edal

ong

with

MD

Rge

nes

byY

rr1/

Yrm

18.

14.

489

8IP

F30

19C

AG

L0A

0165

0gU

ncha

ract

eriz

ed;

nosi

mila

ritie

s7.

24.

456

4IP

F38

75d Y

LR34

6CC

AG

L0G

0112

2gU

ncha

ract

eriz

ed;

synt

enic

but

min

imal

sim

ilarit

ies

3.3

2279

3,80

3IP

F36

55Y

LR17

7WC

AG

L0B

0107

8gU

ncha

ract

eriz

ed2.

72.

5–

IPF

1546

CA

GL0

G09

603g

Unc

hara

cter

ized

;ve

ryw

eak

sim

ilarit

yto

Yor1

86w

2.1

6.1

–IP

F23

82Y

NL2

34W

CA

GL0

J075

02g

Sim

ilar

togl

obin

sw

ithha

em-b

indi

ngdo

mai

n0.

53.

1–

IPF

4149

YO

R05

9CC

AG

L0L1

0318

gU

ncha

ract

eriz

ed0.

52.

4–

IPF

6420

YG

R12

6WC

AG

L0I1

0604

gU

ncha

ract

eriz

ed0.

119

–IP

F22

49Y

HL0

10C

CA

GL0

K02

563g

Unc

hara

cter

ized

;m

amm

alia

nB

RA

P2

hom

olog

ue0.

15.

0–

IPF

9234

CA

GL0

M07

766g

Unc

hara

cter

ized

;no

sim

ilarit

ies

0.1

4.0

–

a.P

aren

thes

esin

dica

tea

prev

ious

lyna

med

C.g

labr

ata

gene

.b

.E

xpre

ssio

nin

C.g

labr

ata

6603

2is

repr

esen

ted

inar

bitr

ary

mic

roar

ray

units

(for

com

paris

on,

actin

and

b-tu

bulin

gene

hom

olog

ues

AC

T1

and

TU

B2

had

aver

age

expr

essi

onle

vels

of66

and

8.6

resp

ectiv

ely)

.F

15/6

6032

repr

esen

tsth

era

tioof

expr

essi

onin

the

fluco

nazo

le-r

esis

tant

mut

ant

vs.

itspa

rent

(for

com

paris

on,

AC

T1

and

TU

B2

had

ratio

sof

0.6

and

0.8

resp

ectiv

ely)

.c.

Pro

mot

erre

gion

s(9

00bp

)w

ere

sear

ched

for

mat

ches

toth

eS

.cer

evis

iae

PD

RE

cons

ensu

sT

CC

RY

GS

R.N

umbe

rsin

dica

teth

edi

stan

ce(in

bp)

upst

ream

ofth

eA

TG

star

tcod

onof

the

PD

RE

;hy

phen

s(–

)in

dica

teth

eab

senc

eof

aP

DR

E.

d.

Gen

essi

mila

rlyup

regu

late

din

S.c

erev

isia

eP

dr1–

Pdr

3ga

in-o

f-fu

nctio

nm

utan

ts(D

eRis

iet

al.,

2000

;D

evau

xet

al.,

2001

).



acid efflux protein genes QDR2 and YBT1. Additional lipidmetabolism genes include ARE1 whose disruption inS. cerevisiae confers azole hypersensitivity (T. Edlind,unpubl. data) and ATF2 involved in fatty acid and steroiddetoxification. A third group of well-represented genes areinvolved in the cell stress response, including membraneprotein gene HSP12, and YML131W-YNL134C; the lattertwo are unrelated by BLAST but share an ADH_zinc_Ndomain (identified by CD-search; Marchler-Bauer andBryant, 2004) characteristic of zinc-dependent alcoholdehydrogenases-oxidoreductases. YML131W-YNL134Care also coordinately upregulated in S. cerevisiae inresponse to diverse stresses including heat, oxidizingagents, ethanol, nitrogen depletion and stationary phase(Gasch et al., 2000; see Expression Connection athttp://www.yeastgenome.org). A similarly regulatedS. cerevisiae gene is GRE2, also encoding an oxi-doreductase and among the genes upregulated in Pdr1–Pdr3 gain-of-function mutants (DeRisi et al., 2000;Devaux et al., 2001). This provides an example of analo-gous but non-homologous genes upregulated inC. glabrata F15 and S. cerevisiae Pdr1–Pdr3 mutants.

Notable among the remaining upregulated genes withsignificant expression levels are: SUT1 encoding a tran-scription factor involved in sterol uptake and hypoxic geneexpression, YIM1 implicated in DNA damage response,OCH1 and GSF2 involved in Golgi-ER functions,proteasome-related genes UFD1 and RPN8, putativemitochondrial protein kinase gene FMP48, a quinonereductase-like gene curiously lacking in other fungalgenomes but present in many bacteria and vertebrates,and the uncharacterized YIL077C whose product hasbeen mitochondria-localized but interacts with a nucleartranscriptional complex.

Microarray analysis: downregulated genes

There were 31 genes downregulated � twofold in F15relative to parent 66032 (Table 2). Only one of these wasalso downregulated in S. cerevisiae Pdr1–Pdr3 gain-of-function mutants: membrane transporter gene PDR12involved in efflux of weak organic acids such as sorbate.Additional genes with significantly downregulated expres-sion include zinc transporter gene ZRT1, major facilitatorgenes including FLR1 implicated in fluconazole efflux, andhomologues of cell surface protein genes MUC1-EPA2 andMKC7 implicated in adhesion and aspartic protease activ-ity respectively. Finally, a gene was downregulated whoseproduct has clear homology to the WRY family of putativemembrane-anchored proteins previously identified only inC. albicans (unpublished annotation in NCBI database).This family has nine paralogues in C. albicans and seven inC. glabrata but none, surprisingly, in S. cerevisiae, sug-gesting a possible role in mammalian colonization.

Confirmation of microarray results

As our studies represent the first application of theseC. glabrata microarrays, it was important to validate theresults by independent methods. RNA blots or real-timereverse transcription (RT)-PCR were used to examine theexpression of selected genes identified as upregulated inthe microarray (other than already confirmed CDR1,PDH1 and PDR1). For five of seven genes tested(YLR346C, YOR1, YNL134C, YML131W and RTA1),RNA blots confirmed F15 upregulation relative to theparent 66032 strain (Fig. 6A). The two exceptions (MEC3and YJL163C) represent genes whose expression in bothparent and F15 were below the level of detection by RNAblot (not shown).

For all nine genes tested by real-time RT-PCR (YOR1,RTA1, RPN4, QDR2, MET8, BAG7, CSR1, PDR1 andYBT1), the upregulation observed by microarray wasconfirmed (Fig. 5B). For most of these, the results werequantitatively similar; e.g. YOR1 was upregulated16-fold by microarray and 17-fold by RT-PCR and CSR1was upregulated 3.8-fold by microarray and 2.9-fold byRT-PCR. Expression of ERG11 encoding the azoletarget lanosterol demethylase was essentially unalteredby RT-PCR (not shown), in agreement with microarrayanalysis (F15/66032 = 0.6) and RNA hybridization (Ver-mitsky and Edlind, 2004). One anomaly in the microar-ray analysis was the relatively low upregulation of CDR1(2.5-fold) compared with its high upregulation (c. 20-fold)in both RNA blots and RT-PCR (Fig. 2). Furthermore,Cdr1 was strongly upregulated on the protein level, asshown by SDS-PAGE of membrane preparations fol-lowed by mass spectrometric identification of elutedbands (Rogers et al., submitted for publication). Poten-tial explanations for this anomaly include degradation ormasking of the CDR1 mRNA region targeted by themicroarray, or a saturation effect due to the relativelyhigh CDR1 basal expression.

Promoter sequence analysis

To identify a candidate C. glabrata Pdr1 responseelement (PDRE), we took advantage of the F15 microar-ray data, the available genome sequence, and theevolutionary relatedness of this yeast to S. cerevisiae.The promoter regions (900 bp upstream of the startcodon) for all genes listed in Tables 1 and 2 weresearched for a match to the consensus S. cerevisiaePDRE (DeRisi et al., 2000; Devaux et al., 2001):TCC(GA)(CT)G(GC)(AG). At least one match to thissequence was identified in 31 of the 78 genes (40%)upregulated � twofold. Moreover, one or more PDREwere identified in all nine genes upregulated in bothC. glabrata and S. cerevisiae gain-of-function mutants, in




Tab

le2.

C.g

labr

ata

gene

sdo

wnr

egul

ated

�tw

ofol

din

fluco

nazo

le-r

esis

tant

mut

ant

F15

com

pare

dw

ithpa

rent

6603

2.

Gro

upS

yste

mat

icna

me

S.c

erev

isia

eho

mol

ogue

aC

.gla

brat

ade

sign

atio

nD

escr

iptio

n

Exp

ress

ionb

PD

RE

c66

032

F15

/660

32

Sm

allm

olec

ule

tran

spor

tIP

F55

05IP

F43

92Z

RT

1F

LR1

CA

GL0

E01

353g

CA

GL0

H06

017g

Hig

h-af

finity

zinc

tran

spor

ter

Mul

tidru

gef

flux

pum

pof

maj

orfa

cilit

ator

supe

rfam

ily10 6.

10.

50.

4– –

IPF

6218

d PD

R12

CA

GL0

M07

293g

AB

Ctr

ansp

orte

rof

wea

kor

gani

cac

ids

4.5

0.2

–IP

F78

08Y

HR

048W

CA

GL0

J003

63g

Unc

hara

cter

ized

;m

ajor

faci

litat

orsu

perf

amily

2.2

0.4

–IP

F86

7A

TR

1C

AG

L0B

0234

3gM

ultid

rug

efflu

xpu

mp

ofm

ajor

faci

litat

orsu

perf

amily

1.8

0.4

–IP

F15

68S

NG

1C

AG

L0G

0927

3gN

itros

ogua

nidi

nere

sist

ance

;pu

tativ

etr

ansp

orte

r1.

30.

1–

Cel

lstr

ess

IPF

3249

CTA

1C

AG

L0K

1086

8gC

atal

ase

A8.

80.

4–

IPF

1102

1C

RS

5C

AG

L0H

0425

7gC

oppe

r-bi

ndin

gm

etal

loth

ione

in-li

kepr

otei

n3.

40.

4–

IPF

9018

LTV

1C

AG

L0J0

0891

gR

equi

red

for

grow

that

low

tem

pera

ture

1.0

0.4

656

IPF

9641

SLG

1C

AG

L0F

0150

7gS

enso

rof

stre

ss-a

ctiv

ated

Pkc

1-S

lt2pa

thw

ay0.

80.

5–

IPF

5937

CU

P2(

AM

T1)

CA

GL0

I041

80g

Met

al-a

ctiv

ated

tran

scrip

tiona

lfac

tor

0.9

0.2

–C

arbo

hydr

ate

met

abol

ism

IPF

986

IPF

6117

SD

H2

DO

G2

CA

GL0

C03

223g

CA

GL0

M12

925g

Suc

cina

tede

hydr

ogen

ase

iron-

sulp

hur

prot

ein

subu

nit

2-de

oxyg

luco

se-6

-pho

spha

teph

osph

atas

e4.

91.

60.

40.

3– –

IPF

290

PC

K1

CA

GL0

H06

633g

Pho

spho

enol

pyru

vate

carb

oxyk

inas

e1.

20.

3–

Cel

lcyc

leco

ntro

lIP

F25

55S

DS

22C

AG

L0D

0026

4gN

ucle

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14 of 15 genes in the small molecule transport and lipidmetabolism groups, and in 26 of 34 genes with expressionlevel � 3 (arbitrary microarray units). Conversely, onlyone PDRE was identified among the 31 downregulatedgene promoters (Table 2); similarly, none was identified inthe promoters of representative housekeeping genes(ACT1, TEF1, TDH3) or azole target gene ERG11. Thisanalysis therefore identifies TCC(AG)(TC)G(GC)(AG) asa strong candidate for the C. glabrata PDRE. More spe-cifically, we note a clear preference for G as the penulti-mate base (95% of PDREs) and A as the final base (84%),although two of the four PDREs within the CDR1 promoterhave G as the final base.

Two exceptions warrant discussion. The promoters ofC. glabrata upregulated genes RPN8 and YOR052C lacka PDRE but include perfect matches to the S. cerevisiaeRpn4 transcription factor-binding site GGTGGCAAA(Mannhaupt et al., 1999); perfect or near-perfect matchesare also found in the promoters of their S. cerevisiaehomologues. As noted above, Rpn4 is upregulated inboth C. glabrata Pdr1 and S. cerevisiae Pdr1–Pdr3gain-of-function mutants. Thus, RPN8-YOR052C upregul-ation is likely Rpn4-mediated and only indirectly Pdr1-mediated.

Candida glabrata F15 exhibits additional phenotypespredicted by microarray analysis which may altervirulence

As coordinate CDR1-PDH1 upregulation is commonlyobserved in C. glabrata azole-resistant clinical isolates

(Bennett et al., 2004; Vermitsky and Edlind, 2004; San-guinetti et al., 2005), the responsible mutations musthave minimal effects on fitness. On the other hand,these mutations could alter C. glabrata in subtle waysthat affect, for example, its relative virulence in thebloodstream versus mucosa. The F15 microarray resultsprovided us with an opportunity to begin to test thisgeneral hypothesis. Specifically, we looked for pheno-types other than azole susceptibility predicted to beassociated with altered expression of genes coregulatedwith CDR1-PDH1.

Upregulation of the YOR1 transporter 11-fold (Table 1)predicts that azole-resistant F15 should be cross-resistant to oligomycin, an inhibitor of mitochondrialF1F0 ATPase and known S. cerevisiae Yor1 substrate.This was confirmed by broth microdilution assay(MIC = 0.5 mg ml-1 for F15 vs. 0.125 mg ml-1 for parent66032), using medium with glycerol as respiratorycarbon source. Yor1 also confers tolerance in S. cerevi-siae to a wide range of organic anions such as lepto-mycin B and acetic acid, along with cadmium (Cui et al.,1996). PDR12, downregulated fivefold in F15, similarlyencodes an efflux pump with specificity for organicacids, in particular sorbic acid (Piper et al., 1998). Spotassays (Fig. 7) confirmed sorbate hypersensitivity ofF15, although the effects on MIC were modest (4 mM forF15 vs. 8 mM for 66032). With respect to organic acidsensitivity, PDR12 downregulation may be largely offsetby YOR1 upregulation. There was no detectable changein sensitivity to acetic, boric, or lactic acids (MICs = 64,16 and 250 mM respectively).

Fig. 6. Confirmation of F15/66032 microarrayresults for selected genes by RNAhybridization and real-time RT-PCR.A. RNA was isolated from log phase cultures,slot-blotted to membranes, and hybridized tothe indicated gene probes; ACT1 served asloading control.B. Quantitative real-time RT-PCR analysis ofrelative gene expression in F15 versus 66032.Data are shown as mean ± SD.

A

B



YML131W and YNL134C homologues were similarlyupregulated c. ninefold in F15. As noted above, theproducts of these uncharacterized genes share adomain characteristic of alcohol dehydrogenases/oxidoreductases; furthermore, they are coregulated inresponse to environmental stresses including heat shockand treatment with reactive oxygen species or ethanol(Expression Connection, SGD website). Conversely, cata-lase gene CTA1 was downregulated 2.5-fold. Consistentwith this, F15 demonstrated hypersensitivity to hydrogenperoxide by spot assay (Fig. 7) and broth microdilution(MIC = 16 mM vs. 32 mM for 66032). Similarly, F15 dem-onstrated hypersensitivity to ethanol (Fig. 7; MIC = 2% vs.4% for 66032). Equivalent results were obtained with F15PDR1 replacement clone F15R as compared with wild-type PDR1 replacement clone 66032R (Fig. 7), confirm-ing that these altered phenotypes resulted from the PDR1gain-of-function mutation. (Note that the ura3 phenotypeof 66032R can account for its variable growth relative to66032, an effect also observed with the 66032 ura3 strain;not shown.) Finally, we examined sensitivity to heat shockby exposing mid-log or early stationary phase cultures to50°C for 10 min, following by plating on YPD with incuba-tion at 35°C for 3 days to obtain colony counts. For F15versus 66032, viability was 6 versus 0.3% and 16 versus5% in log and stationary phase cultures respectively.Taken together, these data suggest that regulatory muta-tions conferring azole resistance in C. glabrata may haveboth positive and negative effects on fitness andvirulence.

Conclusions

An important ‘virulence factor’ for the emerging opportunistC. glabrata appears to be its capacity for intrinsic low-level

and acquired high-level azole resistance. The studies com-pleted here with laboratory mutant F15, and initial studieswith representative clinical isolates, identify the zinc clustertranscriptional activator Pdr1 as a key regulator of azole/multidrug transporter genes CDR1 and PDH1. Constitutiveupregulation of these genes is observed in most azole-resistant clinical isolates; furthermore, they are transientlyupregulated in sensitive isolates following azole exposure.Consistent with this, in PDR1 disruptants acquired resis-tance was reversed and intrinsic resistance was reduced.We have shown that F15 Pdr1 has a gain-of-functionmutation analogous to those previously characterized inS. cerevisiae Pdr1–Pdr3, and this mutation is sufficient toconfer azole resistance. Pdr1 mutation is not, however,necessary for resistance, because at least one resistantstrain analysed had unchanged PDR1 (Vermitsky andEdlind, 2004). Azole resistance may potentially arise frommutations in upstream signalling proteins or transcriptioncofactors, both of which remain to be defined (althoughhistone modifying enzymes represent likely cofactors).Moreover, we observed here that PDR1 disruptants,although azole hypersensitive, continued to yield sponta-neous azole-resistant mutants at reduced frequency.These Pdr1-independent resistance mechanisms, andtheir clinical relevance, warrant further study.

Microarray analysis of genome-wide gene expressionhas become a central tool in molecular genetics, and thearrays developed and tested here should be particularlyuseful in studies of C. glabrata in large part because ofits close evolutionary relatedness to S. cerevisiae.Most genes with altered expression in F15 had well-characterized S. cerevisiae homologues. This allowedus to predict F15 phenotypes, a number of which weretested including sensitivity to organic acids, alcoholsand oxidants. Ultimately, these data should help us to

Fig. 7. Spot assays examining sensitivity of66032 and its azole-resistant mutant F15 tohydrogen peroxide, ethanol and sorbic acid.Approximately 300 cells were spotted on YPDagar with the indicated inhibitor. Plates wereincubated for 2–4 days at 35°C. Forcomparison, 66032Dpdr1, F15Dpdr1 and the66032Dpdr1-PDR1 replacement strains66032R and F15R were examined in parallel.



understand and possibly exploit the consequences forC. glabrata of regulatory mutations leading to azoleresistance. F15 hypersensitivity to hydrogen peroxide isof particular interest, because this implies hypersensitiv-ity to immune cells such as neutrophils and environ-ments such as the lactobacillus-colonized vaginal tract inwhich hydrogen peroxide plays an important role.Although the relatedness of C. glabrata and S. cerevi-siae is invaluable in terms of predicting gene function,microarray analysis indicated that the Pdr1 and Pdr1-Pdr3 gain-of-function mutants of these yeast are moredifferent than similar. This no doubt reflects the very dif-ferent pressures placed on these organisms by theirvery different niches; e.g. the skin of a grape versus thehuman mucosa.

Following submission of this manuscript, Tsai et al.(2006) reported results that parallel and complementthose described here. Specifically, a C. glabrata labora-tory strain with transposon-disrupted PDR1 exhibited flu-conazole hypersensitivity and diminished CDR1-PDH1expression. Importantly, two fluconazole-resistant clinicalisolates with increased CDR1-PDH1 expression wereshown to harbour PDR1 mutations, and integrativetransformation of these alleles conferred fluconazoleresistance and upregulated CDR1-PDH1 expression onthe pdr1::Tn strain. These results confirm the relevanceof laboratory mutant F15 as a model for clinicalresistance.

Experimental procedures

Media, inhibitors and strains

For most experiments, the medium employed was YPD (1%yeast extract, 2% peptone, 2% dextrose). Gene disruptantsand ura3 mutants were selected on DOB (synthetic definedmedium with dextrose) with complete supplement mixture(CSM) or CSM lacking uracil/uridine (-URA) (Qbiogene/BIO101). Drugs were obtained from the following sources: flu-conazole (Pfizer), itraconazole (Janssen), terbinafine (Novar-tis); caspofungin (Merck), amphotericin B, miconazole andcyloheximide (Sigma-Aldrich). They were dissolved in dim-ethyl sulphoxide (DMSO); the final DMSO concentration was� 0.5% in all experiments which had no detectable effect ongrowth. Sorbic acid, lactic acid, acetic acid and hydrogenperoxide (Sigma) were diluted as necessary in water. Strainswere previously described (Vermitsky and Edlind, 2004) orconstructed as described below.

Isolation of ura3 strains

Wild-type URA3 yeast strains are sensitive to 5FOA. Toisolate 5FOA-resistant mutants, a single colony from a freshYPD plate was streaked on DOB + CSM agar containing0.1% 5FOA (Research Products International) and incu-bated at 35°C for 3 days. Colonies were streaked for isola-

tion on YPD and DOB-URA; those that failed to grow on thelatter were then tested for URA3 complementation by trans-formation with pRS416 (shuttle vector with S. cerevisiaeURA3) and selection on DOB-URA plates. Yeast transfor-mations employed the Frozen-EZ Yeast Transformation IIKit (Zymo Research) as previously described (Edlind et al.,2005).

Gene disruption and replacement

The PRODIGE method for PCR product-mediated gene dis-ruption was employed (Fig. 1A; Edlind et al., 2005). Briefly,primers (80 mers; Table 3) were designed to preciselyreplace, after homologous recombination, a C. glabratacoding sequence (CDS) with the selection marker CDS.These primers consisted of c. 60 nucleotides at the 5′ endcomplementary to C. glabrata sequences directly upstreamand downstream of the targeted CDS and c. 20 nucleotidesat the 3′ end complementary to the S. cerevisiae URA3CDS contained in plasmid template pRS416. PCR productsgenerated with these primers were used to transformC. glabrata ura3 strains. Following selection on DOB-URAmedium, transformants were screened by PCR with specificprimer pairs (Table 3; Fig. 1A) to confirm replacement of thetargeted CDS with URA3 CDS. DNA was generally pre-pared by phenol extraction of glass bead-disrupted cells(Edlind et al., 2005); some screens employed colony PCRin which a small volume of cells was added directly to thePCR mix.

For PDR1 replacement, a PCR product representing thePDR1 CDS plus 430–680 bp upstream and downstreamsequence was amplified with primers PDR1uF-PDR1dR(Table 3) from 66032 or F15 genomic DNA. These productswere used to transform 66032Dpdr1 strain with selection on1 mg ml-1 cycloheximide-containing YPD plates. Colonieswere screened as above with primer pair PDR1uF2-PDR1iR(Table 3; Fig. 4A).

Broth microdilution assay

Fresh overnight cultures from a single colony were diluted1 : 100 in YPD, incubated for 3 h with aeration, and thencounted in a haemocytometer and diluted again to1 ¥ 104 cells ml-1. Aliquots of 100 ml were distributed to wellsof a 96-well flat-bottomed plate, except for row A whichreceived 200 ml. Inhibitor was added to row A to the desiredconcentration and then serially twofold diluted to rows Bthrough G; row H served as inhibitor-free control. Plates wereincubated at 35°C for the indicated times. Absorbance at630 nm was read with a microplate reader; background dueto medium was subtracted from all readings. The MIC(minimum inhibitory concentration) was defined as the lowestconcentration inhibiting growth at least 80% relative to thedrug-free control.

RNA hybridization

Log phase cultures in YPD at 35°C were adjusted to 3 ¥ 106

cells ml-1 and incubated for an additional 3 h. In somestudies, cultures were divided into equal portions to which



Table 3. Primers used in this study (grouped by application).

PRODIGE-based gene disruptionPDR1-URA3F 5′-GCCTTTTTTTTTAGAATATATTGGTAAAGTCATTCTTTAGC

TACGTTATTGAGAGAATATGTCGAAAGCTACATATAAGG-3′PDR1-URA3R 5′-TGATTTTTCAGATTAAATATAAAATTATACAGGCTATGCACA

CTGTCTAAATTAATAGCATTAGTTTTGCTGGCCGCATC-3′CDR1-URA3F 5′-TACTTACAGGAAAAAGAATTTACAACTCTTGATATATACAA

AGTAAAGAAAAGTAACAATGTCGAAAGCTACATATAAGG-3′CDR1-URA3R 5′-TTTTCCGAATGCAATATGTATTAATACCAGAGCCAGATTATG

AGCGCAGGCTAAATAAATTAGTTTTGCTGGCCGCATC-3′PCR screening and PDR1 replacement

PDR1uF 5′-GGCGTATTCATAGAATCCGAA-3′PDR1uF2 5′-GGTCCTTCTAATAGTCATCTTT-3′PDR1iR 5′-CCATAGTATTCGTCGAGAGCA-3′PDR1dR 5′-GACCTCTGTGAAAAGCTACTG-3′URA3iR 5′-CAGCAACAGGACTAGGATGAG-3′CDR1uF 5′-GCAGCTATGAGTTGAGGAAG-3′CDR1iR 5′-ACGCCACATCGGCATCCTT-3′

DNA Probes for RNA hybridizationACT1F 5′-TTGACAACGGTTCCGGTATG-3′ACT1R 5′-CCGCATTCCGTAGTTCTAAG-3′CDR1F 5′-ACAATGTCTCTTGCAAGTGAC-3′CDR1R 5′-AAGTGTTTTCTGATGTGCTTT-3′PDH1F 5′-GTGATGAACCCCGATGA-3′PDH1R 5′-TTCTTGATCTCGTTGGGCGT-3′PDR1F 5′-AGTGCCACCACTAAGTCACT-3′PDR1R 5′-CCATAGTATTGCTGCAGAGCAYLR346F 5′-GGAACTGAAACGCAGAACCA-3′YLR346R 5′-ATCCTTCCATGTGTCGGCAT-3′YOR1F 5′-GAACAAGCCACAGACGTATC-3′YOR1R 5′-CAAATTGCCAAGATGGCTGG-3′YNL134F 5′-CCACCATGAAAGCTGCTGTA-3′YNL134R 5′-AACTTAGGATCAGCTGGCAG-3′YML131F 5′-AATGAACCCACACCGGGTTA-3′YML131R 5′-TTCACCAGTTGCATCAACCAT-3′RTA1F 5′-CGTTCGCGGTGTTGTTTCTT-3′RTA1R 5′-CATCTTCAATATCGGCTTCGA-3′MEC3F 5′-TAGCGTCATTACGGAGCCTT-3′MEC3R 5′-TATCGGGACCGCTTTCTTGT-3′YJL163F 5′-TAGGTGCCTCGCATTCTGAT-3′YJL163R 5′-ATCTTGCCAGCTAATCCAGG-3′

Real-time RT-PCR18SrtF 5′-TCGGCACCTTACGAGAAATCA-3′18SrtR 5′-CGACCATACTCCCCCCAGA-3′CDR1rtF 5′-CATACAAGAAACACCAAAGTCGGT-3′CDR1rtR 5′-GAGACACGCTTACGTTCACCAC-3′PDH1rtF 5′-ACGAGGAGGAAGACGACTACGA-3′PDH1rtR 5′-CTTTACTGGAGAACTCATCGCTGTT-3′CSR1rtF 5′-TGGATTTTTTCTCCCATCTGGA-3′CSR1rtR 5′-ACCACAGGGTCAAGCCATTTT-3′PDR1rtF 5′-TTTGACTCTGTTATGAGCGATTAC-3′PDR1rtR 5′-TTCGGATTTTTCTGTGACAATGG-3′KAD2rtF 5′-AACCCGCAGTCATCGTGG-3′KAD2rtR 5′-CCTGTCTCTCAGTTCTTGGAAACC-3′YOR1rtF 5′-CCATCGGTGCTTGTGTAATGTTA-3′YOR1rtR 5′-TTGAGAGGCGTGGAAAAAATG-3′RTA1rtF 5′-TCCTGTTTGTCATTAGGGTTAGGG-3′RTA1rtR 5′-TGGCAATTTTGTTCTTATTCCTCAG-3′QDR2rtF 5′-GACGAATGAGGACGAGGCTG-3′QDR2rtR 5′-GGTTGGACCTGGTTCTGTAAATAGG-3′SUT1rtF 5′-ACGAGAGCCAGAAGTTGATGG-3′SUT1rtR 5′-TGGAGGCGATAGGAATTGGT-3′RPN4rtF 5′-AGCCAGTATGCTGACCCGAG-3′RPN4rtR 5′-ACACGCCACATCGCCC-3′SAC7rtF 5′-CGCTGGAGACGCCTGG-3′SAC7rtR 5′-TCGTATCCGCTTGCTGTTCC-3′YBT1rtF 5′-AAGTGCTTCTTCCGCCTCATT-3′YBT1rtR 5′-AACAGGAGCTGGTGTAGTACCCA-3′MET8rtF 5′-TCCACCGCTATGCGATTTCT-3′MET8rtR 5′-GGAGATGACCCATTGGATGAA-3′



either fluconazole or a comparable volume of DMSO wasadded, followed by incubation for the indicated times. In allstudies, culture volumes corresponding to 3 ¥ 107 cells wereremoved and centrifuged to pellet cells. RNA preparation andhybridization analysis were as previously described (Smithand Edlind, 2002). Briefly, cell pellets were suspended insodium acetate-EDTA buffer and stored frozen. After thawing,RNA was extracted by vortexing in the presence of glassbeads, SDS and buffer-saturated phenol alternating withincubation at 65°C for 10–15 min. Samples were cooled onice and centrifuged, and RNA was ethanol precipitated fromthe aqueous phase. RNAs were dissolved in water and dena-tured in formaldehyde-SSPE with incubation for 15 min at65°C. Either 40 ml (for ACT1 probing) or 200 ml (for otherprobes) of denatured RNA (approximately 4 or 20 mg, respec-tively) was applied to nylon membrane by using a slot blotapparatus. Membranes were rinsed in SSPE, UV cross-linked, hybridized to purified PCR products (see Table 3 forprimers) labelled with 32P by random priming (Takara), andexposed to film.

Construction of C. glabrata microarrays

The nucleotide sequences corresponding to 5272 C. gla-brata ORFs were downloaded from the Génolevures Con-sortium (http://cbi.labri.fr/Genolevures/about.php, Build 2).Following the Affymetrix Design Guide, two separate probesets for each ORF were designed, each consisting of 13perfect match and 13 mismatch overlapping 25 base oligo-nucleotides targeted to the 3′ 600 bp region. For ORFs� 600 bp the sequence was divided in two equal segmentsfor subsequent design procedures. For quality control andnormalization purposes, we designed two to three additionalprobe sets spanning the C. glabrata 18 s rRNA, TDH1 andACT1 genes in addition to standard Affymetrix controls(BioB, C, D, cre, DAP, PHE, LYS, THR). The probe selec-tion was performed by the Chip Design group at Affymetrix,using their proprietary algorithm to calculate probe setscores, which includes a probe quality metric, cross-hybridization penalty, and gap penalty. The probe sets werethen examined for cross-hybridization against all othersequences in the C. glabrata genome as well as a numberof constitutively expressed genes and rRNA from othercommon organisms. Duplicate probesets are made to dis-tinct regions of the ORF, thereby allowing two independentmeasurements of the mRNA level for that particular gene.C. glabrata custom Affymetrix NimbleExpress Arrays weremanufactured by NimbleGen Systems (Albert et al., 2003)per our specification.

RNA preparation for microarrays

Total RNA was isolated using the hot SDS-phenol method(Schmitt et al., 1990). Frozen cells were suspended in 12 mlof 50 mM sodium acetate (pH 5.2), 10 mM EDTA at roomtemperature, after which 800 ml of 25% sodium dodecyl sul-phate and 12 ml of acid phenol (Fisher Scientific) wereadded. This mixture was incubated 10 min at 65°C with vor-texing each minute, cooled on ice for 5 min, and centrifugedfor 15 min at 12 000 g. Supernatants were transferred to new

tubes containing 15 ml of chloroform, mixed and centrifugedat 200 ¥ g for 10 min. The aqueous layer was removed tonew tubes, RNA was precipitated with 1 vol isopropanol and0.1 vol 2 M sodium acetate (pH 5.0), and then collected bycentrifugation at 17 000 g for 35 min at 4°C. The RNA pelletwas suspended in 10 ml of 70% ethanol, collected again bycentrifugation, and suspended in diethyl pyrocarbonate-treated water.

cRNA synthesis and labelling

Immediately prior to cDNA synthesis, the purity and concen-tration of RNA samples were determined from A260/A280

readings and RNA integrity was determined by capillaryelectrophoresis using the RNA 6000 Nano Laboratory-on-a-Chip kit and Bioanalyzer 2100 (Agilent Technologies) as perthe manufacturer’s instructions. First and second strandcDNA was synthesized from 15 mg total RNA using theSuperScript Double-Stranded cDNA Synthesis Kit (Invitro-gen) and oligo-dT24-T7 primer (PrOligo) according to themanufacturer’s instructions. cRNA was synthesized andlabelled with biotinylated UTP and CTP by in vitro transcrip-tion using the T7 promoter-coupled double stranded cDNAas template and the Bioarray HighYield RNA TranscriptLabelling Kit (ENZO Diagnostics). Double stranded cDNAsynthesized from the previous steps was washed twice with70% ethanol and suspended in 22 ml of Rnase-free water.The cDNA was incubated as recommended with reactionbuffer, biotin-labelled ribonucleotides, dithtiothreitol, Rnaseinhibitor mix and T7 RNA polymerase for 5 h at 37°C. Thelabelled cRNA was separated from unincorporated ribo-nucleotides by passing through a CHROMA SPIN-100column (Clontech) and ethanol precipitated at -20°Covernight.

Oligonucleotide array hybridization and analysis

The cRNA pellet was suspended in 10 ml of Rnase-free waterand 10 mg was fragmented by ion-mediated hydrolysis at95°C for 35 min in 200 mM Tris-acetate (pH 8.1), 500 mMpotassium acetate, 150 mM magnesium acetate. The frag-mented cRNA was hybridized for 16 h at 45°C to theC. glabrata NimbleExpress GeneChip arrays. Arrays werewashed at 25°C with 6 ¥ SSPE, 0.01% Tween 20 followed bya stringent wash at 50°C with 100 mM MES, 0.1 M NaCl,0.01% Tween 20. Hybridizations and washes employed theAffymetrix Fluidics Station 450 using their standard EukGE-WS2v5 protocol. The arrays were then stained withphycoerythrein-conjugated streptavidin (Molecular Probes)and the fluorescence intensities were determined using theGCS 3000 high-resolution confocal laser scanner(Affymetrix). The scanned images were analysed using soft-ware resident in GeneChip Operating System v2.0 (GCOS;Affymetrix). Sample loading and variations in staining werestandardized by scaling the average of the fluorescentintensities of all genes on an array to a constant targetintensity (250). The signal intensity for each gene wascalculated as the average intensity difference, representedby [S(PM – MM)/(number of probe pairs)], where PM andMM denote perfect-match and mismatch probes.



http://cbi.labri.fr/Genolevures/about.php

Microarray data analysis

The scaled gene expression values from GCOS softwarewere imported into GeneSpring 7.2 software (Agilent Tech-nologies) for preprocessing and data analysis. Probesetswere deleted from subsequent analysis if they were calledabsent by the Affymetrix criterion and displayed an absolutevalue below 20 in all experiments. The expression value ofeach gene was normalized to the median expression of allgenes in each chip as well as the median expression forthat gene across all chips in the study. Pairwise comparisonof gene expression was performed for each matchedexperiment (F15 vs. 66032). Genes were included in thefinal data set if their expression changed by at least twofoldbetween strain F15 and strain 66032 in two independentexperiments.

Quantitative real-time RT-PCR

First strand cDNAs were synthesized from 2 mg total RNA ina 21 ml reaction volume using the SuperScript First-StrandSynthesis System for RT-PCR (Invitrogen) as per the manu-facturer’s instructions. Quantitative real-time PCR was per-formed in triplicate using the 7000 Sequence DetectionSystem (Applied Biosystems). Independent amplificationswere performed using the same cDNA for both the gene ofinterest and 18S rRNA, using the SYBR Green PCR MasterMix (Applied Biosystems). Gene-specific primers weredesigned for the gene of interest and 18S rRNA usingPrimer Express software (Applied Biosystems) and theOligo Analysis and Plotting Tool (Qiagen). The PCR condi-tions consisted of AmpliTaq Gold activation at 95°C for10 min, followed by 40 cycles of denaturation at 95°C for15 s and annealing/extension at 60°C for 1 min. A dissocia-tion curve was generated at the end of each cycle to verifythat a single product was amplified using software providedwith the 7000 Sequence Detection System. The change influorescence of SYBR Green I dye in every cycle was moni-tored by the system software, and the threshold cycle (CT)above background for each reaction was calculated. The CT

value of 18S rRNA was subtracted from that of the gene ofinterest to obtain a DCT value. The DCT value of an arbitrarycalibrator (e.g. untreated sample) was subtracted from theDCT value of each sample to obtain a DDCT value. The geneexpression level relative to the calibrator was expressed as2–DDCT.

Acknowledgements

We thank V. Pirrone for assistance with the heat shock assay,and J. Rex and B. Cormack for providing strains. Support wasprovided by NIH Grant AI047718 (to T.D.E.) and AI058145 (toP.D.R.).

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