Screening for effectors that modify multidrug resistance in yeast

Zuzana Kozovska´, Julius Subik

*

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

Abstract

The yeast transcription factors Pdr1p and Pdr3p regulate the expression of several genes that encode energy-dependent efflux

pumps involved in multidrug resistance. They recognize specific pleiotropic drug resistance elements in the promoters of the target
gene such as PDR5 coding for a major multidrug transporter. Gain-of-function mutations in Pdr1p/Pdr3p result in over-expression
of transporter genes and establishment of multidrug resistance. We developed a novel yeast-based screening procedure designed to
detect compounds that specifically modify multidrug resistance due to an interference with the expression of drug efflux transporter
genes. The screening is based on the ability to abrogate the growth defect of cells suffering from the galactose induced Pdr3p driven
over-expression of a dominant-lethal allele of the PMA1 gene placed under the control of the PDR5 promoter. Validation of the
assay was achieved by showing that growth inhibition was relieved by mutant Pdr3p devoid of activation domain. This screening
system may also be used to select the loss-of-function pdr3 (or pdr1 ) mutants and to identify specific gene(s) whose over-expression
or deletion will suppress the expression of multidrug transporters and increase the susceptibility of yeast cells to antifungals.
#

2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Keywords: Multidrug resistance; PDR3 ; PMA1 ; Saccharomyces cerevisiae ; Transcription factor; Yeast

1. Introduction

The chemotherapy of tumors, bacterial and fungal

infections constitute a serious problem of growing
importance in clinical medicine due to the appearance
of multidrug resistant cancer cells and resistant strains
of microbial pathogens. Multidrug resistance which
render cells simultaneously resistant to several structu-
rally and functionally unrelated drugs, is conserved in
evolution from bacteria to human and is associated with
an increased efflux of drugs from cells due to over-
expressed membrane transporters

[1,2]

.

Although several genes coding for eukaryotic drug

efflux pumps were already identified in mammalian cells

[3,4]

, pathogenic yeast Candida albicans

[5,6]

and related

non-albicans Candida species

[7,8]

, the molecular me-

chanisms of multidrug resistance in eukaryotes are best
understood in the yeast Saccharomyces cerevisiae . Both
YAP and PDR family of genes contribute to the
establishment of this phenomenon

[9

/

11]

. Most impor-

tantly, well characterized efflux pumps are represented

by encoded genes such as PDR5 , SNQ2 and YOR1 . The
main and best described transcription factors of zinc
cluster proteins regulating the expression of efflux
pumps involved in multidrug resistance are encoded by
the PDR1 and PDR3 genes. Whereas the deletion of
both genes renders yeast cells hypersensitive to drugs

[12]

their gain-of-function mutations result in multidrug

resistance due to an increased expression of PDR5 and
other genes encoding drug efflux pumps; these are
detected by the level of their transcripts and abundance
of corresponding protein products

[13

/

17]

.

To overcome multidrug resistance in microbial and

cancer cells several strategies have been developed.
Some of them are based on the inhibition of activity
of membrane bound drug efflux transporters

[1,18,19]

,

others try to use the antisense RNA or ribozymes to
modulate the expression of genes encoding these pumps

[20,21]

. Due to a continual increasing appearance of

fungal strains with multidrug resistance in clinical
practice it has become imperative to find novel fungal
cell targets, new antifungals as well as multidrug
resistance reversion agents that would render resistant
strains sensitive to commercial antifungals.

We present a novel strategy for screening compounds

that can suppress multidrug resistance in yeast by the

* Corresponding author. Tel.:

/

42-12-6029-6631; fax:

/

42-12-

6542-9064.

E-mail address:

[e-mail]

(J. Subik).

International Journal of Antimicrobial Agents 22 (2003) 284

/

290

www.ischemo.org

0924-8579/03/$30 # 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
doi:10.1016/S0924-8579(03)00216-4

interference with drug efflux transporter gene expres-
sion. The developed yeast-based sensor is a simple
growth assay designed to detect compounds that mod-
ulate multidrug resistance and sensitize drug resistant
strains. The screening system is based on the ability of
specific compounds to abrogate the growth defect of the
budding yeast S. cerevisiae suffering from galactose
induced Pdr3p driven over-expression of the dominant-
lethal pma1 -D378N allele of the essential plasma
membrane ATPase gene placed under the control of
the PDR5 promoter.

2. Materials and methods

2.1. Strains and culture conditions

Experiments were performed using the S. cerevisiae

strain FY1679-28C/TDEC (MATa ura3 -52 trp1 -63
leu2 -1 his 3 -200 pdr1::TRP1 pdr3 ::HIS3 )

[12]

. Escher-

ichia coli strain XL1 blue was used for plasmid
amplification. Yeast cells were grown at 30 8C in
complex medium (YPD Broth, DIFCO) containing
glucose (1% yeast extract, 2% bacteriological peptone,
2% glucose) or in minimal medium (0.67% Yeast
Nitrogen Base without amino acids) (YNB, DIFCO)
containing either 2% glucose or 2% galactose. The
appropriate nutritional requirements and drugs were
added at indicated concentrations. E. coli cells were
grown at 37 8C in Luria-Bertani (DIFCO) medium (1%
tryptone, 1% NaCl, 0.5% yeast extract, pH 7.5) supple-
mented with 100 mg/l ampicillin (SIGMA) for selection
of transformants.

2.2. Plasmids and molecular biology

The pdr3 -7 and pdr3 -9 mutant alleles of the PDR3

gene were cloned in a centromeric plasmid pFL38-PP3
(ARS1 CEN4 URA3 ) under the control of its own
promoter

[14]

. The multi-copy plasmid encoding PDR3

under the control of the GAL1 promoter, called pYE-
P

GAL1

-PDR3, contains the PDR3 gene in pYE-DP1/8-2

vector (2 mm URA3 )

[22]

. YCp2HSE-PMA1 and

YCp2HSE-pma1(D378N) are plasmids generously pro-
vided by C.W. Slayman, of Yale University, USA and
contain a LEU2 marker, ARS1 , CEN4 , the wild-type
PMA1 and pma1 -D378N mutant allele, respectively,
under the control of two copies of the heat-shock
elements inserted into the CYC1 upstream region

[23]

.

The plasmid pSK-P

PDR5

PPUS carries the PDR5 pro-

moter fused to the two copies of the PDR5 terminator
separated by the URA3 gene

[24]

. pRS306K is a

multicopy (ARS1 URA3 KARS2 ) plasmid used as an
empty vector in control experiments. The transforma-
tion of E. coli and all DNA manipulations were carried
out as described by Sambrook et al.

[25]

. Yeast cells

were transformed by electroporation

[26]

. Plasmid DNA

was extracted from yeast cells as described by Alister
and Ward

[27]

.

2.3. Drug sensitivity assay

The sensitivity of yeast cells to cycloheximide

(SIGMA) and fluconazole (SLOVAKOFARMA) was
assayed by determination of their individual minimal
inhibitory concentration (MIC) on solid minimal med-
ium

[14]

. Drug sensitivity was scored after 5 days of

growth at 30 8C.

2.4. Determination of plasmid loss

Transformants were grown in liquid YPD medium.

Appropriate dilutions of the cell suspension were plated
onto solid YPD media. After 3 days of growth at 30 8C,
colonies were replica-plated onto minimal medium
supplemented with either uracil or leucine for the
evaluation of the plasmid-containing fraction.

3. Results

3.1. pma1-D378N mediated growth defect induced by
over-expression of the PDR3 gene

The pma1 -D378N allele expressed under the control

of the GAL1 or heat shock promoters even in the
presence of a wild-type copy of the PMA1 gene, has a
dominant-negative effect on growth when galactose is
used as a carbon and energy source or when the culture
is grown at 37 8C

[23,28]

. We used this phenomenon to

develop a yeast strain showing similar growth defect due
to a regulated over-expression of the PDR3 gene. In the
first step we constructed the plasmid YCpP

PDR5

-

pma1(D378N) in which the pma1 -D378N mutant allele
was placed under the control of the PDR5 promoter
containing several pleiotropic drug response elements
(PDRE) recognized by Pdr3p (

Fig. 1

). The plasmid

YCp2HSE-pma1(D378N) was restricted by XhoI and
HindIII and the excised CYC1 -2HSE promoter was
replaced by the XhoI-HindIII DNA fragment from
pSK-P

PDR5

PPUS containing the PDR5 promoter. The

plasmid

YCpP

PDR5

-PMA1

bearing

the

wild-type

P

PDR5

-PMA1 fusion gene and used in control experi-

ments was constructed by similar way from YCp2HSE-
PMA1.

When the plasmid YCpP

PDR5

-pma1(D378N) was

transformed

into

the

hypersensitive

S.

cerevisiae

FY1679-28/TDEC host strain that was deleted in the
chromosomal PDR1 and PDR3 genes and bearing the
PDR3 gene expressed from the GAL1 promoter on
pYE-P

GAL1

-PDR3 plasmid, the resulted double trans-

formants were able to grow as prototrophic cells on

Z. Kozovska´, J. Subik / International Journal of Antimicrobial Agents 22 (2003) 284

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solid minimal medium containing glucose. However,
when such transformants containing the P

PDR5

-pma1 -

D378N and P

GAL1

-PDR3 fusion genes on a centromeric

and multi-copy plasmids, respectively, were transferred
to minimal medium containing galactose instead of
glucose they failed to develop colonies (

Fig. 2

A). This

is due to the growth defect caused by abnormality of
protein folding and biogenesis of the yeast Pma1p
ATPase

[29]

. Conversely, transformants of the same

Dpdr1 Dpdr3 host strain bearing the P

GAL1

-PDR3

fusion gene and the wild-type PMA1 placed under the
control of the PDR5 promoter grew well on media
containing either glucose or galactose. These results
demonstrated the successful construction of the yeast

strain, which we now name ZK11-1, which exhibits the
growth defect due to galactose induced expression of the
PDR3 gene. Such a strain may be used as a bio-sensor
that reports the down-regulation of multidrug resistance
efflux pumps like Pdr5p as changes in growth of
galactose-sensitive yeast cells.

3.2. Physiological properties of the Dpdr1 Dpdr3
transformants bearing the P

PDR5

-pma1-D378N and

P

GAL1

-PDR3 fusion genes

The strain ZK11-1 bearing the P

PDR5

-pma1 -D378N

and P

GAL1

-PDR3 fusion genes on a centromeric and

multi-copy plasmid, respectively, exhibited a growth

Fig. 1. Construction of a centromeric plasmid containing the pma1 -D378N mutant allele under the control of the PMA1 promoter.

Z. Kozovska´, J. Subik / International Journal of Antimicrobial Agents 22 (2003) 284

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defect which was fully developed after two to four
generations when cultured on either solid or liquid
minimal media containing galactose (

Fig. 2

). The

doubling time of the strain growing in minimal medium
containing glucose was 5.75 h. When 10

7

of its cells were

directly plated on minimal medium containing galac-
tose, individual galactose positive spontaneous mutants
developed as colonies in the background of a non-
growing cell population, which appeared with a fre-
quency of 1.8

/

10

6

. This low frequency of galactose-

positive clones, however, did not interfere with the
growth arrest of the culture monitored in a liquid
medium containing galactose until to 102 h of cultiva-
tion at 30 8C (

Fig. 2

B).

Cells of the strain ZK11-1 were found to lose the

plasmids bearing the fusion genes during the cultivation
under selective growth conditions. The number of
colonies after plating on minimal medium containing

glucose usually reached only a one third of that
appeared on complex YPD medium. The frequency of
plasmid loss (plasmid instability) was even higher when
the strain ZK11-1 was grown under non-selective
conditions in a complex YPD medium containing
glucose. After 24 generations only 2.7% of cells in the
culture retained both plasmids (

Table 1

).

The susceptibility of the strain ZK11-1 to cyclohex-

imide and fluconazole is shown in

Table 2

. Along with

the growth defect of galactose the strain was highly
sensitive to drugs when tested on minimal medium
containing glucose. Its sensitivity to drugs was similar
to that of the transformants containing an empty vector
pRS306K instead of the plasmid bearing the galactose
induced and glucose repressed P

GAL1

-PDR3 fusion

gene. However, when the pma1 mutant allele on plasmid
was replaced by the wild-type PMA1 gene the corre-
sponding transformants were able to grow on medium

Fig. 2. Transformants of the S. cerevisiae strain FY1679-28C/TDEC bearing the P

GAL1

-PDR3 and P

PDR5

-pma1 -D378N fusion genes exhibit

galactose induced growth defect. (A) growth on solid minimal medium, (B) growth in liquid medium.

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containing galactose and exhibited a slightly decreased
sensitivity to both cycloheximide and fluconazole. These
results indicate that drug sensitivity assay performed on
minimal medium containing glucose or galactose can
discriminate between the changes in the expression/
activity of the PDR3 or pma1 -D378N genes in galactose
positive mutant clones issued spontaneously from
ZK11-1. Loss-of-function pdr3 mutants are expected
to grow on galactose and be sensitive to drugs irrespec-
tive of the carbon source (glucose or galactose) used in
minimal medium.

3.3. Incompatibility of gain-of-function pdr3 mutations
with P

PDR5

-pma1-D378N and selection of loss-of-function

pdr3 mutants

Galactose-induced and Pdr3p-mediated expression of

the dominant-negative pma1 -D378N mutant allele in
the strain ZK11-1 results in growth defect because newly
synthesized mutant and wild-type Pma1p molecules are
retained and degraded in the endoplasmic reticulum

[28,29]

. A similar effect is expected on medium contain-

ing even glucose with yeast cells expressing a hyperactive
Pdr3p from gain-of-function pdr3 mutant alleles. In fact
we failed to transform the Dpdr1 Dpdr3 host strain
harboring YCpP

PDR5

-pma1(D378N) with the centro-

meric plasmids containing either the pdr3 -7 or pdr3 -9
mutant allele. On the other hand, when the C-terminal
activation domain of Pdr3p was eliminated from the
wild-type PDR3 gene cloned on the pYEP

GAL1

-PDR3

plasmid by deletion of the corresponding EcoRI-EcoRI
DNA fragment, the truncated P

GAL1

-PDR3 DAD trans-

formed into the Dpdr1 Dpdr3 host strain harboring
YCpP

PDR5

-pma1(D378N) did not cause galactose-in-

duced growth defect of transformants (

Fig. 3

). More-

over,

such

transformants

were

hypersensitive

to

cycloheximide on minimal media containing galactose.
The MIC of this antibiotic against this transformant was
similar to that determined with medium containing
glucose.

The ZK11-1 galactose positive clones appear with a

relatively low frequency. A large number of these clones
(25 and 68% in two independent experiments) was found
to be sensitive to 0.05 mg/l of cycloheximide even in the
presence of galactose thereby pointing to an alteration
of Pdr3p function. The properties of ten independent
galactose positive and cycloheximide sensitive sponta-
neous mutants of the strain ZK11-1 were studied in
more

detail.

After

the

loss

of

the

YCpP

PDR5

-

pma1(D378N) plasmid induced by growth of transfor-
mants under non-selective conditions, the plasmid con-
taining the P

GAL1

-PDR3 fusion gene was extracted from

yeast cells, amplified in E. coli and used to transform the
Dpdr1

Dpdr3

host

strain

bearing

YCpP

PDR5

-

pma1(D378N). The results of this retransformation
experiment showed that in all cases the double trans-
formants selected on minimal glucose medium were also
able to grow in the presence of galactose (

Fig. 3

). In

addition, their sensitivity to cycloheximide in the pre-
sence of glucose or galactose was as low as that of the
primary clones from which the pYE-P

GAL1

-PDR3

originated. These results proved that galactose positive
phenotype of the tested transformants clearly resulted
from the plasmid pYE-P

GAL1

-PDR3 born mutations.

Therefore, loss-of-function pdr3 mutants can be easily
selected by growth of the strain ZK11-1 on galactose.
Their mutations may diminish the expression of the
PDR3 gene, inactivate the encoded Pdr3p or lead to a
premature termination of its translation. A more

Table 1
Retention of the pYE-P

GAL1

-PDR3 and YCpP

PDR5

-pma1(D378N) plasmids in the strain ZK11-1 during growth under non-selective conditions

Number of generations

Phenotype (%)

Total scored colonies

Ura

Leu

Ura

Leu

Ura

Leu

Ura

Leu

12

6.9

14.4

11.5

67.2

201

24

2.7

5.5

6.9

84.7

72

Table 2
Susceptibility to drugs of the S. cerevisiae FY1679-28C/TDEC double transformants containing different combinations of plasmids

Plasmids

MIC (mg/l)

Cycloheximide

Fluconazole

Glucose

Galactose

Glucose

Galactose

pYEP

GAL1

-PDR3

YCpP

PDR5

-pma1

0.05

No growth

10

No growth

pYEP

GAL1

-PDR3

YCpP

PDR5

-PMA1

0.20

0.60

30

60

pRS306K

YCpP

PDR5

-pma1

0.05

0.05

5

5

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sophisticated strategies must be developed to discrimi-
nate easily among these possibilities.

4. Discussion

We described the development of a novel biological

screening system for the positive selection of natural or
synthetic compounds affecting multidrug resistance in S.
cerevisiae at the level of transcription factor(s) activat-
ing the expression of a set of genes coding for multidrug
resistance efflux pumps. A designed yeast bio-sensor is
constructed within the genetic background of a hyper-
sensitive strain deleted in the PDR1 and PDR3 genes
encoding the main transcriptional factors involved in the
control of multidrug resistance

[9

/

12]

. The screen is

based on the restoration of yeast growth on medium
containing galactose used to induce the Pdr3p activated
expression of a dominant-negative pma1 -D378N allele
placed under the control of the PDR5 promoter.

A positive screening system can be carried out on

both solid and liquid media containing galactose thereby
allowing a high throughput screening of libraries of
compounds for specific inhibitors of Pdr1p/Pdr3p tran-
scription factors which exhibit a low toxicity and permit
cell growth on galactose. Along with the identification
of new compounds that render multidrug resistant
strains sensitive to antifungals the screening system is
also suitable for selection of loss-of-function pdr3
mutants. After minor modifications it can even be
used for selection of multicopy suppressors or disrup-
tants (after transposon mutagenesis) in novel genes
essential for the establishment and maintenance of
multidrug resistance in yeast. The products of such
novel genes may represent potential targets for a new
generation of antifungals.

In the developed yeast-based screening system for

compounds suppressing simultaneously the expression
of several drug transporter genes instead of the function
of a single multidrug resistance pump

[30]

, the pma1

mutant allele can be alternatively replaced by any other
gene or DNA sequence

[31]

of over-expression in which

the PDR5 promoter (or a similar promoter recognized
by Pdr1p/Pdr3p) arrests cell growth in response to
regulated PDR3 expression. Since Pdr1p and Pdr3p
recognize the same PDRE in the promoters of their
target genes the P

GAL1

-PDR1 fusion gene can also be

used instead of P

GAL1

-PDR3 .

Homologues of S. cerevisiae membrane efflux pumps

that decrease intracellular concentrations of antifungals
below effective levels were also found in pathogenic
yeasts such as C. albicans , C. dubliniensis and C.
glabrata

[5

/

8]

. Subsequent to the identification of the

major transcription factors that control their expression,
a similar experimental strategy consisting of an analo-
gous heterologous screening system can be designed for
inhibitors of specific transcription factors of pathogenic
yeast species.

Unfortunately, the lead compounds that bind to

Pdr3p (Pdr1p) transcription factor(s) and inhibit its
(their) function have not yet been identified. Therefore,
we validated our yeast-based screen using a truncated
Pdr3p. In fact, deletion of the C-terminal activation
domain of Pdr3p resulted in growth of the indicator
strain on a medium containing galactose. Similarly, we
proved by retransformation experiments that all galac-
tose positive and at the same time cycloheximide
sensitive clones issued spontaneously from the strain
ZK11-1 contained only plasmid born mutations that
altered the expression or function of Pdr3p transcription
factor and hence the expression of its target genes
involved in multidrug resistance.

A yeast-based biosensor system has been developed

and described that affords the reporting of down-
regulation of multidrug resistance efflux pumps as
changes in the growth of galactose sensitive cells. The
biosensor can be used in positive screening for the
effectors that suppress multidrug resistance in yeast and
restore their sensitivity to antifungals.

Fig. 3. Transformants of the S. cerevisiae strain FY1679-28C/TDEC bearing the P

PDR5

-pma1 -D378N and plasmid with either the truncated

(P

GAL1

-PDR3 DAD ) or spontaneously mutated (P

GAL1

-pdr3

) fusion gene are able to grow on galactose.

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Acknowledgements

We thank C.W. Slayman and C. Jacq for plasmids

and helpful discussions. This work was supported in
part by grants from the Slovak Grant Agency of Science
(VEGA.1/0019/03), from Science and Technology Assis-
tance Agency (APVT-51-000502), from Slovak Ministry
of Education, from Comenius University and from
European Commission (QLK2-2001-02377 and COST
B16).

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