Antagonistic Changes in Sensitivity to Antifungal Drugs
by Mutations of an Important ABC Transporter Gene in a
Wenjun Guan1,2, Huifeng Jiang2, Xiaoxian Guo1, Eugenio Mancera3, Lin Xu4, Yudong Li1, Lars
Steinmetz3, Yongquan Li1*, Zhenglong Gu2*
1College of Life Sciences, Zhejiang University, Hangzhou, People’s Republic of China, 2Division of Nutritional Sciences, Cornell University, Ithaca, New York, United States
of America, 3European Molecular Biology Laboratory, Heidelberg, Germany, 4Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United
States of America
Fungal pathogens can be lethal, especially among immunocompromised populations, such as patients with AIDS and
recipients of tissue transplantation or chemotherapy. Prolonged usage of antifungal reagents can lead to drug resistance
and treatment failure. Understanding mechanisms that underlie drug resistance by pathogenic microorganisms is thus vital
for dealing with this emerging issue. In this study, we show that dramatic sequence changes in PDR5, an ABC (ATP-binding
cassette) efflux transporter protein gene in an opportunistic fungal pathogen, caused the organism to become
hypersensitive to azole, a widely used antifungal drug. Surprisingly, the same mutations conferred growth advantages to
the organism on polyenes, which are also commonly used antimycotics. Our results indicate that Pdr5p might be important
for ergosterol homeostasis. The observed remarkable sequence divergence in the PDR5 gene in yeast strain YJM789 may
represent an interesting case of adaptive loss of gene function with significant clinical implications.
Citation: Guan W, Jiang H, Guo X, Mancera E, Xu L, et al. (2010) Antagonistic Changes in Sensitivity to Antifungal Drugs by Mutations of an Important ABC
Transporter Gene in a Fungal Pathogen. PLoS ONE 5(6): e11309. doi:10.1371/journal.pone.0011309
Editor: Deb Fox, The Research Institute for Children at Children’s Hospital New Orleans, United States of America
Received July 14, 2009; Accepted May 11, 2010; Published June 25, 2010
Copyright: ? 2010 Guan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by the faculty startup funds from Cornell University awarded to Z.G. and the Future Academic Stars project of Zhejiang
University to W.G. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (YL); firstname.lastname@example.org (ZG)
Opportunistic fungal infections are a global health threat .
Widespread use of antifungal drugs in the immunocompromised
population has been associated with the emergence of clinically
significant drug resistance among patients who have been exposed
to such antimycotics for prolonged periods [2,3]. Understanding
the processes that underlie the emergence of such resistance is vital
for dealing with this critical issue. Known molecular mechanisms
of drug resistance in fungi include overexpression of genes that
encode drug-efflux pumps belonging to the ABC (ATP-binding
cassette) family of transporter proteins , overexpression or
mutation of the target enzyme, and alteration of other enzymes in
the same biosynthetic pathway as the target enzyme .
The budding yeast, Saccharomyces cerevisiae, widely used in baking
and ethanol production for industrial usage and human consump-
tion, in general is non-pathogenic. Strain YJM789, however, was
derived from a clinical S. cerevisiae isolate (YJM128) collected from
the lung of an AIDS patient [6,7]. The YJM789 strain has many
phenotypes that are relevant to its pathogenicity, including high-
temperature growth , pseudohyphae , and deadly virulence
in mouse models [7,10]. Its capability of crossing with laboratory
strains of S. cerevisiae makes it an excellent tool to study genetic
systems that underlie these complex phenotypes . As fungal
infections are common among immunocompromised individuals,
AIDS patients are routinely treated with antifungal drugs in
general clinical therapy [11,12]. Therefore, YJM789 represents an
excellent tool for understanding how an organism can survive in
antifungal drug environments.
Our initial determination of its genome sequence showed that
the PDR5 gene is highly polymorphic in YJM789 . It has an
amino acid difference of 5.3% from the lab strain, S288c, whereas
at the whole genome level the difference is only 0.43%. It is
particularly noteworthy that most of the amino acid differences
occurred in two transmembrane domain regions (TMDRs). In
yeast, PDR5 is an important ABC transporter that actively exports
various xenobiotic compounds [13,14,15,16], such as azole
antifungal drugs. Loss-of-function mutants for the PDR5 gene in
lab strains show hypersensitivity to a spectrum of antifungal drugs
and overexpression of this gene product results in resistance to a
variety of chemicals [17,18,19,20]. The mechanism by which
Pdr5p recognizes so many structurally and functionally unrelated
substrates remains an enigma . Previous studies indicate that
the transmembrane domains of the ABC transporters may play
major roles in recognizing substrates [22,23]. Because the hyper-
variable regions co-localize with the transmembrane regions of
Pdr5p from YJM789, we have investigated the functional
consequence of these dramatic changes in this important gene.
In this study, several representative antifungal drugs were used
to treat YJM789 and BY4741 (a lab strain that is isogenic with
S288c) and the outcomes after drug treatment were compared. In
contrast to BY4741, deletion of the PDR5 gene in YJM789
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appeared to have no impact on growth in the presence of both
azole and polyene, two antifungal drugs widely used in clinical
practice. Interestingly, although YJM789 is hypersensitive to
azoles, as expected by loss of Pdr5p-exporter function, it is
hyposensitive to polyene antimycotics. Our experiments, using
both a laboratory strain (BY4741) and YJM789, show that loss of
the PDR5 gene indeed confers a small, but significant growth
advantage to the organism in the presence of polyenes.
Dramatic sequence divergence in YJM789 PDR5 gene
Pdr5p belongs to the ABC gene family, a large and important
group of proteins that are conserved from bacteria to humans .
As shown in Figure 1A, Pdr5p has two transmembrane domain
regions (TMDRs) and two nucleotide-binding domains (NBDs).
Each TMDR has six stretches of amino acids that span cell
membranes and five linkers that connect the transmembrane
segments. According to genomic sequences, PDR5 is a highly
polymorphic ORF in YJM789 . Table 1 shows the number of
amino acid changes in each part of the gene. Consistent with our
previous report, TMD regions have significantly more amino acid
changes than do NBD regions in Pdr5p of YMJ789 (chi-square
test, P,0.0001). Interestingly, within TMDR2, the transmem-
brane segments also have higher percentage of amino acid changes
than the linker regions (Fisher’s Exact Test, P=0.04).
To see if PDR5 is uniquely different in YJM789, we compared
the polymorphism and divergence of PDR5 genes among S.
cerevisiae strains and other Saccharomyces species. Figure 1B and 1C
show the pairwise Pdr5p amino acid differences among eight
sequenced S. cerevisiae strains and S. paradoxus, S. bayanus, two
species in the Saccharomyces sensu stricto complex. For the entire
Pdr5p sequence, all seven S. cerevisiae strains except YJM789
shared more than 99% similarity, but the similarities of YJM789
Pdr5p to those of the other S. cerevisiae strains were less than 95%
(bright green lane), which are even lower than those between S.
paradoxus and other S. cerevisiae strains (.96%, Figure 1B).
Figure 1C shows pairwise differences for the transmembrane
domain regions. The sequence similarities are about 100% among
seven S. cerevisiae strains except YJM789, which had similarities of
only ,87% to the other strains. In comparison, the average
sequence similarities for S. paradoxus and S. bayanus to other S.
cerevisiae strains were 96% and 94%, respectively. It is noteworthy
also that, except for YJM789, the TMDRs in Pdr5p were more
conserved than the rest of the gene within or between species,
indicating that TMDRs are more important for gene function.
Our results imply that functions of Pdr5p in YJM789 might have
been dramatically changed during its evolution.
Figure 1. Sequence differences between eight S. cerevisiae strains, S. paradoxus and S. bayanus. A: Schematics of PDR5 gene regions; B:
Amino acid difference for whole PDR5 sequence; C: Amino acid difference for transmembrane domain regions of PDR5 sequence. The topology
information for the WT Pdr5p was downloaded from UniProtKB/Swiss-Prot database (http://www.uniprot.org/uniprot/P33302). PDR5 DNA sequences
of eight strains of S. cerevisiae (DBVPG1788, DBVPG1853, K11, NCYC361, S288c, YJM789, YJM981 and YPS606) were downloaded from a recent study
. Only the DNA sequences without any frame-shift mutations were used in this study. DNA sequences of PDR5 gene in S. paradoxus and S.
bayanus were downloaded from NCBI database. The phylogenetic tree of these species was adapted from Fitzpatrick et al. . The data were
analyzed by Matlab and the different color schemes represent levels of amino acid similarity.
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YJM789 is hypersensitive to azole antifungal drugs
In the United States, approximately ten antifungal drugs are
currently approved by the Food and Drug Administration (FDA)
for the therapy of systemic fungal infections. Azoles and polyenes
are two principal classes . The azoles can block the ergosterol
biosynthesis pathway by inhibiting the enzyme 14-a-demethylase
(ERG11), which converts lanosterol to ergosterol and is required in
fungal cell membrane synthesis. The polyene antimycotics bind to
sterol (ergosterol preferentially) in cell membranes and create holes
that lead to ion leakage and fungal death.
It is well known that azoles are substrates of Pdr5p in yeast
[25,26]. To investigate the functional consequence of dramatic
changes in the YJM789 PDR5 gene, we compared the growth of
YJM789 with that of BY4741 in three selected azoles: itracona-
zole, ketoconazole, and fluconazole. As shown in Figure 2,
YJM789 was much more sensitive than BY4741 to all these drugs.
We tagged PDR5 genes with GFP in both BY4741 and YJM789.
Although Pdr5p still localizes in cell membranes and has an intact
protein expression (Figure 3), growth patterns of YJM789 were
similar to those of the BY4741Dpdr5 strain when grown in the
presence of azoles (Figure 2), which would be expected if Pdr5p
drug efflux functions were impaired in YJM789. Considering that
YJM789 and BY4741 have significant genetic differences, it is
risky to conclude that from this evidence that hypersensitivity of
YJM789 to azoles is solely due to its highly divergent PDR5 gene.
Azole-efflux function of PDR5 in YJM789 might be
To determine if the PDR5 gene is responsible for the observed
drug phenotype in YJM789, we conducted the following two
experiments, with fluconazole used to represent azole antimycot-
ics. First, growth profiles of YJM789 were measured before and
after the deletion of the PDR5 gene. If Pdr5p had indispensable
drug-exporter function, deletion of the gene would lead to reduced
growth of YJM789 in fluconazole. As shown in Figure 4, however,
YJM789 and YJM789Dpdr5 grew similarly in fluconazole. The fact
that Pdr5p played no significant role in YJM789 for resistance to
fluconazole suggests that the protein might have lost its ability to
export azoles. In the presence of fluconazole, both YJM789 and
YJM789Dpdr5 grew slightly better than BY4741Dpdr5 (Figure 4),
indicating that YJM789 may have acquired residual azole-export
capability elsewhere in the genome.
Second, to test if BY4741 Pdr5p could accomplish its normal
function in the YJM789 background, we replaced the YJM789
PDR5 gene with the allele from BY4741. The growth profiles for
BY4741, YJM789 and YJM789 expressing BY4741 PDR5 were
compared. As shown in Figure 4, YJM789 with BY4741 PDR5
grew as well as BY4741 WT over the range of fluconazole
concentrations, indicating that in the YJM789 background,
Table 1. Amino acid changes in each part of PDR5 gene
between YJM789 & S288c (isogenic to BY4741).
Length492 121 155137 125
Amino acid changes7 10 122311
% changes 1.42 8.267.7416.8 8.8
a: NBD: Nucleotide Binding Domain.
b: TMDR: Trans-Membrane Domain Region.
Figure 2. Azole sensitivity of YJM789, BY4741 and BY4741 PDR5 gene deletion strain. The strains were grown in YPD overnight at 30uC
and reinoculated to OD600=0.1. 90 mL media of strains were treated with 10 mL of water or a pharmacological compound (A: itraconazole, B:
ketoconazole, C: fluconazole), respectively, and then grown for 24 h. Only OD600values at 24 h are shown in the figure. Measurements were made in
triplicate with standard deviations shown in the figures. D. Strains were grown overnight and reinoculated to OD600=0.2, then 4 mL of ten-fold serial
dilutions were spotted onto YPD agar containing one of the drugs (itraconazole: 2 mg/mL, ketoconazole: 1 mg/mL, fluconazole 5 mg/mL), and the
plates were incubated at 30uC for 2 days.
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BY4741 Pdr5p confers normal azole-exporter function. It also
indicates that hypersensitivity of YJM789 to azoles was not due to
inherent changes in the plasma membrane of YJM789. Our
additional results indicated that it is more likely that the TMDR2
domain in the YJM789 PDR5 gene cannot carry out its original
function (Figure 5), which is consistent with the observation that
the transmembrane segments in this region have a higher
percentage of amino acid changes than other regions (Table 1).
Mutations in PDR5 gene lead to gain of resistance to
Resistance to antifungal drugs represents a serious health threat.
YJM789 is a common yeast strain increasingly isolated from AIDS
patients who have received prolonged courses of prophylactic
antifungal drug treatment . Because these isolates of YJM789
presumably have survived intensive exposure to antimycotic drugs,
particularly the azoles, it was intriguing to observe that YJM789
lost the ability to grow in the presence of antifungal drugs that
target ergosterol synthesis.
To further investigate the reason underlying the high mutations
of PDR5 gene in YJM789, we examined the influence of another
group of antifungal drugs: the polyenes. Polyene antimycotics can
bind to sterols (ergosterol preferentially) in fungal cell membranes,
promoting leakage that contributes to cell death. Amphotericin B
(AmB) and nystatin are commonly used polyene antimycotics .
Interestingly, YJM789 is much more resistant to AmB (Figure 6A,
6C) and nystatin (data not shown) than is BY4741. Several lines of
evidence indicate that mutations in PDR5 gene of YJM789 are
related to its improved growth in this drug environment. First, the
BY4741Dpdr5 (green bars in Figure 6A) strain was more resistant to
AmB than BY4741WT (yellow bars in Figure 6A), indicating that
loss of PDR5 gene function led to a growth advantage in the
Figure3.Pdr5plocalizes andexpressesinYJM789.A.ThestrainscarryingtheGFP-taggedversion ofPdr5pwereexponentiallygrownin YPDmedia
and visualized by tagged-GFP signal. Fluorescence (left) and DIC (right) images were background-subtracted and scaled identically. The results clearly
show that Pdr5p localizes at plasma membrane in YJM789 strain. B. Western blot analysis of Pdr5p (GFP-tagged) in YJM789 strain by using anti-GFP
Figure 4. Growth differences between YJM789 and BY4741 in
the presence of fluconazole. BY4741, YJM789,their PDR5 null strains
and YJM789Dpdr5::BPDR5(GY02) were grown in YPD overnight and
reinoculated to OD600=0.1. 90 mL of the above media with strains were
treated with 10 mL of water or different concentrations of fluconazole,
and then grown for 24 h at 30uC. OD600values at 24 h are shown in the
figure. Measurements were made in triplicate with standard deviations
shown in the figures.
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presence of AmB. Second, strain YJM789 showed similar growth
profiles both before and after PDR5 deletionin the presenceof AmB
(Figure 6A, 6C). Third, and most importantly, YJM789 strains
containing a functional BY4741 PDR5 gene became more sensitive
to AmB than did YJM789 strains without a functional PDR5 gene
(Figure 6B). Interestingly, this growth difference was significant only
at 37uC, the temperature of the environment from which YJM789
was isolated, whereas it was difficult to see this difference at 30uC.
Of note, YJM789 with a functional BY4741 PDR5 gene showed a
growth advantage over wild-type YJM789 in the presence of
fluconazole at both at 30uC (Figure 4) and 37uC (Figure S1).
Genetic variation among individuals contributes to the fitness
landscape of a population. Loss of gene function can have
dramatic fitness consequences for individuals. Can loss of a gene
be adaptive? The antagonistic pleiotropy hypothesis states that
certain genes, when functional, are beneficial in some conditions
and deleterious in others [28,29,30,31]. This genetic pleiotropic
effect is a result of interactions between genes and environments,
which can lead to a trade off for organismal fitness under certain
conditions. When environments change, the antagonistic pleiot-
ropy of gene functions can lead to adaptive gene loss in evolution.
Indeed, this ‘‘less is more’’ scenario was proposed as one model for
phenotypic evolution [32,33]. Accumulating evidence for adaptive
gene loss indicates that antagonistic pleiotropy of gene function
may play an important role in species adaptation [34,35,36,37].
In this study, we discovered an interesting case of possible
adaptive functional loss that has clinical relevance. Our data show
that the PDR5 gene in YJM789 has lost the ability to facilitate
growth in the presence of azoles, presumably due to the inability to
Figure 5. Drug resistance assays with PDR5 variants. A. Cell growth on Fluconazole. 4 ml five-fold serial dilutions of BY4741 WT cells, pdr5 null
mutant cells, and cells expressing PDR5-1, which was reconstructed with YPDR5 TMDR1 and BPDR5 TMDR2 and PDR5-2, which was reconstructed with
BPDR5 TMDR1 and YPDR5 TMDR2, were spotted on SC-uracil drug agar plates. Before spotting, all strains were grown to exponential phase, diluted to
0.2 OD600. Plates were incubated for 3 days at 30uC. The results indicate that the TMDR1 in YJM789 PDR5 is functional (row #1 vs. row #3), but the
TMDR2 in YJM789 PDR5 cannot conduct its original function (row #1 vs. row #4). B. Expression of hybrid constructs. Electrophoresis result for RT-
PCR products was depicted. Total RNA (lane 1) and cDNA (lane 2) of pdr5D null mutant cells harboring YEplac195-PDR5-1, total RNA (lane 3) and cDNA
(lane 4) of pdr5D null mutant cells harboring YEplac195-PDR5-2 were amplified by specific primer pairs. The result indicates that both constructs can
be expressed successfully in the BY4741Dpdr5 background.
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recognize and export members of this class of antifungal drug. It
has been reported that mutations in PDR5 gene in YJM789 may
be responsible for its hypersensitivity to cycloheximide . We
showed that deletion of PDR5 gene, however, improves organism
growth in the presence of polyenes. Interestingly, among recently
genotyped S. cerevisiae strains , several different pathogenic S.
cerevisiae isolates showed large sequence divergence in the PDR5
gene, which was not observed in any non-pathogenic strains,
indicating that dramatic changes in this gene could be important
for the pathogenicity of S. cerevisiae (Figure S2). We are currently
conducting experiments to address this issue in these pathogenic
Because at least two azole drugs were brought to market
(ketoconazole and miconazole) before the isolation of YJM789 and
other clinical strains , it is intriguing to observe that a yeast
strain with high azole sensitivity could still survive in the
pathogenic S. cerevisiae population. One possibility is that the
patients from whom the strains were isolated didn’t receive
intensive treatment with azole antifungal drugs. However, if the
gene has been in the population for a considerable time, it seems
less likely that azoles were not used extensively in this population.
YJM789 is a haploid derivative of the heterozygous diploid clinical
isolate YJM128 . The highly mutated allele we observed could
be protected by another copy due to the rare occurrence of meiosis
in these clinical strains. Indeed, both the complicated life history of
pathogenic yeast strains and adaptation to antifungal drugs
(polyenes) might function together to keep this highly mutated
PDR5 allele at a certain frequency in pathogenic population
of S. cerevisiae.
The origin of the highly polymorphic regions in the YJM789
PDR5 gene is not clear. The synonymous distance (KS) of two
transmembrane domain regions between S288c and YJM789 are
0.3 and 0.5, respectively (Figure 7A), which are similar to the
synonymous distance between S288c and S. paradoxus, another
Saccharomyces species, whereas KSfor the rest of the gene between
S288c and YJM789 is 0.071, similar to the genome average .
AmB, a polyene antimycotic, was made available in the early
1960s, and is widely used in human immunodeficiency virus
(HIV)-seropositive patients . Since antifungal drug therapy has
a short history, we have to emphasize that adaptation to polyenes
by modification of the PDR5 gene, on the assumption that it did
occur, likely represents an evolutionary force that selected a pre-
existing genetic variation.
The closest ABC-family member to the PDR5 gene in S. cerevisiae
genome (PDR15 gene) has a 24% amino acid difference from
Pdr5p and the synonymous differences between PDR5 and PDR15
genes are saturated (Ks.5), indicating that the two TMDRs in the
YJM789 PDR5 gene could not have originated from ectopic gene
conversion within the same species. A phylogenetic tree was built
for the two TMD regions within orthologs of Pdr5p for all
sequenced strains of S. cerevisiae and S. paradoxus . As shown in
Figure S3, TMD regions in Pdr5p from YJM789 have the most
divergent sequences among strains of S. cerevisiae, which is not the
case for other genes in the YJM789 genome . Furthermore,
Figure 6. Drug susceptibility of YJM789 and BY4741 in AmB. A. All strains were grown in YPD overnight at 30uC and reinoculated to
OD600=0.1. 90 mL of media with strains were treated with 10 mL of water or different concentrations of AmB, and then grown for 20 h at 30uC. The
values are the averages from three experiments. B. YJM789Dpdr5 (GY03) and YJM789Dpdr5:: BPDR5 (GY02) mutants grew in YPD medium overnight
at 30uC and reinoculated to OD600=0.1. 90 mL of media with strains were treated with 10 mL of water or different concentrations of AmB, and then
grown for 20 h at 37uC. Measurements were made in triplicate with standard deviations shown in the figures. C. BY4741, YJM789, BY4741Dpdr5 and
YJM789Dpdr5 were grown overnight and reinoculated to OD600=0.2, then 4 mL of ten-fold serial dilutions were spotted onto YPD agar containing
AmB (5 mg/mL, 10 mg/mL), and the plates were incubated at 30uC for 2 days.
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TMDRs in the YJM789 PDR5 gene also show great sequence
divergence from strains of S. paradoxus, indicating that these two
domains were not from S. paradoxus, or at least from strains of that
species with available genome sequences. No matter what are the
origins of these two TMDRs in the YJM789 PDR5 gene, the ratio
of nonsynonymous to synonymous changes across the whole PDR5
gene significantly increased in YJM789 (Figure 7B), indicating
functional relaxation of this gene, which is consistent with our
experimental data for inactivation of important functions in Pdr5p
of strain YJM789.
The observation of a gain in AmB resistance as a result of the
loss of PDR5 gene is surprising. Ergosterol is an important
component of fungal membranes and serves the same function as
cholesterol in animal cells. When ergosterol content decreases in
fungal plasma membranes, AmB-binding sites might decrease,
leading to AmB resistance [42,43]. If this is indeed the mechanism
for AmB resistance after PDR5 deletion, our results imply that
Pdr5p is involved in ergosterol homeostasis. Consistent with our
speculation, some studies have reported that Pdr5p may transport
lipids, such as phospholipids , sphingolipids  or glycer-
ophospholipids , although the mechanisms of transport are
unclear. There are no reports of ergosterol transport by Pdr5p.
Further experiments are needed to elucidate the functional role of
Pdr5p in ergosterol homeostasis.
It is important to point out that the PDR5 gene in YJM789
might not be correctly called a pseudogene. The fact that Pdr5p
could localize in cell membranes and has an intact protein
expression in YJM789 indicates that the pleiotropic protein Pdr5p
might conduct unrecognized functions. Because Pdr5p is a multi-
substrate transporter , our observation could be caused by
changes in substrate specificity as a result of mutations in the
Pdr5p TMDRs of YJM789. Indeed, this conclusion is supported
by the observation that the ratio of non-synonymous to
synonymous distances between S288c and YJM789 is less than
0.2 for the PDR5 gene (Figure 7B).
It is also important to note that other genetic changes contribute
to the YJM789 AmB-resistance phenotype. At higher concentra-
tions of AmB, growth of BY4741 and BY4741Dpdr5 was inhibited,
whereasYJM789 and YJM789Dpdr5stillshowed significantgrowth,
indicating that changes in other genes in YJM789 contributed to
AmB adaptation. It was shown that mutations in ERG6, an
important gene in the ergosterol biosynthesis pathway, can lead to
AmB resistance in various pathogenic species of yeast [48,49]. Our
analysis indicated that ERG6 gene and its ,300 bp 59 UTR region
areidentical in BY4741 and YJM789, indicating that the gene is not
likely involved in AmB resistance of YJM789. By using a genotyped
progeny panel(,200 strains)from a cross between BYand YJM789
, we are currently conducting QTL-mapping to identify genetic
loci that underlie this interesting phenotype.
Regardless of the evolutionary history of the PDR5 gene and the
mechanism for the AmB-sensitivity change in YJM789, our
experimental results might imply a new drug-resistance strategy in
pathogenic yeasts, i.e., sacrificing an important function in one
drug condition for a minor fitness gain in other drug conditions.
This clinically important showcase with interesting evolutionary
implication will, hopefully, lead to better understanding of the
emergence of drug resistance not only in pathogenic fungi, but also
in microbes in general.
Materials and Methods
All antifungal drugs were obtained from Sigma-Aldrich and
Fisher Scientific. AmB, fluconazole, cycloheximide, nystatin
dihydrate and itraconazole were reconstituted with water to
appropriate concentrations. Ketoconazole was prepared in
dimethyl sulfoxide. All stock dilutions were stored at –20uC for
up to 2 months.
Strains, media and growth conditions
The strains of S. cerevisiae, listed in Table 2, were grown at 30uC
and maintained on yeast extract/peptone/dextrose medium
(YPD). YPD and synthetic media (SD) were prepared as described
by Rose et al. , YPD media containing G418 (200 mg/mL) was
used for selection of strains with kanMX4 dominant drug-
resistance markers. SD uracil-deficient medium was used for
selection of strains with URA3 marker.
Transformation of yeast with plasmid DNA was achieved
according to the procedure previously described . Bacterial
transformation was performed using the calcium chloride
procedure as described by Sambrook et al. .
The strains GY01 (YJM789Dpdr5) and GB01 (BY4741Dpdr5)
were constructed by replacing the PDR5 gene from YJM789 and
Figure 7. Synonymous evolutionary distances (A) and ratio of non-synonymous to synonymous evolutionary distances (B) in
different parts of PDR5 gene. The distances were calculated using PAML . The bars to the left indicate the distances (ratio) between S288c and
YJM789 while the bars to the right measure the distances (ratio) between S288c and S. paradoxus.
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BY4741 parental strains using a URA3 marker, respectively.
Correct integration of the deletion constructs and proper looping-
out were confirmed by PCR analysis.
Strain GY02 was constructed by inserting BY4741 PDR5 gene
(BPDR5) into the PDR5 locus of GY01. The transformants were
selected on 5-FOA plates . Because the viability of the mutants
(GY02) in antifungal drugs might be influenced by 5-FOA, the
BPDR5 gene was disrupted in GY02 by targeted insertion with
URA3 as the selection marker to reconstruct the pdr5 null mutants
Agar-plate drug-sensitivity assays
Fresh S. cerevisiae colonies were inoculated in 5 mL of YPD or
SD selective medium, and grown overnight at 30uC. The cells
were diluted to an OD600of 0.2, and 4 mL was spotted with serial
dilutions on solid medium containing drugs (AmB, fluconazole,
itraconazole or ketoconazole) in the agar. The plates were
incubated at 30uC for 48 h.
Real-time drug-sensitivity assay
To test drug resistance, strains were grown overnight, diluted
in 90 mL of rich medium (YPD), treated with 10 mL of water or
a pharmacological compound, and then grown for 20 to 24 h.
Samples were grown in a microplate
(OD 600) (model Um-MQX200R; Bio-T Instrument Inc.).
Drug resistance was estimated from
after incubating for 20 to 24 h (20 h for AmB and 24 h for
the optical density
Plasmids construction and transformation
The 5.18 kb PDR5 fragment including 404 bp upstream and
224 bp downstream sequences was amplified from YJM789 or
BY4741 genomic DNA (using primers Xba I_upper/Xba
I_lower) and then inserted into the Xba I site of vector
BPDR5. For the replacement of YJM789 PDR5 transmembrane
domains (TMDR1 or TMDR2) with the BY4741 PDR5 TMDRs,
2.7KB (1–2764 bp) and 1.8 KB (2764–4535 bp) fragments of
PDR5 ORF which include BY4747 PDR5 TMDR1 or TMDR2
encoding sequences were amplified by using primer combinations
Sac I-upper/BstP I-lower and BstP I-upper/Sal I-lower, and then
cloned into Sac I-BstP I and BstP I-Sal I digested YEplac195-
YPDR5, respectively. All the above reconstructed plasmids were
confirmed by colony PCR and DNA sequencing. Primer
sequences used in this work (the underlined sequences indicate
restriction sites, the restriction site of BstP I is inside the PCR
product) are listed in Table 3.
Construction of GFP-tagged yeast strains and
Pdr5p-GFP protein fusions were constructed as previous
described. The 2.6kb GFP-kanMX6 cassette was amplified
from pFA6a-GFP(S65T)-kanMX6 with a pair of chimeric primers:
TCTCGGAATTCTTTCGGACcc aatacgcaaaccgcctct. The PCR
product was transformed into YJM789 WT and the transformants
were selected on YPD plates containing G418. Integration of the
GFP fusion at the PDR5 locus was confirmed by PCR using
combinations of primers flanking PDR5 gene and GFP-kanMX6
cassette-specific primers. Fluorescence analysis was done using
exponentially growing cells. Cells expressing Pdr5p-GFP fusion
protein were examined by using fluorescence and differential
interference contrast (DIC) microscopy (OLYMPUS BX51
RNA preparation and RT-PCR
To confirm PDR5-1 (YPDR5 TMDR1 + BPDR5 TMDR2) and
PDR5-2 (BPDR5 TMDR1 + YPDR5 TMDR2) could express in
BY4741Dpdr5 background, total RNA was prepared as described
in an earlier study. The cDNA was synthesized by
PrimeScriptTMRTase (Takara). Primer pairs (TGAGGTCTT-
CATCTCCTT/AAACCAACCTCTCCGTGT for PDR5-1, the
PCR product length is 1111 bp, GCAAGACCTTCCTCTCCT/
CAATTTCTCCGTAAGT ATCG for PDR5-2, the PCR product
length is 1114 bp) were used to detect transcription of TMDR1
and TMDR2 constructs.
Substitution rate analysis
To calculate evolutionary distance between two strains, we used
PAML program to calculate the substitution rates of synonymous
Table 2. Yeast strains and plasmids used in this study.
StrainGenotype Parental StrainSource
BY4741MATa ho::KanMX his3 leu2 met15 ura3 Yeast Knock-out (YKO) deletion collection
YJM789 MATa ho::hisG lys2 cyh
GY00 MATa ho::hisG lys2 cyh ura3::KanMXYJM789 This study
GB01MATa ho::KanMX his3 leu2 met15 ura3 pdr5::URA3 BY4741This study
GY01GY00, pdr5::URA3YJM789 This study
GY02 GY01, ura3::BPDR5 YJM789This study
GY03GY02, bpdr5::URA3 YJM789 This study
Table 3. Primers used in PDR5 plasmid construction.
Xba I_lower gctctagaACCGATGAGATAACCTAGAAAT
Sac I_ upper ggtgagctcCACGATTCAGCACCCTTTG
BstP I_lower GATTTATCACGGGGAATACCAT
BstP I_upper TATCAATCCGTTGGCTTACTT
Sal I_lower cgcgtcgacACCGATGAGATAACCTAGGAAT
Antifungal Drug Resistance
PLoS ONE | www.plosone.org8 June 2010 | Volume 5 | Issue 6 | e11309
sites (KS) and nonsynonymous sites (KA) as previously described
Phylogenetic tree reconstruction
All sequenced S. cerevisiae strains and four S. paradoxus strains, one
from each of four clades in this species , were used for tree
reconstruction. S. bayanus sequence was used as outgroup. The
combined TMDRs in each strain were aligned by Muscle  and
tree was reconstructed by Clustalw . The phylogenetic tree is
shown in Figure S3.
(GY02) and YJM789Dpdr5 (GY03) mutants in media containing
fluconazole at 37uC. GY02(N) and GY03(-) were grown in YPD
overnight at 30uC and reinoculated to OD600=0.1. 90 ml above
media of strains were treated with 10 ml of water containing
fluconazole (Final concentration is 20 mg/ml), and then grown for
24 h at 37uC in a microplate spectrophotometer (model Um-
MQX200R; Bio-T Instrument Inc.). OD600 value was sampled
every 20 minutes.
Found at: doi:10.1371/journal.pone.0011309.s001 (0.03 MB
Growth difference between YJM789Dpdr5::BPDR5
divergent sequence in PDR5 gene. SNP distributions for PDR5
gene from all 63 strains were generated from http://gbrowse.
princeton.edu/cgi-bin/gbrowse/yeast_strains_snps/ (YSB, Yeast
SNPs Browser ). Eleven strains were shown in this figure, and
the pathogenic strains were highlighted in red rectangular. Among
all 63 strains, five pathogenic strains (including YJM145 that is
isogenic to YJM789), which were from different clinical isolation
, display dramatic sequence divergence in PDR5 gene, while
the remaining clinical strains (YJM421 was shown as an example)
show only a few SNPs. In contrast, none of the non-clinical strains
Multiple pathogenic S. cerevisiae strains have
(five were shown as example) show dramatic sequence divergence
in PDR5 gene.
Found at: doi:10.1371/journal.pone.0011309.s002 (0.04 MB
brane domain regions (TMDR) in PDR5 (YOR153W) gene. All
sequenced S. cerevisiae strains and four S. paradoxus strains
(marked in green), one from each of four clades in this species ,
were used for tree reconstruction. S. bayanus sequence was used as
outgroup. The combined TMDRs in each strain were aligned by
Muscle  and tree was reconstructed by Clustalw .
Bootstrap values are shown on the tree. As indicated in the
figure, the PDR5 gene in YJM789 (indicated by red arrow) is
clearly an outgroup in all S. cerevisiae strains, which is not true for
whole genome sequence comparison(3). The PDR5 gene in
YJM789 is also very divergent from all S. paradoxus strains
(including all sequenced strains, only four were shown here). For S.
mikatae, S. kudriavzevii, S. cariocanus, as they either are more
divergent than S. paradoxus from S. cerevisiae, or their PDR5
gene sequences are not available, we didn’t include them in the
Found at: doi:10.1371/journal.pone.0011309.s003 (0.05 MB
Phylogenetic tree for the combined two transmem-
We thank Hong Chen, Wu Wei for help and discussions, and Andre ´
Goffeau, Paul Soloway, Lixin Zhang, and Allan Eaglesham for reading the
manuscript. Helps during experiments from Eric Alani and Koodali
Nishant are also very much appreciated.
Conceived and designed the experiments: WG HJ LMS YL ZG.
Performed the experiments: WG HJ XG LX. Analyzed the data: WG
HJ LX YL ZG. Contributed reagents/materials/analysis tools: WG HJ
EM LX YL ZG. Wrote the paper: WG HJ ZG.
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