Cryptococcus neoformans Overcomes Stress of Azole
Drugs by Formation of Disomy in Specific Multiple
Edward Sionov., Hyeseung Lee., Yun C. Chang., Kyung J. Kwon-Chung*
Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, United States of
Cryptococcus neoformans is a haploid environmental organism and the major cause of fungal meningoencephalitis in AIDS
patients. Fluconazole (FLC), a triazole, is widely used for the maintenance therapy of cryptococcosis. Heteroresistance to FLC,
an adaptive mode of azole resistance, was associated with FLC therapy failure cases but the mechanism underlying the
resistance was unknown. We used comparative genome hybridization and quantitative real-time PCR in order to show that C.
neoformans adapts to high concentrations of FLC by duplication of multiple chromosomes. Formation of disomic
chromosomes in response to FLC stress was observed in both serotype A and D strains. Strains that adapted to FLC
concentrations higher than their minimal inhibitory concentration (MIC) contained disomies of chromosome 1 and stepwise
exposure to even higher drug concentrations induced additional duplications of several other specific chromosomes. The
number of disomic chromosomes in each resistant strain directly correlated with the concentration of FLC tolerated by each
strain. Upon removal of the drug pressure, strains that had adapted to high concentrations of FLC returned to their original
level of susceptibility by initially losing the extra copy of chromosome 1 followed by loss of the extra copies of the remaining
disomic chromosomes. The duplication of chromosome 1 was closely associated with two of its resident genes: ERG11, the
target of FLC and AFR1, the major transporter of azoles in C. neoformans. This adaptive mechanism in C. neoformans may play
an important role in FLC therapy failure of cryptococcosis leading to relapse during azole maintenance therapy.
Citation: Sionov E, Lee H, Chang YC, Kwon-Chung KJ (2010) Cryptococcus neoformans Overcomes Stress of Azole Drugs by Formation of Disomy in Specific
Multiple Chromosomes. PLoS Pathog 6(4): e1000848. doi:10.1371/journal.ppat.1000848
Editor: Scott G. Filler, David Geffen School of Medicine at University of California Los Angeles, United States of America
Received October 8, 2009; Accepted March 5, 2010; Published April 1, 2010
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This study was supported by funds from the intramural program of the National Institute of Allergy and Infectious Diseases, NIH. 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: firstname.lastname@example.org
. These authors contributed equally to this work
Cryptococcus neoformans is the most common cause of fungal
meningoencephalitis world-wide. A major predisposing factor is
the profound cellular immune defect caused by HIV infection or
other underlying conditions. Cryptococcal meningoencephalitis is
fatal unless treated and even with the most advanced treatment it
is known for its high mortality rates [1,2]. Fluconazole (FLC), a
triazole antifungal drug, has been the agent most widely used for
prophylactic therapy as well as for the long term management of
common mycoses such as candidiasis and cryptococcosis owing to
its efficacy and safety . Long-term maintenance therapy with
azoles creates favorable conditions for the emergence of resistance
to the drug and increased azole resistance in vitro has been shown
to be predictive of treatment failures and infection relapses .
The molecular basis of resistance to azole antifungals has been
studied extensively in Saccharomyces cerevisiae and pathogenic Candida
species such as C. albicans and C. glabrata which are phylogenetically
distant from C. neoformans [5–13]. In these fungi, resistance is known
to emerge via (1) increased production of multidrug transporters
[14–16], (2) mutations in ergosterol biosynthetic pathway genes
[17,18], (3) amplification of genomic regions that contain ergosterol
biosynthetic pathway genes and transcription factors that positively
regulate a subsets of efflux pump genes [19,20] and (4) activation of
Hsp90 that may facilitate the cells to respond to drug stress [21,22].
In C. neoformans, FLC resistant strains have rarely been reported and
the emergence of resistance has most often been documented with
clinical outcomes of AIDS patients receiving azole maintenance
therapy [23–27]. The mechanism of resistance in C. neoformans
during maintenance therapy is poorly understood.
An intriguing pattern of intrinsic azole resistance termed
‘heteroresistance’ was reported in 1999 among C. neoformans strains
isolated from AIDS patients undergoing FLC maintenance therapy
 and has only recently been characterized further . This
phenomenon of heteroresistance has been described as the
emergence of a resistant minor subpopulation, within the single
higher than the strain’s MIC. The resistant subpopulations can
adapt to increasing concentrations of the drug in a stepwise manner.
However, this acquired resistance to high concentrations of FLC is
lostduringrepeated passageindrugfree mediaand theclones return
to their original level of heteroresistance. The level of hetero-
resistance to FLC (LHF) was defined as the lowest concentration
of the azole drug at which resistant subpopulations emerge . All
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different LHF regardless of whether they are pre- or post therapy
strains and the frequency of resistant subpopulations that emerge at
each LHF ranged between 0.3 and 10% depending on strains .
Purification of a homogeneously sensitive subpopulation was not
achieved at each strain’s LHF while a homogeneous population of
resistant cells could readily be obtained by exposure to FLC
concentrations equal to or higher than its initial LHF. This acquired
resistance to high concentrations of FLC, however, was lost during
repeated passage in drug free media and the clones returned to the
original LHF at which only 0.3 to 10% of the subpopulations grew.
The molecular mechanism involved in this unique pattern of azole
resistance remains an enigma.
In this paper, we employed a genomic approach to uncover the
mechanism by which C. neoformans cells acquire resistance to high
concentrations of FLC and then subsequently lose the resistance
when the drug stress is removed. We demonstrate that the adaptive
resistance to higher concentrations of FLC was achieved by
duplications of multiple chromosomes in response to drug pressure.
Upon repeated transfer in drug free media, cells with multiple
disomic chromosomes lose duplicated copies of the chromosomes
sequentially and return to their original levels of drug tolerance.
Such genomic fluidity that enables the cells to cope with the drug
stress was observed in C. neoformans strains of both serotypes, A and
D. Our results provide an explanation as to the mechanism
governing the transiently high azole resistance observed in C.
neoformans. We propose that this mechanism contributes to the
failure of FLC therapy that results in the recurrent infection
reported in patients undergoing prolonged azole therapy .
Characterization of heteroresistance to FLC in strain H99
All strains of C. neoformans tested in our laboratory displayed the
intrinsic adaptive heteroresistant phenotype to FLC . Since
serotype A strains of C.neoformans are the most prevalent of all the
four serotypes in clinical settings, we chose the strain H99, a
genome sequenced reference strain of serotype A, to study the
mechanism of heteroresistance. Equal numbers of colonies were
observed on YPD agar media with or without 16 mg/ml FLC.
However, growth of the colonies on 16 mg/ml FLC was slightly
slower with heterogeneity in size. On YPD media containing
32 mg/ml FLC, only 0.3–0.6% of the input cells consistently
formed colonies within 72 h. Therefore, the intrinsic level of H99
FLC heteroresistance was determined to be 32 mg/ml .
Exposure of these subclones resistant at 32 mg/ml FLC to stepwise
increases in FLC concentration generated clones resistant to
64 mg/ml (strain H99R64) and 128 mg/ml (strain H99R128).
Conversely, repeated transfer in drug free media of cells that
had adapted to FLC at concentrations .32 mg/ml resulted in
their reversal to original levels of heteroresistance. For instance,
the H99Rvt16strain derived from 16 daily transfers of the strain
H99R64in drug-free media displayed a FLC resistance phenotype
intermediate between H99R64and H99. Its colony size on YPD
with 32 mg/ml FLC was larger and its FLC E-test value was higher
(48 mg/ml) than the parental H99 strain (E-test MIC=24 mg/ml,
Figure 1). In contrast, the H99Rvt26strain similarly derived from
26 daily transfers of the strain H99R64in drug free media
completely reverted back to the parental type.
Genome analysis of FLC resistant strain H99R64
It is possible that the resistant strain H99R64may express a
different set of genes compared to H99. Thus, we compared the
gene expression profiles of H99R64and H99 using microarray
analysis. Of the 6719 detectable genes analyzed, 4149 genes were
identified as significant by a mean false discovery rate (FDR) of 5%
with significance analysis of microarray (SAM) as described in
Material and Methods. We found 763 genes to be up or down
regulated at least 1.8-fold in H99R64compared to H99. As
expected, some of the differentially regulated genes are annotated
for drug-related functions such as ABC transporter, multidrug
resistance protein and enzymes involved in the ergosterol
biosynthetic pathway. More significantly, among the 491 genes
observed to be upregulated in H99R64, 308 (63%) are located on
chromosome 1 (Chr1) and 143 (29%) on chromosome 4 (Chr4),
which in collectively comprises 92% of the upregulated genes in
H99R64(Figure 2A). Having a majority of the upregulated genes
distinctly clustered in two chromosomes, we suspected some
chromosomal anomaly in H99R64. To examine global genomic
changes in H99R64, we performed comparative genome hybrid-
ization (CGH). Interestingly, CGH analysis of the H99R64strain
revealed that the average log2ratio of hybridization signals for
Chr1 and Chr4 was significantly above zero (0.84 and 0.89,
Figure 1. FLC E-tests. Approximately 16106cells of each strain were
plated on YPD media and E-test strips were placed on the media. The
plates were incubated at 30uC for 72 h. Strains: H99 (wild type), H99R64
(resistant at 64 mg/ml of FLC), H99Rvt16, and H99Rvt26(H99R64derivatives
obtained by daily transfer of H99R64on drug-free media for 16 and 26
Cryptococcus neoformans is an environmental fungus that
causes life threatening brain disease, primarily in AIDS
patients. The disease is estimated to claim 700,000 lives
annually world-wide but most heavily in Africa. Fluconazole
(FLC), a fungistatic antifungal drug, is commonly used to
treat patients for long term maintenance therapy. Recur-
rence of cryptococcosis in AIDS patients undergoing FLC
maintenance therapy has been increasingly reported.
Heteroresistance, an adaptive azole resistance, was associ-
ated with FLC therapy failure cases but the mechanism
underlying the resistance was unknown. We previously
described that C. neoformans strains are innately hetero-
resistant to FLC; each strain producing a fraction of
subpopulation that can tolerate a high concentration of
the drug. These resistant subpopulations revert to original
phenotype during maintenance in drug free media. Various
methods including cDNA microarrays, comparative genome
hybridization and quantitative PCR have been applied to
uncover the mechanism involved in the adaptation of C.
neoformans to high concentrations of FLC and subsequent
loss of resistance upon the removal of drug pressure. We
discovered that C. neoformans adapts to high concentration
of FLC by formation of disomy in multiple chromosomes.
The removal of drug pressure results in a sequential loss of
the extra chromosomal copies. It is likely that this novel
mechanism of adaptation contributes to the failure of FLC
therapy for cryptococcosis.
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Figure 2. Chromosome 1 and 4 are duplicated in H99R64. (A) Gene expression profiles. Gene expression patterns of H99R64were compared to
those of the wild type strain, H99. A total of eight arrays including three biological repeats and dye-reversed sets were performed as described in
materials and methods. Each column (lane 1 to 8) represents a microarray experiment and each row represents the expression of a gene on the array
arranged by its chromosomal position. A total of 4149 significant genes were plotted after SAM analysis with a mean FDR of 5%. Chromosomes where
upregulated genes are clustered are indicated. The relative expression levels are represented by color as shown in the bar. (B) CGH plot of H99R64.
The genomic DNA of the experimental strain was hybridized against the genomic DNA of the reference strain, H99. Each panel represents the CGH
plot of each chromosome. Chromosome number is indicated in the right side corner of each panel. The x-axis represents the position of each gene
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respectively) across the entire chromosome as shown in Figure 2B.
The simplest explanation for this observation was that Chr1 and
Chr4 had duplicated in the cells of H99R64. We also analyzed H99
and H99R64by flow cytometry and the data suggested that
H99R64is not a diploid strain harboring trisomic Chr1 and Chr4
(Figure S1). Consequently, the observed overexpression of the
genes on Chr1 and Chr4 in cDNA microarray was due to the
increase in copy number of the genes on the two chromosomes.
To confirm this phenomenon of multiple chromosome
duplications revealed by the CGH data, quantitative real time
PCR (qPCR) of genomic DNA was performed. Four probes
representing the four genes at different locations of chromosome 1
that span the left and the right arm (chr1A, chr1B, chr1C, and chr1D)
were chosen for qPCR using the same genomic DNA used for
CGH analysis. qPCR results of each probe on Chr1 were
compared to those of the probe on either Chr3 (chr3A) or Chr11
(chr11A), which served as unduplicated internal controls. As shown
in Figure 2C, the copy number of all tested genes at different
locations on Chr1 was close to two fold higher than the genes on
Chr3 or Chr11 in H99R64(P,0.001), while the relative copy
number of those genes was close to 1 in H99. This indicated that
H99R64has two copies for each of the four genes on Chr1.
Similarly, the qPCR results from probes representing two genes on
Chr4 (chr4A and chr4B) showed the dosage of each gene in H99R64
to be two fold of that in H99 (Figure 2D; P,0.001). These qPCR
results corroborated with the CGH data and suggested that
chromosomes 1 and 4 in the strain H99R64were disomic and the
disomy of these two chromosomes was associated with resistance at
64 mg/ml FLC.
The number of disomic chromosomes correlates with the
level of FLC resistance
Since the resistant clones can adapt to different levels of FLC
concentration, it is possible that the FLC resistance level of each
clone positively correlates with the number of disomic chromo-
somes. To test this hypothesis, we analyzed by CGH array six
other H99-derived strains that had adapted to different levels of
FLC concentration. First, we tested two of the aforementioned
reverted strains, H99Rvt16and H99Rvt26, which had resulted from
repeated transfer of H99R64in drug free media for 16 days and 26
days respectively (Figure 1). CGH data revealed the intermediate
revertant H99Rvt16to be monosomic for Chr1 but still disomic for
Chr4 while the complete revertant, H99Rvt26, contained no
disomic chromosomes (Figure 3A and Figure S2). These results
suggested that removal of drug pressure caused a loss of the
duplicated copies of chromosomes in the cells starting with that of
Chr1 and then eventually return to the wild type status. Second,
we performed CGH analysis of the H99R32and H99R128strains
which were resistant to 32 mg/ml and 128 mg/ml FLC, respec-
tively. CGH plots revealed that only Chr1 was duplicated in
H99R32, while four chromosomes (Chr1, 4, 10, and 14) were
duplicated in H99R128(Figure 3B and Figure S2). Third, we
analyzed strain H99R64L, a clone of H99R64that was maintained
for an additional two weeks on the media with 64 ug/ml FLC.. As
was the case with H99R64, Chr1 and Chr4 were duplicated in
H99R64L. Interestingly, Chr10 was also duplicated in H99R64Land
the copy number of many genes on Chr14 increased although
not quite two-fold compared to that of H99 (Figure 3B). It appears
that prolonged incubation of cells at high FLC concentrations
results in the emergence of additional disomic chromosomes.
CGH results were confirmed by qPCR analysis of a gene chosen
from each of the four chromosomes 1, 4, 10 and 14. As shown in
Figure 3C, relative copy numbers of each gene against the internal
control gene on Chr3 corroborated the CGH analysis. All four
genes located on different chromosomes were duplicated in the
strain H99R128while no gene duplication was evident in the
complete revertant strain H99Rvt26(P,0.001). In the strain
H99R64L, the gene copy number on Chr10 and Chr14 was close
to 2 and 1.5, respectively. Furthermore, chromosome duplication
was also verified by quantitative Southern blot analysis using a
probe from each of the four affected chromosomes (Figure S3 and
Table S1). Collectively, these data strongly suggested that the
number of disomic chromosomes positively correlated with the
levels of FLC resistance of the strain and with the duration of
exposure to FLC.
Genome fluidity reflected in the gene dosages at colony
Since CGH experiments require relatively large amounts of
genomic DNA, each strain was allowed to proliferate for many
generations on the drug media in order to obtain enough cells.
The CGH data, therefore, represents the average status of the
whole population grown on the media containing a certain
concentration of FLC for many generations. qPCR was performed
to examine gene dosages in the small number of individual
resistant clones immediately after their emergence on plates
containing high concentrations of FLC. This would determine
whether gene duplication occurred during the early stages of
growth in which resistance was initially observed at the single
colony level. We chose 4 different colonies that appeared 4 days
after plating naive H99 cells on media with 32 mg/ml FLC. Four
independent colonies resistant at 64 and/or 128 mg/ml of the drug
(derived from four different 32 and/or four different 64 mg/ml
FLC resistant clones, respectively) were also isolated and analyzed.
The CGH data suggested that the chromosome duplication occurs
primarily in Chr1, 4, 10 and 14 and thus we focused our colony
qPCR analysis only on these four chromosomes, although the
duplication event might not be limited to these chromosomes.
Interestingly, variations in the gene duplication events on different
chromosomes was observed among the independent colonies
grown on 32 mg/ml and 128 mg/ml FLC, respectively, but not on
64 mg/ml FLC (0 passage in Figure 4A, 4B, and 4C). To test
whether prolonged drug-exposure would alter the outcome of gene
duplication, the same sets of the four clones from each
concentration of FLC were streaked on media with the same
FLC concentration for 4 and 8 passages. Single colonies were then
subjected to qPCR which exposed the tremendous variability in
the duplication of genes representing different chromosomes. For
example, one clone from 32 mg/ml FLC plate (clone #2) initially
had duplication of a gene on Chr1. After 4 passages, genes on
Chr4 and Chr10 were also duplicated in addition to Chr1 and the
status of gene duplication in these chromosomes was the same
when tested after 8 passages (Figure 4A). Clone #3 from the
32 mg/ml plate, however, appeared to have genes on Chr1 and
arranged in the order of its chromosomal location. The y-axis plots gene copy number as a running average over seven genes calculated from the
log2ratio of relative hybridization intensity. (C) Copy number of four genes on Chr1 determined by qPCR. Four probes (chr1A, chr1B, chr1C, and
chr1D) at different locations on Chr1 were compared to two control probes; one located on Chr3 (chr3A) and the other on Chr11 (chr11A) in H99 and
H99R64, respectively. (D) Copy number of Chr4 genes (chr4A and chr4B) was compared to that of the probe (chr3A) on the endogenous control Chr3
in H99 and H99R64.
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Figure 3. Gain and loss of FLC resistance positively correlates with the number of chromosomes duplicated. (A) CGH plots of Chr1, 4,
and 3 for strains H99R64, H99Rvt16, and H99Rvt26. (B) CGH plots of Chr1, 4, 10, 14, and 3 for strains H99R32, H9964L, and H99R128. (C) Copy number of four
genes in H99-derived strains with different levels of resistance. The relative gene copy number of four probes (chr1A, chr4A, chr10A and chr14A)
located on Chr1, 4, 10, and 14 was compared to the control probe (chr3A) on Chr3. The same genomic DNA from the strains used for CGH arrays was
used for qPCR assays. Only the chromosomes involved in duplication are shown. Chr3 serves as a non-duplicated chromosome control.
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Chr14 duplicated initially, but the genes on Chr14 did not remain
duplicated after longer exposure to the drug. In contrast, clone #4
did not show the gene on Chr1 duplicated until after 4 passages on
32 mg/ml FLC media while gene on Chr4 was duplicated from the
beginning and remained duplicated throughout the 8 passages.
Generally, a more consistent pattern of gene duplication was
observed with independent clones isolated from the plates
containing FLC 64 mg/ml compared to those isolated from
32 mg/ml FLC plates (Figure 4B). Fluctuations in the pattern of
gene duplication, however, were also obvious among the colonies
grown on FLC 128 mg/ml (Figure 4C). These data clearly showed
the plasticity of gene duplication patterns at the single colony level,
which could not be depicted clearly in CGH data. It is likely that
CGH results represent the average status of chromosomes in the
whole population and the system is not sensitive enough to allow
detection of transient chromosomal duplication events in individ-
ual colonies. However, the CGH results of H99R64Lapparently
revealed the intermediate process of Chr14 duplication in which
the gene copy number of Chr14 was 1.5 as verified by qPCR using
the same batch of DNA (Figure 3B and 3C). Taken together, our
data suggested that when C. neoformans was treated with FLC, the
process of multiple chromosome duplication may vary among
individual cells and the status of chromosome copy number
determined by CGH appears to be an average of the whole
population from cells grown in the presence of FLC for many
ERG11 is important for chromosome 1 duplication under
It was plausible that formation of disomic chromosomes in
association with FLC resistance was due to the presence of certain
genes on the duplicated chromosomes which plays crucial role in
the survival of cells under the drug stress. Since Chr1 was
universally duplicated in the resistant clones, we focused on Chr1
as the first step to determine whether each duplicated chromosome
carries genes that confer selective advantage under azole drug
stress. Among the annotated genes on Chr1, AFR1 and ERG11
were the two candidate genes that had already been characterized
involving FLC resistance in C. neoformans. AFR1 is an ATP binding
cassette (ABC) transporter-encoding gene and have shown to play
an important role in azole susceptibility [29,30]. ERG11 encodes
lanosterol-14-a-demethylase, the target of FLC, and increased
expression levels of ERG11 is associated with increased FLC
resistance in several fungi [15–18]. ERG11, the target of FLC, has
been proposed to contribute to isochromosome formation in C.
albicans , so we chose it to address its role in Chr1 duplication.
If the presence of ERG11 were the main cause of Chr1 duplication,
ERG11-containing chromosome would primarily duplicate re-
gardless of the location of the gene. On the other hand, if other
genes besides ERG11 were equally or more important for the
survival in the presence of FLC, Chr1 would remain duplicated
even if ERG11 is relocated from Chr1 to other chromosomes.
Since ERG11 is most likely essential, we first inserted an extra copy
of ERG11 on Chr3, which had not duplicated under any level of
drug stress and then deleted ERG11 from its native location on
Chr1 (Figure 5). Strain C1345, which contained two copies of
ERG11 – one on Chr1 and the other on Chr3, exhibited elevated
resistance to FLC according to E-test (Figure 5) as well as by
growth analysis (100% growth at 32 mg/ml and 0.1% growth on
64 mg/ml in contrast to 0.3–0.6% growth at 32 mg/ml and 0 %
growth at 64 mg/ml of H99) compared to H99. These data
indicate that the extra copy of ERG11 inserted onto Chr3
conferred increased FLC resistance. Since C1345 had two copies
of ERG11 mimicking the effect of Chr1 duplication regarding
ERG11 copy number, it was of great interest to determine the
status of chromosome duplication in C1345 upon exposure to high
concentration of FLC. First, qPCR was performed on two
independent colonies of C1345 isolated immediately after
emerging on the plate containing 32 mg/ml FLC. We detected
close to two copies of ERG11 (chr1A probe) but only one copy of
other genes on Chr1, Chr3, and Chr4 suggesting no duplication of
chromosomes of C1345 at 32 mg/ml FLC (Figure 6A). This is in
contrast to H99 subclones resistant to 32 mg/ml FLC in which
Chr1 is duplicated (Figure 3B and 3C). However, qPCR of two
C1345 colonies isolated directly from 64 mg/ml of FLC plate
showed the existence of three copies of ERG11 (chr1A probe) and
two copies of both chr1D and chr4A, but only one copy of chr3A
(Figure 6A). These data suggested that both Chr1 and Chr4 were
duplicated in the C1345 colonies grown in 64 mg/ml of FLC.
CGH analysis of the entire cell population harvested from 64 mg/
ml FLC (C1345R64) clearly showed duplication of Chr1 and Chr4
and not Chr3 (Figure 6B). In addition, C1345R128, the C1345
Figure 4. Gene duplication determined by colony qPCR. Gene dosage was determined by qPCR in four independent colonies of H99 derived
strains by stepwise selection on YPD agar plates containing increasing concentrations of FLC: (A) 32 mg/ml; (B) 64 mg/ml; (C) 128 mg/ml. The relative
gene copy number of four probes (chr1A, chr4A, chr10A and chr14A) located on Chr1, 4, 10, and 14 was compared to the control probe on Chr3
(chr3A). The 0 passage samples represent initial colonies that appeared 4 days after plating the parental strains on plates containing FLC. The gene
duplication in those colonies was also determined following 4 and 8 passages on agar media with the same concentration of FLC.
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strain grown on128 mg/ml FLC showed similar chromosome
duplication patterns as C1345R64maintaining duplication of Chr1
and Chr4. It is intriguing, however, to observe intermediate
hybridization signal for Chr3 in C1345R128(average log2ratio of
20.035 and 0.317 for C1345R64and C1345R128, respectively).
qPCR using the same DNA showed that the copy number of chr3A
probe located on Chr3 was 1.2260.06, confirming the CGH
results. This data suggests that a proportion of the cells in the
population of C1345R128strain may have an extra copy of Chr3.
Colony PCR from two independent colonies of C1345R128
supported duplication of the gene on Chr3 (Figure 6A). The
second copy of ERG11 resides on Chr3 in C1345R128which has
not been observed to be duplicated in other strains tested so far.
These results showed that two copies of ERG11, one on Chr 1 and
the other on Chr3 prevented disomy formation of Chr1 at 32 mml
FLC but did not prevent disomy formation of Chr1 and Chr4 at
FLC 64 mg/ml, a concentration which is 2-fold higher than the
level tolerated by C1345. Furthermore, Chr1 was preferentially
duplicated over Chr3 at high concentrations of FLC when the
ERG11 existed on both chromosomes.
The ERG11 gene on the Chr1 was subsequently deleted from
C1345 leaving only one copy of ERG11 inserted on Chr3. The
FLC resistance levels of three independent transformants (C1347,
C1348, and C1350) were comparable with H99 (100% growth at
16 mg/ml and 0.3–0.6% growth at 32 mg/ml), indicating that
translocation of ERG11 from Chr1 to Chr3 did not alter the
strain’s FLC resistance level. Clones of these three independent
transformants grown in 32 mg/ml FLC were subjected to colony
qPCR. Noticeably, the copy number of ERG11 (chr1A probe) and
chr3A were close to two fold, while the copy number of chr1D
remained close to one suggesting duplication of Chr3 but not Chr1
(Figure 6C). CGH analysis of C1347R32, C1348R32, and C1350R32
showed Chr3 was duplicated in all three strains (Figure 6D).
Interestingly, Chr4 was also duplicated in all three strains although
colony PCR results suggested duplication of a gene on Chr4 only
in C1347R32and C1348R32(Figure 6C and 6D). This result was
different from H9932Rin which only Chr1 duplication was
observed at 32 mg/ml FLC (Figure 3B). These data suggested that
when only one copy of ERG11 is present in the genome, the
ERG11 bearing chromosome is the primary one to be duplicated
at 32 mg/ml FLC. However, additional chromosome duplication
(Chr4) was required to tolerate the stress exerted by FLC when
ERG11 was moved from its native location to Chr3. Additional
CGH was performed using strains derived from C1347, C1348,
Figure 5. The correlation between ERG11 location and the phenotype. The diagram on the right represents the chromosomal location of
ERG11 and its corresponding phenotype is shown on the left (FLC E-tests). The position of 4 tested genes (chr1A, chr1B, chr1C, and chr1D) on Chr1 is
indicated at the top of the diagram.
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and C1350 resistant to 64 mg/ml FLC (Figure 6D). C1347R64and
C1348R64displayed disomies of Chr1, Chr3 and Chr4 while
C1350R64showed duplication only in Chr3 and Chr4 but not in
Chr1. Single colony qPCR of these three strains supported the
CGH results although the copy number of chr1D in C1347R64was
only 1.32 (60.05), suggesting that Chr1 amplification occurred in
Figure 6. The importance of ERG11 and AFR1 in Chr1 duplication. (A) Gene duplication patterns in C1345 derived strains. Gene copy number
was determined by qPCR in resistant colonies derived from C1345 which contains two copies of ERG11, one on Chr1 and the other on Chr3. Two
different colonies resistant to FLC 32, 64 and 128 mg/ml are shown respectively. Gene copy number was quantified by measuring four probes
(ERG11=chr1A, chr1D, chr3A and chr4B) located on Chr1, 3 and 4, respectively, in comparison to the control probe (chr5A) on Chr5. (B) CGH plots of
Chr1, 3, and 4 for C1345R64and C1345R128that tolerate 64 and 128 mg/ml FLC, respectively. Only the chromosomes involved in duplication are shown.
(C) Gene copy number was determined by qPCR in resistant colonies derived from three independent transformants (C1347, C1348, and C1350), each
with a single copy of ERG11 on Chr3. Strains resistant to 32 and 64 mg/ml FLC are shown. The probes located on Chr1, 3, and 4 were compared to the
control probe (chr5A) on Chr5. (D) Heat map of CGH results: lanes 1–3 represent the CGH array of three independent strains, C1347, C1348, and
C1350, resistant at 32 and 64 mg/ml FLC, respectively. The relative copy number is represented by color as shown in the bar. Red indicates that the
copy number of the genes is close to 2. (E) CGH plots of Chr1, 4, 5, 9, 10, and 3 for the C1371 (afr1DR1) strain resistant at 1 mg/ml of FLC.
Disomy Formation in Azole Resistant C. neoformans
PLoS Pathogens | www.plospathogens.org8 April 2010 | Volume 6 | Issue 4 | e1000848
a certain portion of the clonal population (Figure 6C). These data
indicated that when a single copy of ERG11 gene exists in the
genome, the chromosome carrying ERG11 is consistently dupli-
cated in all subsequently derived FLC-resistant strains. However,
our data also pointed out that ERG11 was not the sole reason for
the Chr1 duplication and duplication of other genes on Chr1 and
those on Chr4 also appeared to have contributed to the survival of
cells at 64 mg/ml FLC.
AFR1 also plays a role in chromosome 1 duplication
under FLC stress
In an attempt to investigate other genes on Chr1 that confer
resistance to FLC via chromosome duplication, we investigated
AFR1. Several lines of evidence have indicated that AFR1 plays an
important role in FLC resistance. First, AFR1 expression was
upregulated in both H99R64and the H99 strains treated with
FLC. Second, deletion of AFR1 resulted in a drastic decrease in
FLC resistance in H99 . Third, high expression level of AFR1
resulted in the increased level of FLC resistance . The afr1D
strain (C1371) was used to determine the possible involvement of
AFR1 in Chr1 duplication under FLC stress. If AFR1 were
important for duplication of Chr1, we would not expect Chr1 to
be duplicated in afr1D strains resistant to FLC. The H99 afr1D
strain is extremely sensitive to FLC (MIC 0.38 mg/ml) and its level
of heteroresistance was reduced from 32 mg/ml to 1 mg/ml .
CGH analysis of the subpopulation resistant at 1 mg/ml FLC
(afr1DR1) clearly showed that Chr1 was not duplicated in the
afr1DR1strain (Figure 6E). Instead, Chr4 and Chr5 were
duplicated along with short segmental duplications of Chr9 and
Chr10. Thus, absence of AFR1 on Chr1 not only abrogated Chr1
duplication but also caused whole duplications or segmental
duplication in other chromosomes at 1 mg/ml FLC. Such a
chromosomal duplication pattern was presumably due to the
presence of genes on these duplicated chromosomes which might
compensate for the effect of AFR1 deletion from Chr1 in afr1DR1.
It is noteworthy that although ERG11 is present on Chr1 in
afr1DR1, disomy formation of Chr1 does not occur at 1 mg/ml
FLC. However, CGH analysis of the subpopulation resistant at
8 mg/ml (afr1DR8) showed that Chr1 was duplicated along with an
additional four chromosomes (Chr 4, 5, 6, and 10; Figure S4).
These findings underscore the importance of both ERG11 and
AFR1 in the formation of Chr1 disomy under FLC stress.
Disomic chromosome formation is a common
phenomenon in strains of C. neoformans
All strains of C. neoformans tested thus far displayed the FLC
heteroresistant phenotype . Although different strains dis-
played heteroresistance at different concentrations of FLC, the
stepwise exposure to higher concentrations of FLC allowed the
strains to adapt to levels of FLC that are higher than their original
MIC. These resistant strains all reverted to the original level of
resistance upon removal of drug pressure. To investigate whether
chromosome duplication associated with FLC resistance was an
H99-specific event, we analyzed a number of matched pairs of
naive vs. FLC-adapted resistant isolates in both serotype A and D
backgrounds. Consistent with the observation in H99, FLC-
resistant strains derived from both serotype backgrounds con-
tained disomic chromosomes according to CGH analysis (Figure
S5), even though the duplicated chromosomes were not always
identical in these strains. These results demonstrated that
chromosome duplication associated with FLC resistance is a
general mechanism employed by C. neoformans to overcome the
stress exerted by FLC.
We report here that C. neoformans consistently forms disomies in
multiple chromosomes in response to high level of azole pressure
in both serotype A and D strains. Duplicated copies of the disomic
chromosomes are lost as the drug pressure is removed. While there
can be minor variations in the number of duplicated chromosomes
among individual colonies grown on the same FLC media, the
number of disomic chromosomes in the population of the over-
all cultures positively correlates with the adaptation to stepwise
increase in FLC concentration.
Aneuploidy associated with azole resistance was reported in
Candida albicans where a substantially higher frequency of
aneuploidy was found among azole resistant strains compared to
susceptible strains . In addition, chromosome instability,
specific segmental aneuploidy, translocation of chromosomal arms
and whole chromosome duplication have been previously reported
in Candida species [20,31,32].
One could argue that the clones with disomy observed in the
subpopulation of H99 under FLC stress may comprise a normal
population that is selected by the drug rather than the drug
induced chromosome amplification. There are three reasons for
this argument being unlikely. First, aneuploidy caused by
chromosome missegregation occurs once every 56105
divisions in yeast  and once every 104to 105cell divisions in
mammalian cells . The frequency of FLC resistant clones of
H99 (0.3 to 0.6%) that emerged on drug containing media is too
high to be the result of spontaneous chromosomal missegregation.
Furthermore, the frequency of FLC resistant clones in different
strains can be as high as 10% . The frequency at which
aneuploidy occurs in C. neoformans under FLC stress, therefore, is
several logs higher than the frequency of spontaneous aneuploidy
formation in other eukaryotes. Second, H99 is the most widely
studied strain of C. neoformans and yet a clone derived from H99
that contains disomic chromosomes in a stress-free environment
has never been reported. Third, we observed disomy formation in
H99 only when exposed to FLC but not other xenobiotics such as
trichostatin A, gliotoxin or rhizoxin (data not shown). Aneuploidy
is reported to have multiple effects on cellular physiology and cell
division in haploid yeast . Consistent with findings in yeast,
disomic chromosomes in C. neoformans result in a proliferative
disadvantage as evidenced by the retarded growth rate of H99R64
which harbors extra copies of Chr1 and 4, and exhibits lower
virulence in mice compared to the wild type strain (Figure S6).
Although many fungi undergo chromosome length polymor-
phisms, chromosomal loss  or gain of minichromosomes 
under different environmental stress, the degree of consistency and
reproducibility of genomic fluidity observed in the present work
has not been reported in other fungi.
Since genetically identical cells of a single C. neoformans colony
exposed to a high concentration of FLC can produce small
subpopulations that show a marked difference in FLC suscepti-
bility, we can speculate that this variability is linked to stochasticity
in gene expression . The genes that govern the capacity to
differentiate into heteroresistant subtypes are not known. Although
the CGH data show an increase of specific disomic chromosomes
when C. neoformans is challenged by increasing drug pressure,
minor variations in duplicated chromosomes appear to occur
among individual colonies. Such plastic outcomes of duplication
events can be advantageous for C. neoformans since it can provide
the flexibility required for the cells to respond to various kinds of
sudden stress it encounters either in the environment or in the
host. The extra copy of a disomic chromosome may have resulted
from non-disjunction, which occurs commonly in eukaryotes
Disomy Formation in Azole Resistant C. neoformans
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under different stresses [39,40]. In mammalian systems, inhibition
of cholesterol biosynthesis by blocking sterol 14 a-demethylase
(ERG11 ortholog) induces the formation of polyploid cells and
mitotic aberrations . Since ergosterol, the counterpart of
cholesterol in fungi, is the essential molecule for maintaining
membrane integrity, depletion of ergosterol in nuclear and cell
membranes due to FLC treatment may jeopardize normal
patterns of cytokinesis and enhance the frequency of chromosomal
non-disjunction. For example, the spindle pole body (SPB), a
fungal equivalent of the centrosome is closely associated with the
outer nuclear membrane in C. neoformans . Once integrity of
the nuclear membrane is compromised by depletion of ergosterol
in FLC treated cells, segregation of the SPB may become irregular
and enhance the chromosomal instability during cell division .
Gene duplication is known to be one of the key mechanisms
which allows fungi to be selected during evolution . Aneuploidy
resulting in gene duplication has been reported to be the initial
evolutionary change in S. cerevisiae selected in vitro to overcome loss
of the myosin II protein which is crucial for normal cytokinesis .
In response to drug pressure, disomic chromosomes that contain
genes relevant to ergosterol synthesis and drug transport could be
beneficial for the survival of C. neoformans. Our hypothesis on the
crucial roles of ERG11 and AFR1 in the occurrence of Chr1
duplication in clones resistant to high drug concentrations was
borne out. When grown on 32 ug/ml FLC, the drug level at which
Chr1 disomy occurs in H99, the strain with ERG11 translocated
from Chr1 to Chr3 showed duplication only in Chr3 but not in
Chr1. However, an extra copy of ERG11 on Chr3 in addition to
the native copy on Chr1 was not enough to prevent Chr1
duplication at FLC concentrations higher than 32 mg/ml. This
indicated that multiple copies of ERG11 alone can not meet the
challenge of very high FLC stress. Similarly, Chr1 was not
duplicated when AFR1 was deleted and grown on 1 mg/ml FLC
(the strain’s initial heteroresistance level). However, Chr4 and
Chr5 were duplicated along with short segmental duplications of
Chr9 and Chr10, which most likely compensate for the loss of
AFR1. These findings underscore the important roles of ERG11
and AFR1 in Chr1 duplication under drug stress. Afr1 is related to
Snq2 of C. glabrata which is known to function as a transporter for
several compounds including FLC . In our test, afr1D was also
sensitive to cycloheximide and rhizoxin treatment suggesting that
AFR1 may function as a transporter for these drugs (data not
shown). An ideal experiment to test the hypothesis that duplication
of Chr1 causes drug resistance would be to construct a strain in
which only Chr1 is duplicated without exposure to azoles and then
test the FLC resistance level of the strain. In S. cerevisiae, strains
containing duplicated chromosomes could be constructed and the
effect of aneuploidy tested . Currently, construction of such
strains, however, is technically not feasible in C. neoformans.
Duplication of Chr1 has never been observed in H99 prior to
the acquisition of FLC resistance. Since the resistance persisted as
long as Chr1 disomy remained but was lost simultaneously after
prolonged maintenance in drug free media, we are convinced that
the two genes contribute to disomy of Chr1.
The C. neoformans genome contains all the genes known to be
associated with ergosterol biosynthesis and has twice as many
drug-related transporters as S. cerevisiae. These genes are
distributed widely among 14 chromosomes and it is possible that
some of them play a role in azole tolerance. It remains to be
determined whether any other gene and its regulator necessitate
duplication of the chromosome on which it resides. C. neoformans
strains, regardless of the chronology of isolation either before or
after the launch of azole drugs, showed that 0.3 to 10% of the
subpopulations consistently resisted FLC concentrations higher
than their MICs . This number did not vary significantly
during repeated tests. Although FLC resistant strains of C.
neoformans have been increasingly reported from azole therapy
failure cases [24,26,28,29,47–49], the number of stable FLC
resistant mutants among clinical isolates is rare compared to other
pathogenic fungi [15,50]. One reason for the rarity in isolating
FLC resistant C. neoformans mutants may be that heteroresistance
masks mutation. The regular mutation rate is 1025to 1026and
such a low population would be masked by the adaptive
heteroresistant population. Our results provide the foundation
for a mechanistic understanding of transient high azole resistance
to FLC which might occur during prolonged maintenance therapy
Materials and Methods
Strains and media
C. neoformans isolates H99 and NIH376 are serotype A strains;
NIH429 is serotype D . Table 1 lists all the H99 derived strains
used in this study. Strains were stored in 25% glycerol stocks at
280uC until use and were maintained on YPD (1% yeast extract,
2% peptone, 2% glucose) agar plates at 30uC for routine cultures.
Fluconazole (FLC) was provided as powder by Pfizer Global
Research & Development (Groton, CT). Stock solutions were
Table 1. Strains used in the study.
Strain name Descriptions
H99 wild type; resistant to 16 mg/ml FLC
derived from H99; resistant to 32 mg/ml FLC
derived from H99R32; resistant to 64 mg/ml FLC
H99R64maintained on 64 mg/ml FLC for long period of time
H99R64transferred 16 times in drug free media; resistant to
16 mg/ml FLC (see Materials and Methods)
H99R64transferred 26 times in drug free media; resistant to
16 mg/ml FLC (see Materials and Methods)
derived from H99R64; resistant to 128 mg/ml FLC
C1345two copies of ERG11; one on Chr1 and one on Chr3
derived from C1345; resistant to 64 mg/ml FLC
derived from C1345R64; resistant to 128 mg/ml FLC
C1347 derived from C1345 with ERG11 deletion on Chr1
C1348 derived from C1345 with ERG11 deletion on Chr1
C1350 derived from C1345 with ERG11 deletion on Chr1
derived from C1347; resistant to 32 mg/ml FLC
derived from C1348; resistant to 32 mg/ml FLC
derived from C1350; resistant to 32 mg/ml FLC
derived from C1347R32; resistant to 64 mg/ml FLC
derived from C1348R32; resistant to 64 mg/ml FLC
derived from C1350R32; resistant to 64 mg/ml FLC
C1371derived from H99 with AFR1 deletion
derived from C1371; resistant to 1 mg/ml FLC
derived from C1371; resistant to 8 mg/ml FLC
NIH376 a serotype A environmental isolate from the NIH collection
NIH429a serotype D environmental isolate from the NIH collection
Disomy Formation in Azole Resistant C. neoformans
PLoS Pathogens | www.plospathogens.org 10 April 2010 | Volume 6 | Issue 4 | e1000848
prepared in dimethyl sulfoxide (Sigma) at a concentration of 50
mg/ml. Analysis of FLC heteroresistance was performed by the
method described previously . Briefly, cell suspensions (16103
to 46103CFU/ml) in sterile saline were plated on YPD plates
containing various concentrations of FLC. Growth was recorded
after 72 h incubation at 30uC. Isolates were considered to be
heteroresistant when resistant clonal populations were able to
grow on a plate containing FLC. Resistant subpopulations were
exposed to stepwise increases in FLC concentrations on YPD
Gene expression array analysis
Microarray slides were purchased from the Genome Sequencing
Center at Washington University, St Louis. For cDNA arrays,
overnight cultures were diluted to OD600> 0.2 and grown in YPD
liquid media for 7 hr. RNA was extracted from yeast cells using
Trizol (Invitrogen, Carlsbad, CA), and purified with RNeasy
MinElute cleanup kit (Qiagen, Valencia, CA). RNA was labeled
and hybridized as described previously . Arrays were scanned
on a GenePix 4000B scanner and analyzed using GENEPIX PRO
6.0 (Axon Instruments, Foster City, CA). Data were further
analyzed in mAdb database at http://madb.niaid.nih.gov. Three
biological repeats were performed using three independent RNA
sets isolatedfrom cellsculturedon differentdays and the dye-reverse
hybridizations were performed for all 3 sets. One set of RNA was
also subjected to technical repeats. All statistically significant genes
were identified by significance analysis of microarray using a mean
false discovery rate of less than 5%. Only statistically significant
genes were used for data analysis. Although the microarray slides
used in this study were printed with 70-mers that are designed to
uniquely represent each gene in C. neoformans serotype D, the
oligomers were also optimized for homology to genes predicted in
the serotype A strain, H99 (http://genome.wustl.edu/services/
Comparative genome hybridization
Genomic DNA was prepared from C. neoformans strains grown
overnight in 10 ml YPD medium as described previously .
5 mg DNA was digested with DpnII (10 U/mg DNA, New England
Biolabs, Ipswich, USA) and labeled with dye according to the
BioPrimeHArray CGH Genomic Labeling System protocol
(Invitrogen Life Technologies, Carlsbad, USA). In all CGH
experiments, Alexa647 was used to label DNA from the
experimental strains and Alexa555 was used to label DNA from
the reference control strain (Invitrogen Life Technologies,
Carlsbad, USA). Labeled DNA was purified with the purification
kit from the same manufacturer and subjected to competitive
hybridization with the 70mers microarray. Sample hybridization
and data collection were carried out as described above. Data were
further analyzed in mAdb database after applying 50th percentile
Two parameters were considered for the CGH experiments.
First, we hybridized the slides using H99 genomic DNA as both
the experimental and the reference control samples. The scatter
plot of the normalized log10 signal intensity of both channels
showed tight correlation between two probes attesting to the
reliability of the hybridization patterns of H99 genomic DNA to
the JEC21-based 70mer slides. Second, we tested the reproduc-
ibility between arrays. Data from five independent CGH arrays
were obtained from the H99 control set (H99-Alexa 555 vs. H99-
Alexa 647) as well as from the H99R64set (H99-Alexa 555 vs.
H99R64-Alexa 647). The data were highly consistent indicating
high reproducibility between the arrays. Therefore, in most CGH
studies, only one or two arrays per strain were analyzed.
To visualize the CGH array in a chromosomal context, data
were imported into Excel format from the mAdb database. CGH
data was further normalized by subtracting the average log2signal
ratio of each gene obtained in control experiments (H99 Alexa647
vs. H99 Alexa555) from that of a corresponding gene in the
experimental data set to compensate for the dye and background
bias. Relative hybridization levels were plotted as a running
average over seven ORFs and clipped to the range corresponding
to 1–2 copies (log2ratio of 0–1, respectively). Each ORF was
sorted according to their gene number corresponding to its order
along each chromosome (plotted on the x-axis). Although the
genomes are largely co-linear between the current genomic
assemblies of H99 and JEC21, there are several apparent
inversions and translocations. Due to these alterations, homolo-
gous chromosomes between the H99 and JEC21 assemblies have
been assigned different numbers for some chromosomes . The
chromosomal number assignment of H99 was adopted in our
CGH data. Due to the translocation events in Chr3, Chr4 and
Chr11 of H99, the order of genes on these chromosomes was
manually arranged according to its JEC21 counterparts.
Quantitative real time PCR
To quantify the gene copy number on specific chromosomes in
wild-type and FLC-resistant strains, quantitative real time PCR
(qPCR) assays were performed. For confirmation of CGH data,
the same genomic DNA from strains used in CGH arrays was used
for qPCR assays. For individual colony qPCR, genomic DNA of
selected colonies was used. For colony DNA extraction, a single
colony was picked with a sterile toothpick, suspended in 40 ml of
10 mM EDTA buffer in a microcentrifuge tube, boiled for 6 min
and centrifuged. The supernatant was diluted 1:10 in TE buffer
and 5 ml of diluted DNA template was added to 20 ml of the qPCR
mix (Applied Biosystems, Branchburg, NJ). The reaction was
performed in an Applied Biosystems 7500 Real-Time PCR
System. Each reaction was run in triplicate and the average Ct
value was converted to relative amount of DNA using the relative
standard curve method. The sequences of the primers and probes
used for the qPCR are listed in Table S2. The genes CNAG_02959
on Chr3, CNAG_00869 on Chr5 and /or CNAG_07554 on Chr11
were chosen as endogenous controls. For each specific gene, its
copy number was obtained by comparing its qPCR value with the
endogenous control and expressed as relative gene copy number.
ERG11 was cloned by PCR and sequenced. The NAT selectable
marker was cloned into the 59 flanking region of ERG11 and the
resulting construct was inserted in the intergenic region between
CNAG_03012 and CNAG_03013 on Chr3 which were generated
by PCR and sequenced. The final construct was transformed
into H99 and the transformant containing a second copy of
ERG11 between the intergenic region of CNAG_03012 and
CNAG_03013 on Chr3 was screened by PCR and confirmed by
Southern blot analysis. Subsequently, the ERG11 gene on Chr1
was deleted with the NEO gene from the clone containing two
copies of ERG11 (C1345) by biolistic transformation.
An unpaired t test was used for the statistical analysis of qPCR
data. A P value of less than 0.05 was considered to be significant.
H99R64strains. (A) FACS analysis. Log phase cells of H99,
FACS analysis and morphology of H99, H99R32and
Disomy Formation in Azole Resistant C. neoformans
PLoS Pathogens | www.plospathogens.org11 April 2010 | Volume 6 | Issue 4 | e1000848
H99R32, and H99R64were fixed and subjected to FACS analysis as
described (Lengeler, KB, Cox, GM and Heitman, J 2001. Infect
and Immun. 69:115-122). Blue, H99; red, H99R32; green, H99R64.
(B) Morphology of H99, H99R32, and H99R64. The cell size of
H99R32and H99R64was larger than H99. In addition, elongated
cells were frequently observed in H99R32and H99R64. These
differences might affect the outcome of FACS and caused the
peaks of H99R32and H99R64shifted to the right more than
Found at: doi:10.1371/journal.ppat.1000848.s001 (0.35 MB TIF)
H99R64L, H99R128, H99Rvt16, and H99Rvt26. The genomic DNA
of the experimental strain was hybridized against the genomic
DNA of reference strain, H99. Each panel represents CGH plot of
each chromosome. Chromosome number is indicated in the right
side corner of each panel. X-axis represents the position of each
gene arranged in the order of its chromosomal location. Y-axis
plots gene copy number as a running average over seven genes
calculated from log2ratio of relative hybridization intensity.
Found at: doi:10.1371/journal.ppat.1000848.s002 (1.45 MB XLS)
CGH plots of 14 chromosomes for strains H99R32,
DNAs were digested with the restriction enzyme BglI. After
fractionation on a 0.8% agarose gel the DNA was transferred onto
a Hybond-N nylon membrane (Amersham Biosciences, Buck-
inghamshire, UK). The membrane was hybridized at 65uC with
[a-32P] dCTP labeled probes using StripEZ DNA kit (Ambion Inc,
Austin, TX). PCR was used to generate probes with the primer
pairs listed in Table S3. (B) After hybridization, the membrane was
exposed to a phospho-imager screen and signals were quantified
with ImageQuant (Molecular Dynamics). The relative copy
number of each gene was obtained by comparing the signal
intensity of each gene to that of the internal control probe, chr3.
Found at: doi:10.1371/journal.ppat.1000848.s003 (0.31 MB TIF)
Quantitative Southern blot analysis. (A) Genomic
8 mg/ml FLC, was obtained by exposing C1371 (afr1D) to
increasing concentrations of FLC stepwise. The genomic DNA
of afr1DR8was hybridized against H99 genomic DNA. Data was
normalized by subtracting average log2signal ratio of each gene
obtained in control experiment (H99-Alexa647 vs. H99-Alexa555)
from that of corresponding gene in experimental data set to
compensate for the dye and background bias. Each panel
represents CGH plot of each chromosome from afr1DR8strain.
Chromosome number is indicated in the right side corner of each
Found at: doi:10.1371/journal.ppat.1000848.s004 (0.13 MB TIF)
CGH plots of afr1DR8strain. The afr1DR8, resistant to
serotype A and D strains. NIH376 (serotype A) and NIH429
CGH plot of FLC-resistant strains generated from
(serotype D) are environmental isolates and genetically unrelated
to H99 and JEC21. NIH376R64and NIH429R64are FLC resistant
strains derived from NIH376 and NIH429, respectively. The
genomic DNA of NIH376R64 was hybridized against the NIH376
genomic DNA using the JEC21-based 70mer slides. Data was
normalized by subtracting average log2signal ratio of each gene
obtained in control experiment (NIH376-Alexa647 vs. NIH376-
Alexa555) from that of the corresponding gene in experimental
data set to compensate for the dye and background bias. Same
data normalization procedure was applied to NIH429R64using
NIH429 as the control experiment. Each panel represents CGH
plot of each chromosome from NIH376R64(A), and NIH429R64
(B). Chromosome number is indicated on the side of each panel.
Found at: doi:10.1371/journal.ppat.1000848.s005 (0.35 MB TIF)
lower compare to wild type. (A) In vitro growth kinetics of H99 and
H99R64. An overnight culture of each strain was inoculated in
duplicate into 50 ml YPD broth at a starting OD600of 0.2. The
cells were incubated with shaking at 37uC for 32 h. The OD600of
the cultures was measured at various times after inoculation (0, 2,
4, 6, 8, 10, 12, 24, and 32 h). (B) Virulence study of H99 and
H99R64. The animal study was approved by NIH institutional
animal care and use committee. To compare the virulence
between H99 and H99R64, a murine model of pulmonary
cryptococcosis was established in female BALB/c mice (weight,
20 g). Mice were anesthetized with isoflurane and a 20 ml droplet
containing 56107cells was inoculated by intra-nasal inhalation.
Ten animals were used for each strain. The survival of mice was
recorded daily for a total of 45 days.
Found at: doi:10.1371/journal.ppat.1000848.s006 (0.11 MB TIF)
H99R64growth rate is slower and its virulence is
Found at: doi:10.1371/journal.ppat.1000848.s007 (0.03 MB
Oligonucleotides used for Southern analysis
Found at: doi:10.1371/journal.ppat.1000848.s008 (0.04 MB
Oligonucleotides used for qPCR assays
We thank A. Varma for critical discussions and reading of the manuscript.
Conceived and designed the experiments: ES HL YCC KJKC. Performed
the experiments: ES HL YCC. Analyzed the data: ES HL YCC. Wrote the
paper: ES HL YCC KJKC.
1. Kwon-Chung KJ, Bennett JE (1992) Medical Mycology. Philadelphia: Lea &
Febiger. 866 p.
2. Perfect JR, Casadevall A (2002) Cryptococcosis. Infect Dis Clin North Am 16:
3. Zonios DI, Bennett JE (2008) Update on azole antifungals. Semin Respir Crit
Care Med 29: 198–210.
4. Perfect JR, Cox GM (1999) Drug resistance in Cryptococcus neoformans. Drug Resist
Updat 2: 259–269.
5. Kontoyiannis DP, Sagar N, Hirschi KD (1999) Overexpression of Erg11p by the
regulatable GAL1 promoter confers fluconazole resistance in Saccharomyces
cerevisiae. Antimicrob Agents Chemother 43: 2798–2800.
6. Lamping E, Monk BC, Niimi K, Holmes AR, Tsao S, et al. (2007)
Characterization of three classes of membrane proteins involved in fungal azole
resistance by functional hyperexpression in Saccharomyces cerevisiae. Eukaryot Cell
7. Akins RA (2005) An update on antifungal targets and mechanisms of resistance
in Candida albicans. Med Mycol 43: 285–318.
8. Bennett JE, Izumikawa K, Marr KA (2004) Mechanism of increased fluconazole
resistance in Candida glabrata during prophylaxis. Antimicrob Agents Chemother
9. Brun S, Berges T, Poupard P, Vauzelle-Moreau C, Renier G, et al. (2004)
Mechanisms of azole resistance in petite mutants of Candida glabrata. Antimicrob
Agents Chemother 48: 1788–1796.
10. Helmerhorst EJ, Venuleo C, Sanglard D, Oppenheim FG (2006) Roles of
cellular respiration, CgCDR1, and CgCDR2 in Candida glabrata resistance to
histatin 5. Antimicrob Agents Chemother 50: 1100–1103.
11. Sanglard D, Odds FC (2002) Resistance of Candida species to antifungal agents:
molecular mechanisms and clinical consequences. Lancet Infect Dis 2: 73–85.
12. Tsai HF, Krol AA, Sarti KE, Bennett JE (2006) Candida glabrata PDR1, a transcrip-
tional regulator of a pleiotropic drug resistance network, mediates azole resistance in
clinical isolates and petite mutants. Antimicrob Agents Chemother 50: 1384–1392.
13. White TC, Holleman S, Dy F, Mirels LF, Stevens DA (2002) Resistance
mechanisms in clinical isolates of Candida albicans. Antimicrob Agents Chemother
Disomy Formation in Azole Resistant C. neoformans
PLoS Pathogens | www.plospathogens.org12 April 2010 | Volume 6 | Issue 4 | e1000848
14. Cowen LE, Anderson JB, Kohn LM (2002) Evolution of drug resistance in Download full-text
Candida albicans. Annu Rev Microbiol 56: 139–165.
15. Cowen LE, Steinbach WJ (2008) Stress, drugs, and evolution: the role of cellular
signaling in fungal drug resistance. Eukaryot Cell 7: 747–764.
16. Lupetti A, Danesi R, Campa M, Del Tacca M, Kelly S (2002) Molecular basis of
resistance to azole antifungals. Trends Mol Med 8: 76–81.
17. Marichal P, Koymans L, Willemsens S, Bellens D, Verhasselt P, et al. (1999)
Contribution of mutations in the cytochrome P450 14alpha-demethylase
(Erg11p, Cyp51p) to azole resistance in Candida albicans. Microbiology 145:
18. Sanglard D, Ischer F, Calabrese D, Micheli M, Bille J (1998) Multiple resistance
mechanisms to azole antifungals in yeast clinical isolates. Drug Resist Updat 1:
19. Selmecki A, Forche A, Berman J (2006) Aneuploidy and isochromosome
formation in drug-resistant Candida albicans. Science 313: 367–370.
20. Selmecki A, Gerami-Nejad M, Paulson C, Forche A, Berman J (2008) An
isochromosome confers drug resistance in vivo by amplification of two genes,
ERG11 and TAC1. Mol Microbiol 68: 624–641.
21. Cowen LE, Lindquist S (2005) Hsp90 potentiates the rapid evolution of new
traits: drug resistance in diverse fungi. Science 309: 2185–2189.
22. Cowen LE, Carpenter AE, Matangkasombut O, Fink GR, Lindquist S (2006)
Genetic architecture of Hsp90-dependent drug resistance. Eukaryot Cell 5:
23. Armengou A, Porcar C, Mascaro J, Garcia-Bragado F (1996) Possible
development of resistance to fluconazole during suppressive therapy for AIDS-
associated cryptococcal meningitis. Clin Infect Dis 23: 1337–1338.
24. Berg J, Clancy CJ, Nguyen MH (1998) The hidden danger of primary
fluconazole prophylaxis for patients with AIDS. Clin Infect Dis 26: 186–187.
25. Birley HD, Johnson EM, McDonald P, Parry C, Carey PB, et al. (1995) Azole
drug resistance as a cause of clinical relapse in AIDS patients with cryptococcal
meningitis. Int J STD AIDS 6: 353–355.
26. Paugam A, Dupouy-Camet J, Blanche P, Gangneux JP, Tourte-Schaefer C,
et al. (1994) Increased fluconazole resistance of Cryptococcus neoformans isolated
from a patient with AIDS and recurrent meningitis. Clin Infect Dis 19: 975–976.
27. Venkateswarlu K, Taylor M, Manning NJ, Rinaldi MG, Kelly SL (1997)
Fluconazole tolerance in clinical isolates of Cryptococcus neoformans. Antimicrob
Agents Chemother 41: 748–751.
28. Mondon P, Petter R, Amalfitano G, Luzzati R, Concia E, et al. (1999)
Heteroresistance to fluconazole and voriconazole in Cryptococcus neoformans.
Antimicrob Agents Chemother 43: 1856–1861.
29. Sionov E, Chang YC, Garraffo HM, Kwon-Chung KJ (2009) Heteroresistance
to fluconazole in Cryptococcus neoformans is intrinsic and associated with virulence.
Antimicrob Agents Chemother 53: 2804–2815.
30. Posteraro B, Sanguinetti M, Sanglard D, La Sorda M, Boccia S, et al. (2003)
Identification and characterization of a Cryptococcus neoformans ATP binding
cassette (ABC) transporter-encoding gene, CnAFR1, involved in the resistance to
fluconazole. Mol Microbiol 47: 357–371.
31. Polakova S, Blume C, Zarate JA, Mentel M, Jorck-Ramberg D, et al. (2009)
Formation of new chromosomes as a virulence mechanism in yeast Candida
glabrata. Proc Natl Acad Sci U S A 106: 2688–2693.
32. Rustchenko E, Sherman F (2002) Genetic instability of Candida albicans. In:
Howard DH, ed. Fungi Pathogenic for Humans and Animals. New York:
Marcel Dekker, Inc. pp 723–776.
33. Hartwell LH, Dutcher SK, Wood JS, Garvik B (1982) The fidelity of mitotic
chromosome reproduction in S. cerevisiae. Rec Adv Yeast Mol Biol 1: 28–38.
34. Rosenstraus MJ, Chasin LA (1978) Separation of linked markers in Chinese
hamster cell hybrids: mitotic recombination is not involved. Genetics 90:
35. Torres EM, Sokolsky T, Tucker CM, Chan LY, Boselli M, et al. (2007) Effects of
aneuploidy on cellular physiology and cell division in haploid yeast. Science 317:
36. Zolan ME (1995) Chromosome-length polymorphism in fungi. Microbiol Rev
37. Varma A, Kwon-Chung KJ (1994) Formation of a minichromosome in
Cryptococcus neoformans as a result of electroporative transformation. Curr Genet
38. Kaern M, Elston TC, Blake WJ, Collins JJ (2005) Stochasticity in gene
expression: from theories to phenotypes. Nat Rev Genet 6: 451–464.
39. Stern C (1958) The nucleus and somatic cell variation. J Cell Physiol Suppl 52:
1–27; discussion 27-34.
40. Kafer E (1976) Mitotic crossing over and nondisjunction in translocation
heterozygotes of Aspergillus. Genetics 82: 605–627.
41. Fernandez C, Lobo Md Mdel V, Gomez-Coronado D, Lasuncion MA (2004)
Cholesterol is essential for mitosis progression and its deficiency induces
polyploid cell formation. Exp Cell Res 300: 109–120.
42. Yamaguchi M, Biswas SK, Ohkusu M, Takeo K (2009) Dynamics of the spindle
pole body of the pathogenic yeast Cryptococcus neoformans examined by freeze-
substitution electron microscopy. FEMS Microbiol Lett 296: 257–265.
43. Ganem NJ, Godinho SA, Pellman D (2009) A mechanism linking extra
centrosomes to chromosomal instability. Nature 460: 278–282.
44. Wapinski I, Pfeffer A, Friedman N, Regev A (2007) Natural history and
evolutionary principles of gene duplication in fungi. Nature 449: 54–61.
45. Rancati G, Pavelka N, Fleharty B, Noll A, Trimble R, et al. (2008) Aneuploidy
underlies rapid adaptive evolution of yeast cells deprived of a conserved
cytokinesis motor. Cell 135: 879–893.
46. Torelli R, Posteraro B, Ferrari S, La Sorda M, Fadda G, et al. (2008) The ATP-
binding cassette transporter-encoding gene CgSNQ2 is contributing to the
CgPDR1-dependent azole resistance of Candida glabrata. Mol Microbiol 68:
47. Bicanic T, Harrison T, Niepieklo A, Dyakopu N, Meintjes G (2006)
Symptomatic relapse of HIV-associated cryptococcal meningitis after initial
fluconazole monotherapy: the role of fluconazole resistance and immune
reconstitution. Clin Infect Dis 43: 1069–1073.
48. Friese G, Discher T, Fussle R, Schmalreck A, Lohmeyer J (2001) Development
of azole resistance during fluconazole maintenance therapy for AIDS-associated
cryptococcal disease. AIDS 15: 2344–2345.
49. Yamazumi T, Pfaller MA, Messer SA, Houston AK, Boyken L, et al. (2003)
Characterization of heteroresistance to fluconazole among clinical isolates of
Cryptococcus neoformans. J Clin Microbiol 41: 267–272.
50. Coste A, Selmecki A, Forche A, Diogo D, Bougnoux ME, et al. (2007)
Genotypic evolution of azole resistance mechanisms in sequential Candida albicans
isolates. Eukaryot Cell 6: 1889–1904.
51. Lee H, Bien CM, Hughes AL, Espenshade PJ, Kwon-Chung KJ, et al. (2007)
Cobalt chloride, a hypoxia-mimicking agent, targets sterol synthesis in the
pathogenic fungus Cryptococcus neoformans. Mol Microbiol 65: 1018–1033.
52. Chang YC, Kwon-Chung KJ (1994) Complementation of a capsule-deficient
mutation of Cryptococcus neoformans restores its virulence. Mol Cell Biol 14:
53. Kavanaugh LA, Fraser JA, Dietrich FS (2006) Recent evolution of the human
pathogen Cryptococcus neoformans by intervarietal transfer of a 14-gene fragment.
Mol Biol Evol 23: 1879–1890.
Disomy Formation in Azole Resistant C. neoformans
PLoS Pathogens | www.plospathogens.org13 April 2010 | Volume 6 | Issue 4 | e1000848