Microevolution of Cryptococcus neoformans Driven by
Massive Tandem Gene Amplification
Eve W.L. Chow,1 Carl A. Morrow, 1 Julianne T. Djordjevic, 2 Ian A. Wood, 3 and James A. Fraser* ,1
1
Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland,
Brisbane, Queensland, Australia
2
Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, University of Sydney at Westmead Hospital,
New South Wales, Australia
3
School of Mathematics and Physics, University of Queensland, Brisbane, Queensland, Australia
*Corresponding author: E-mail: [email protected].
Associate editor: James McInerney
The subtelomeric regions of organisms ranging from protists to fungi undergo a much higher rate of rearrangement than is
observed in the rest of the genome. While characterizing these ;40-kb regions of the human fungal pathogen
Cryptococcus neoformans, we have identified a recent gene amplification event near the right telomere of chromosome
3 that involves a gene encoding an arsenite efflux transporter (ARR3). The 3,177-bp amplicon exists in a tandem array of
2–15 copies and is present exclusively in strains with the C. neoformans var. grubii subclade VNI A5 MLST profile. Strains
bearing the amplification display dramatically enhanced resistance to arsenite that correlates with the copy number of the
repeat; the origin of increased resistance was verified as transport-related by functional complementation of an arsenite
transporter mutant of Saccharomyces cerevisiae. Subsequent experimental evolution in the presence of increasing
concentrations of arsenite yielded highly resistant strains with the ARR3 amplicon further amplified to over 50 copies,
accounting for up to ;1% of the whole genome and making the copy number of this repeat as high as that seen for the
ribosomal DNA. The example described here therefore represents a rare evolutionary intermediate—an array that is
currently in a state of dynamic flux, in dramatic contrast to relatively common, static relics of past tandem duplications
that are unable to further amplify due to nucleotide divergence. Beyond identifying and engineering fungal isolates that are
highly resistant to arsenite and describing the first reported instance of microevolution via massive gene amplification in
C. neoformans, these results suggest that adaptation through gene amplification may be an important mechanism that
C. neoformans employs in response to environmental stresses, perhaps including those encountered during infection. More
importantly, the ARR3 array will serve as an ideal model for further molecular genetic analyses of how tandem gene
duplications arise and expand.
Key words: Cryptococcus neoformans, subtelomere, amplification.
Introduction
The haploid basidiomycete yeast Cryptococcus neoformans
is an opportunistic pathogen that causes high morbidity and
mortality among the immunocompromised population,
particularly in the HIV-infected community (Park et al.
2009). Generally acquired from the environment, infection
of a host is accidental and is not required for completion
of the C. neoformans lifecycle (Casadevall 1998). A number
of key virulence traits that enable infection have been extensively characterized, including the antiphagocytic polysaccharide capsule, production of the pigment melanin,
and the ability to grow at mammalian body temperature
(Kwon-Chung 1986). Unlike bacterial virulence factors,
which are tailored specifically for pathogenesis, fungal virulence factors are considered adaptations to environmental
or predatory stresses and are likely co-opted for evasion
of the immune system after evolving under the selective pressure of the environmental niche (Steenbergen et al. 2001).
Previous studies have shown that extensive karyotypic
differences exist between C. neoformans strains collected
not only from the natural environment versus clinical
isolates but also among isolates obtained from the same
patient at different time points during infection (Fries
et al. 1996; Boekhout and van Belkum 1997). Variant karyotypes often predominate during later stages of infection
and potentially confer a selective advantage in the human
host. In support of this theory, emergence of a second karyotype during therapy has been observed coincident with
increased virulence and drug resistance (Fries et al. 1996;
Blasi et al. 2001; Jain et al. 2006). Analysis of initial and relapse
clinical isolates has shown that the vast majority of clinical
recurrences are a result of persistence of the original infection rather than reinfection with a new strain (Fries et al.
1996; Desnos-Ollivier et al. 2010).
Modern population analyses of C. neoformans employing
multilocus sequence typing (MLST) and amplified fragment
length polymorphism have demarcated the species into four
haploid molecular types: C. neoformans var. grubii (VNI, VNII,
and VNB) cause ;95% of infections and C. neoformans var.
neoformans (VNIV) approximately 5% (Xu et al. 2000). Studies have shown that while the genomes of C. neoformans var.
grubii strain H99 (VNI) and C. neoformans var. neoformans
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Mol. Biol. Evol. 29(8):1987–2000. 2012 doi:10.1093/molbev/mss066 Advance Access publication February 14, 2012
1987
Research article
Abstract
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Chow et al. · doi:10.1093/molbev/mss066
strain JEC21 (VNIV) are highly syntenic overall, a number of
translocations, inversions, and duplications are present
(Fraser, Huang, et al. 2005; Kavanaugh et al. 2006; Sun
and Xu 2009). Far more examples of karyotypic change
have been characterized in Saccharomyces cerevisiae, where
they typically correspond with the occurrence of translocations, deletions, insertions, duplications, or changes in
copy number of repetitive DNA sequences (Adams et al.
1992; Ibeas and Jimenez 1996; Fischer et al. 2000). These
are most commonly found near the chromosome ends,
the transitional regions between chromosome-specific sequences and telomeric repeat units (Bosco and Haber 1998;
Kuo et al. 2006; Rehmeyer et al. 2006; Fan et al. 2008). Subtelomeres are generally rich in arrays of nonfunctional repetitive sequences, including degenerate telomeric repeats,
microsatellites, transposable elements, and members of
nonessential gene families (De Bruin et al. 1994; Louis
1995; Flint et al. 1997). Since they undergo high rates of
rearrangement and as one telomere can functionally replace another, the subtelomeres are ideal locations for
gene amplification events through aberrant DNA repair
(Louis 1995; Brown et al. 1998). In Neurospora crassa
and S. cerevisiae, amplified gene arrays are found in multiple
subtelomeres (Brown et al. 1998; Bergthorsson et al. 2007;
Wu et al. 2009; Brown et al. 2010). Over a longer timeframe,
this new genetic material can be used in the generation of
novel genes through accumulation of mutations and domain accretion.
Based on the frequent development of karyotypic variants that occur during cryptococcal infection and the evidence of subtelomeric fluidity in other organisms, we
hypothesized that these genomic regions may be subject
to a higher rate of karyotypic change. Here, we provide evidence that in contrast to the rest of the genome, the gene
content of the subtelomeres of Cryptococcus differs considerably between strains. In addition, we have identified and
characterized a specific example of this change as it is
underway: a gene amplification event that has occurred
in the right subtelomere of chromosome 3 in C. neoformans
var. grubii and is now present in all isolates of a defined subclade with worldwide distribution. Through the amplification of an arsenite efflux transporter gene (ARR3), this
event confers resistance to high concentrations of arsenite
in C. neoformans, producing some of the most arsenic resistant fungi ever reported. This finding is a proof of concept
that adaptation through gene amplification occurs in the
subtelomeric regions and supports the potential use of this
mechanism to promote adaptation during human infection.
Materials and Methods
Bioinformatic Analyses
Analysis of the Cryptococcus subtelomeric regions utilized the
whole-genome shotgun sequences of C. neoformans var. grubii VNI strain H99, Cryptococcus gattii VGII strain R265 and
C. gattii VGI strain WM276 genome downloaded from the
Broad Institute of Harvard and Massachusetts Institute of
Technology (http://www.broadinstitute.org/) (D’Souza et al.
1988
2011), the C. neoformans var. neoformans VNIV strain
JEC21 downloaded from TIGR (http://www.tigr.org/tdb/
e2k1/cna1/), and VNIV strain B3501A downloaded from Stanford Genome Technology Centre (http://www-sequence.
stanford.edu/group/C.neoformans/index.thml) (Loftus et al.
2005). Basic local alignment search tool (BLAST) searches
were used to annotate 40-kb regions from each of the 28
chromosome ends, with the homologs identified in closely
related species (Altschul et al. 1990). Sequence alignments
were generated using ClustalW (Thompson et al. 1994).
Sequence traces for MLST analyses were examined using
Sequencher 4.7 (Gene Codes Corporation, Ann Arbor,
MI) and aligned using ClustalW v1.4 against C. neoformans
MLST genotype sequence data (www.mlst.net) (Litvintseva
et al. 2006).
Statistical Analyses
A variation on the procedure described by Brown et al.
(2010) was used to look for changes in gene density in
the chromosome arms, which might indicate the boundary
of the subtelomeric region. The gene positions in a window
of size 20 kb were compared against a true uniform distribution, and the window was moved in sequential steps of
1 kb from the telomere. A Kolmogorov–Smornov (KS)
P value was calculated for each window. A second method
was also employed to look for a change point in gene density
using the Pruned Exact Linear Time (PELT) algorithm (Killick
et al. 2011) implemented in the R package change point (Killick and Eckley Forthcoming). Intergenic distances were defined as the distances between the centers of consecutive
genes in base pairs. The natural logarithm of the intergenic
distance values was approximately normally distributed. The
PELT algorithm was then applied to the log values to look for
change points in the mean of a normal distribution. For both
methods, three sequenced genomes of Cryptococcus, (H99,
JEC21, and WM276) were pooled to increase sample size. To
determine whether the subtelomeres show gene enrichment, a hypothetical subtelomeric cutoff was utilized by
starting at 5 kb and increasing in increments of 5 kb. The
numbers of hexose transporter genes within and outside
the cutoff were compared against the numbers of all other
genes using Fisher’s exact test. As the previous two analyses
aimed to identify a difference in gene density, the P values
were calculated for a range of possible cutoff values, and
a graph was plotted of the Fisher’s exact test P value against
distance of cutoff from the telomere. In all three analyses, the
final cutoff value used was 110 kb, as the shortest chromosome arm is 111 kb in length.
Fungal Strains and Growth Conditions
Fungal strains used in this study are listed in table 1. Strains
were grown on YPD medium (1% yeast extract, 2% peptone,
and 2% glucose) or on minimal YNB (Becton Dickinson,
Franklin Lakes, NJ) medium (2% glucose as a carbon source)
and supplemented to meet auxotroph requirements. Metalloid sensitivity assays were performed using a method
modified from Wysocki et al. (2001). Cultures were grown
overnight in 100 ml YPD liquid media at 20 °C with
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Massive Gene Amplification in C. neoformans · doi:10.1093/molbev/mss066
Table 1. Fungal Strains Used in the Study.
Strains
Cryptococcus
H99
CM8 (H99 arr3D)
CM48 (H99 arr3D CnARR3)
M8A, M8B
M8A-11
M8A-13
JEC21
R265
F0, F2
B0, B1, B2, B4
M7A, M7B, M7C, M7F
J35a, J35b
G0, G1, G2
I51, I52
I55, I56, I60
I61, I63
I66, I73
I68, I74
I82, I83
I91, I95, I96
I107, I110, I111, I112
A5 35-17
C48
C8
A2 102-5
Ug2463
It754
Tn470
Bt104
123.91
124.91
125.91
A7
A7 35-23
C2
C12
C16
C44
C45
8–1
Ug2472
I57, I59 d-27
Bt33
Bt34
Bt63
Saccharomyces cerevisiae
BY4739
BY4739 arr3D
EC2 (BY4739 arr3D ScARR3)
EC3 (BY4739 arr3D CnARR3)
Genotype/Description
Source
MATa VNI
MATa arr3::NEO
MATa arr3::NEO ARR3:NAT
MATa VNI
Experimentally evolved M8A
Experimentally evolved M8A
MATa VNIV
MATa VGII
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNI
MATa VNII
MATa VNII
MATa VNII
MATa VNII
MATa VNII
MATa VNII
MATa VNII
MATa VNII
MATa VNII
MATa VNII
MATa VNB
MATa VNB
MATa VNB
Perfect Lab
This study
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Fries B. (unpublished data)
This study
This study
Kwon-Chung et al. (1992)
Fraser, Giles, et al. (2005)
Fries B. (unpublished data)
Fries B. (unpublished data)
Fries B. (unpublished data)
Fries B. (unpublished data)
Fries B. (unpublished data)
Jain et al. (2005)
Jain et al. (2005)
Jain et al. (2005)
Jain et al. (2005)
Jain et al. (2005)
Jain et al. (2005)
Jain et al. (2005)
Jain et al. (2005)
Litvintseva et al. (2005)
Litvintseva et al. (2005)
Litvintseva et al. (2005)
Litvintseva et al. (2005)
Litvintseva et al. (2005)
Litvintseva et al. (2005)
Litvintseva et al. (2005)
Litvintseva et al. (2005)
Lengeler et al. (2000)
Lengeler et al. (2000)
Lengeler et al. (2000)
Litvintseva et al. (2005)
Litvintseva et al. (2006)
Litvintseva et al. (2006)
Litvintseva et al. (2006)
Litvintseva et al. (2006)
Litvintseva et al. (2005)
Litvintseva et al. (2005)
Nielsen et al. (2003)
Litvintseva et al. (2006)
Jain et al. (2005)
Litvintseva et al. (2006)
Litvintseva et al. (2003)
Litvintseva et al. (2006)
MATa
MATa
MATa
MATa
Heitman Lab
Heitman Lab
This study
This study
leu2D0 lys2D0 ura3D0
leu2D0 lys2D0 ura3D0 arr3::KanMX
leu2D0 lys2D0 ura3D0 arr3::KanMX CnARR3:URA3
leu2D0 lys2D0 ura3D0 arr3::KanMX ScARR3:URA3
constant agitation. To determine the maximum noninhibitory concentration of arsenic salts, 3 ll of each dilution
series were spotted onto solid YPD media with increasing
concentrations of NaAsO2 (Sigma-Aldrich, St Louis, MO).
Plates were incubated for 3 days at 30 °C. Experimental
evolution assays were performed by sequentially plating
strains on media containing increasing concentrations of
arsenite. Strain M8A (;14 copies of ARR3 amplicon)
was initially plated on media containing 10 mM arsenite.
Colonies that arose after 3 days of incubation at 30 °C were
subcultured onto media containing 15 mM arsenite and
further incubated. This process was repeated in steps of
5 mM arsenite until 35 mM arsenite where no further
growth was observed.
Primers and Sequencing
Primers used in this study are listed in supplementary table
S1 (Supplementary Material online). Primers were designed
using Oligo 6.8 (Molecular Biology Insights, West Casade,
CO) and synthesized by Life Technologies (Carlsbad, CA).
Polymerase chain reaction (PCR) products were purified using a QIAquick gel extraction kit (Qiagen GmbH, Hilden, DE)
1989
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Chow et al. · doi:10.1093/molbev/mss066
Table 2. Plasmids Used in This Study.
Plasmid
pCR2.1 TOPO
YEplac195
pEWC5
pEWC17
pEWC18
pEWC19
pEWC20
pEWC21
pEWC22
Description
Escherichia coli cloning vector, Ampicillin-resistant
Saccharomyces cerevisiae–E. coli shuttle vector, Ampicillin-resistant, URA3
7.46-kb PCR fragment containing Cryptococcus ARR3 cloned in pCR2.1 TOPO
3.1-kb PCR fragment containing Cryptococcus ARR3 CDS cloned in pCR2.1 TOPO
2.16-kb PCR fragment containing Saccharomyces ARR3, ARR2, and a fragment of ARR1 cloned in pCR2.1 TOPO
- kb SacI fragment containing Cryptococcus ARR3 ligated into SacI-digested phosphatased pPZP-NATcc vector
2.27-kb PCR fragment containing Cryptococcus ARR3 CDS flanked by a fragment of Saccharomyces
ARR2 and ARR1 cloned in pCR2.1 TOPO
2.37-kb XbaI–SacI fragment from pEWC20 ligated into the XbaI–SacI cut site of YEplac195
2.26-kb XbaI–SacI fragment from pEWC18 ligated into the XbaI–SacI cut site of YEplac195
and sequenced at the Australian Genome Research Facility
(Brisbane, AU). To identify strains bearing the ARR3 gene
amplification, a diagnostic PCR test was developed using
primers UQ426 and UQ427. Primers UQ436 and UQ437
were subsequently used to amplify the entire amplicon region for sequencing. Amplification of 12 MLST loci (IGS1,
CAP59, CAP10, MP88, GPD1, TOP1, SOD1, URE1, PLB1,
MPD1, TEF1, and LAC1) was performed using standard oligonucleotides (Litvintseva et al. 2006), except for MP88 and
TOP1, which were redesigned to avoid the need for step
down PCR. PCR products were ethanol precipitated and
sequenced.
Molecular Techniques
Cryptococcus neoformans genomic DNA was prepared using the CTAB method (Pitkin et al. 1996). Total RNA was
prepared from C. neoformans strains grown in 100 ml YPD
liquid media for 16 h at 30 °C with constant agitation.
cDNA was generated using the BioLine cDNA Synthesis
Kit (BioLine, London UK) according to the manufacturers’
protocols. Southern hybridizations were performed using
Modified Church buffer (Church and Gilbert 1984). Probes
were produced by PCR amplification from H99 genomic
DNA using primers given in supplementary table S1 (Supplementary Material online) and purified using the QIAquick
gel extraction kit (Qiagen, Valencia, CA). Radiolabeled
probes were prepared using the GE Healthcare Rediprime
II Random Prime Labelling System (GE Healthcare) with
20 lCi a-32P dCTP (Perkin Elmer, Waltham, MA). Hybridizations were performed overnight at 65 °C. Probes were
detected by exposing the blots to Fujifilm Super RX medical
X-ray film (Fujifilm, Tokyo, JA) at 80 °C and developed
using a Konica Minolta SRX-101A Film Processor.
Pulsed-Field Gel Electrophoresis
Intact C. neoformans chromosomal DNA in agarose plugs
was prepared according to the method of Lengeler et al.
(2000). Pulsed-field gel electrophoresis (PFGE) plugs were
digested with restriction enzymes and electrophoresed
in 1% pulsed-field certified agarose (Bio-Rad) using 0.5
Tris-borate-EDTA running buffer using the CHEF DRIII
PFGE system (Bio-Rad, Richmond, CA) as described (Lengeler et al. 2000) with minor modifications. Running conditions are as follows: ramped switch time from 1.5 to 10 s,
120°, 6 V/cm, 24 h, performed at 14 °C. Gels were stained
with ethidium bromide and visualized with a UV transillu1990
Source
Invitrogen
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minator. Southern blotting of pulsed-field gels was performed as previously described (Marra et al. 2004) onto
Hybond-XL nylon membranes (GE Healthcare, Chalfont
St Giles, UK). Blots were UV crosslinked with 100 mJ UV
using a Stratagene UV Stratlinker 2400.
Deletion and Complementation of the ARR3 Gene
The ARR3 (CNAG_06025.2) deletion in strain H99 was created using overlap PCR and biolistic transformation as described (Davidson et al. 2000) using the selectable NEO
marker (Fraser et al. 2003). Recombinants were selected
on media containing G418 (100 lg/ml) and absence of
growth on media containing 1 mM sodium arsenite. Deletion of the gene was confirmed by Southern hybridization.
Plasmids used in this study are listed table 2. To create
the deletion construct and complementation plasmids, primers UQ528 and UQ531 were used to amplify a 7.46-kb
fragment containing the ARR3 gene along with ;1-kb
flanking regions, using H99 genomic DNA as a template.
The PCR product was cloned into pCR2.1 TOPO vector
(Life Technologies) to create pEWC5 and transformed into
Escherichia coli MACH1 cells. The ARR3 construct was then
subcloned onto pPZP-NAT to create pEWC19 (Idnurm
et al. 2004). Complementation of the H99 arr3n strain
was performed using biolistic transformation and selected
on media. Complementation of the ARR3 gene was verified
by Southern hybridization.
To verify the functional role of the ARR3 gene in
C. neoformans, cross-species complementation was performed using the S. cerevisiae arr3n strain from the yeast
deletion library (Winzeler et al. 1999; Giaever et al. 2002).
To create the cross-species complementation plasmid, the
protein-coding open reading frame (ORF) of the C. neoformans var. grubii ARR3 gene (CnARR3) was amplified from
YPD-grown H99 cDNA using primers UQ476 and UQ477
and cloned into pCR2.1 TOPO, yielding the plasmid
pEWC17. The S. cerevisiae ARR3 gene (ScARR3) and flanking
regions were amplified from S. cerevisiae strain BY4739 using
primers UQ809 and UQ812 and cloned into pCR2.1 TOPO
to create pEWC18. The CnARR3 ORF and the ScARR3 promoter and terminator were combined by overlap PCR and
cloned into pCR2.1 TOPO to create pEWC20 and subcloned
into YEplac195 to create pEWC21. This vector was transformed into the S. cerevisiae arr3n strain using the lithium
acetate method (Ghosh et al. 1999). The S. cerevisiae arr3n
strain was also complemented with the native ScARR3 gene
Massive Gene Amplification in C. neoformans · doi:10.1093/molbev/mss066
under the control of its endogenous promoter and terminator by subcloning the gene from pEWC18 into YEplac195 to
create pEWC22 and transforming into the S. cerevisiae
arr3n. Recombinants from both transformations were isolated on appropriate selective media, and the complementation of the CnARR3 and ScARR3 genes were verified by
growth on media containing 1 mM arsenite.
Antifungal Susceptibility Testing
The antifungals amphotericin B, flucytosine, fluconazole,
and intraconazole were obtained from Sigma-Aldrich. Stock
solutions were prepared in dimethyl sulfoxide (amphotericin
B and itraconazole) or water (flucytosine and fluconazole).
Broth microdilutions were performed in accordance with
the CLSI M27-A2 guidelines (Clinical and Laboratory Standards Institute 2002) using YNB medium supplemented
with ammonium sulfate, a final inoculum concentration
of (1.5 ± 1.0) 103 cells/ml and incubation at 35 °C for
72 h (Ghannoum et al. 1992). Concentrations of the antifungal agents were 0.097–50 lg/ml for fluconazole and flucytosine and 0.0156–8 lg/ml for amphotericin B and
itraconazole. Minimal inhibitory concentration (MIC)50 is
defined as the concentration that produced .50% growth
inhibition and MIC90 is defined as the concentration that
produced .90% growth inhibition. Assays were performed
in triplicate. Quality control was performed by testing the
CLSI-recommended strains ATCC6258 (Candida krusei),
ATCC90028 (Candida albicans), and ATCC22019 (Candida
parapsilosis).
Murine Virulence Assays and Organ Burden
Pathogenicity studies were conducted in 6- to 7-week-old
BALB/c mice (Animal Resource Centre, Canning Vale, Australia). Infection was performed under methoxyflurane
anesthesia. Groups of 11 mice were inoculated intranasally
with 1 105 yeast cells in 50 ll phosphate-buffered saline.
All mice were weighed and monitored daily for physical
signs of sickness or distress and humanely euthanized by
CO2 asphyxiation when their body weight had reduced
by 20% from their preinfection weight or upon signs of
debilitating effects. Kaplan–Meier survival curves were
plotted using GraphPad Prism 5.0 and significance was determined using a log-rank test. Values of P , 0.05 were
considered statistically significant.
Murine virulence assays were carried out in strict accordance with the recommendations in the Australian Code of
Practice for the Care and Use of Animals for Scientific Purposes by the National Health and Medical Research Council.
The protocol was approved by the Molecular Biosciences
Animal Ethics Committee of The University of Queensland
(AEC approval number: SCMB/473/09/UQ/NHMRC).
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lomeric regions are gene poor compared to the rest of the
genome (Brown et al. 2010). We performed a statistical
analysis of gene density by comparing the number of genes
within a window with that of a true uniform gene distribution. Comparison across all chromosome arms of
C. neoformans var. grubii strain H99 (VNI), C. neoformans
var. neoformans strain JEC21 (VNIV), and C. gattii strain
WM276 (VGI) revealed no changes in gene density sufficient to define a subtelomeric boundary cutoff (all KS P values .0.35). We also analyzed intergenic distances between
genes as a measure of gene density, moving progressively
along the pooled set of chromosome arms toward the centromere to look for a change point in gene density. This
analysis also did not show a significantly lower gene density
in the subtelomeres compared with the rest of the genomes (results not shown). This suggests that unlike
S. cerevisiae, the subtelomeres of Cryptococcus possess
a gene density similar to the rest of the genome and that
with the exception of the centromeres, gene density is
uniform throughout the Cryptococcus genome.
As gene density did not reveal any unusual characteristics of the chromosome ends, we decided to investigate the
genomic composition of this part of the genome to see if
they are conserved between var. grubii strain H99 and var.
neoformans strain JEC21. Synteny analysis employing dot
plot matrices revealed that while the interstitial chromosomal regions of JEC21 appeared syntenic with H99, the
final 2- to 36-kb regions of each chromosome end were
commonly rearranged or included sequences that were
present only in one or the other strain.
To support this surrogate measure of the region we
would define as the subtelomere, we elected to investigate
the types of gene common to this part of the genome. In
other organisms, subtelomeres are enriched in genes involved in niche adaptation (Adams et al. 1992; De Bruin
et al. 1994; De Las Penas et al. 2003). Analysis of these regions in C. neoformans using BLAST revealed an unusual
number of hexose transporter genes. Whole-genome analysis revealed 34 genes predicted to encode hexose transporters, 12 of which were scattered within the regions
we were investigating. We therefore compared the number
of hexose transporter genes in the region near the telomeres with those in the rest of the genome to see if there
was any statistical evidence of difference in proportion.
Based on a range of possible subtelomeric lengths, we found
that the lowest P value was at the cutoff of 35 kb from the
chromosome end (Fisher’s exact test, P value 5 9.1 107),
indicating that hexose transporter encoding genes are enriched in subtelomeric regions (fig. 1 and supplementary
table S2, Supplementary Material online).
A Focus on the Right Subtelomere of Chromosome 3
Results
A Measure of Subtelomere Length in Cryptococcus
To help us define the regions we would consider subtelomeric in C. neoformans, we first employed an analytical
method based on the observation that S. cerevisiae subte-
Although our study of gene density had not revealed anything unique about the C. neoformans chromosome ends,
our studies of synteny and hexose transporter gene distribution indicated the final ;40 kb exhibit unique characteristics: a higher rate of rearrangement and an enrichment of
certain gene types. We therefore sought to expand this
1991
Chow et al. · doi:10.1093/molbev/mss066
FIG. 1. The chromosome ends of Cryptococcus neoformans are
enriched for hexose transporter genes. By comparing the proportion
of hexose transporter genes at the chromosome ends to total
number of genes in the whole genome, in a 5 kb window and
increasing that window size in steps of 5 kb toward the centromere,
up to 110 kb, the subtelomere in C. neoformans strain H99 is ;35 kb
(dotted line, P value 5 9.1 107). The Fisher’s exact test P value,
between the proportions of hexose transporter genes to those of the
other genes, is compared with the distance from the telomere.
characterization by focusing on one subtelomere across
a range of isolates. Our synteny analysis had shown that
the right subtelomere of chromosome 3 of H99 shared
the least synteny with the corresponding subtelomere in
JEC21, making this a good candidate for further characterization (fig. 2). To more closely scrutinize the gene content
of the chromosome 3 right subtelomere in C. neoformans
var. grubii H99, the final 40 kb of genome sequence from
the end of the chromosome was analyzed bioinformatically
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using BLAST and ClustalW and compared with the homologous telomere from strains JEC21 (VNIV), B3501A (VNIV)
and C. gattii strain R265 (VGII), and WM276 (VGI) (Loftus
et al. 2005; D’Souza et al. 2011). Although the subtelomeric
regions of the three molecular types analyzed were generally
syntenic, there were a number of unique sequences (fig. 2).
For instance, JEC21 possesses a putative polymerase II transcription elongation factor–encoding gene (CNK00050),
which is absent in the other strains, suggesting gene gain
via gene duplication in var. neoformans. An interesting finding made in all five genomes is that the region appears to be
rich in carbohydrate metabolism related genes; of the 20
genes in the 40-kb subtelomeric region analyzed in chromosome 3 of strain H99, 8 are predicted to play a role in carbohydrate metabolism (fig. 2), whereas the other strains
have between 4 and 10 of these genes.
The Chromosome 3 End of C. neoformans var. grubii
Shows Variation in Length among Isolates
A detailed Southern hybridization mapping of the chromosome 3 right subtelomere was performed in a collection of
C. neoformans var. grubii using a combination of three restriction enzymes and multiple gene-specific probes. Subtelomeric regions were liberated via in-gel restriction digest
using a SfiI site 64 kb from the H99 chromosome end and
resolved using PFGE; Southern blotting and subsequent hybridization with a gene-specific probe telomere proximal to
the SfiI cut site revealed a significant increase in distance to
the chromosomal end in 5 of 22 tested isolates compared
with laboratory reference strain H99 (fig. 3). Size differences
become less apparent when a more telomere proximal
ApaI restriction site (31 kb) was employed in the analysis,
FIG. 2. The right telomere of chromosome 3 of var. grubii differs markedly from homologous regions in the different molecular types. Schematic
diagram showing the genome content of the right telomere of chromosome 3 in Cryptococcus neoformans var. grubii strain H99 and the
corresponding regions in C. neoformans var. neoformans strains JEC21 (Chr 11) and B3501A (Chr 11) and C. gattii strains WM276 (Chr 11) and
R265 (Chr 11). The yellow arrows indicate genes predicted to be involved in carbohydrate metabolism.
1992
Massive Gene Amplification in C. neoformans · doi:10.1093/molbev/mss066
MBE
FIG. 3. The right telomere of chromosome 3 differs markedly between strains. (A) Schematic diagram showing the cut sites of the three
restriction enzymes SfiI, ApaI, and SpeI and the gene-specific probes employed in the Southern blot hybridization. (B) Exposure of the three
blots. The SfiI-digested blot was hybridized to the xylulose/fructose phosphoketolase–specific probe (PPK1); the ApaI-digested blot was
hybridized to the Ras GTPase activator–specific probe (IRA2); and the SpeI-digested blot was hybridized to the hexose transporter–specific
probe (HXT5). The presence of a band in each lane indicates the presence of the gene in each isolate. The size of the band in reference to the
band size of Cryptococcus neoformans var. grubii VNI laboratory reference strain H99 indicates the occurrence of subtelomeric length variation.
with variation between strains reduced even further when
a SpeI site only 18 kb from the end of the chromosome—in
this case, almost all strains yielded an equivalent banding
pattern. These results therefore suggest that the most significant variation in the right end of chromosome 3 occurs
not at the very end but between the SfiI (64 kb) and the
ApaI (31 kb) cut sites.
A Subtelomeric Gene Amplification Event on
Chromosome 3
In order to determine the nature of the observed interstrain variability of chromosome 3, we designed probes
specific to the nine genes in the SfiI/ApaI region and sequentially probed genomic digest Southern of these tested
strains. Although most probes gave equivalent hybridization intensity in all 22 strains, a probe targeting
CNAG_06925.2 displayed markedly stronger hybridization
intensity for strains M8A and G0, suggesting an increase
in copy number of the probe DNA in these genomes.
To test this hypothesis, a Southern blot of genomic DNA
from M8A as well as VNI reference strain H99 was performed
using a variety of restriction enzymes that cut in a 10-kb
region around the probe sequence (fig. 4). Rather than observing the fragment sizes observed in H99, each M8A lane
contained either a high molecular weight band of undetermined size or a common ;3.2-kb band when using restric-
tion enzymes in close proximity to CNAG_06925.2. These
results are consistent with strain M8A bearing tandemly
arrayed copies of the region encompassing CNAG_06925.2
(fig. 4).
Based on the predicted existence of a tandem repeat array, diagnostic CNAG_06925.2 primers were designed facing
away from each other, enabling an amplification product
only from strains that carry a tandem array of this gene
(fig. 5A). Subsequent PCR revealed that no product was
obtained from H99, whereas M8A yielded a product consistent with the presence of a tandem array (fig. 5B). The same
amplicon was also observed when DNA from the other
strongly hybridizing strain (G0) was used as the PCR template. Sequencing enabled the repeat boundary to be determined, revealing the repeat unit to be precisely 3,177 bp in
length, comprising the entire CNAG_06925.2 gene, predicted
to be a homolog of S. cerevisiae ARR3 gene, plus a short fragment of the adjacent gene (CNAG_06926.2), predicted to
encode a Major Facilitator Superfamily sugar transporter.
Isolates with the CNAG_06925.2 Gene Amplification
Share a Common Ancestry
During our survey of the right telomere of chromosome
3 in 22 isolates, we identified two strains bearing the
CNAG_06925.2 amplicon repeat. To determine if this amplification event is common in var. grubii, a collection of 70
1993
Chow et al. · doi:10.1093/molbev/mss066
FIG. 4. The ARR3 gene is part of a tandemly amplified array. (A)
Schematic diagram showing the cut sites of the nine restriction
enzymes EcoRI, SnaBI, PshAI, BglII, StuI, SacI, SphI, NdeI, and XhoI
and the location of the ARR3 gene-specific probe (gray box) used.
The strains cut in a ;10-kb region around the probe sequence. (B)
Exposure of the two blots. Genomic DNA of Cryptococcus
neoformans var. grubii VNI laboratory reference strain H99 and
clinical isolate M8A were digested.
strains comprising 12 environmental isolates and 58 clinical
isolates encompassing all known MLST subclades were
tested with our diagnostic PCR for the presence of the tandem array (supplementary fig. S1, Supplementary Material
online). Of those examined, seven isolates tested positive
for the presence of the amplification. Importantly, all seven
isolates shared identical MLST alleles for the 12 loci tested,
revealing they belonged to the previously described VNI
subclade A5 (ST5) (Litvintseva et al. 2006). The remaining
63 amplicon-negative strains all belonged to other subclades.
To determine the approximate amplicon copy number
in the positive isolates, whole chromosomal preparations of
these isolates were digested in-gel with XhoI, a restriction
enzyme that cuts outside the amplicon boundaries and
Southern blotted (fig. 5C). As the size of the ARR3 amplicon
unit is known, the number of tandem repeat units in the
isolates could be determined based on band size. Copy
number was found to vary from 3 to 19 copies, suggesting
that strains bearing the amplification may undergo expansion and contraction of the repeat (fig. 5C).
CNAG_06925.2 Encodes an Arsenite Efflux
Transporter Whose Copy Number Correlates with
Arsenical Resistance
BLASTx analysis suggested that the CNAG_06925.2 ORF
encodes an arsenite efflux transporter homologous to
S. cerevisiae Arr3p (Wysocki et al. 1997). To explore the
function of this gene in C. neoformans, the gene was deleted
by homologous recombination in the strain H99 background. Deletion of CNAG_06925.2 resulted in sensitivity
to arsenite; subsequent complementation with the wild-type
1994
MBE
gene restored arsenical resistance (fig. 6). These phenotypic
results are consistent with CNAG_06925.2 encoding an arsenite efflux transporter. To confirm this role, we introduced CNAG_06925.2 into the S. cerevisiae arr3n mutant,
which effectively restored arsenite resistance to the mutant
(fig. 6). Functional complementation thus confirmed the bioinformatically predicted function of the gene product as an
arsenite efflux transporter, and we have therefore named the
gene ARR3.
Retention of the ARR3 amplicon in all tested strains belonging to clade VNI A5 suggests that it may confer a selective advantage. To determine if increasing copy number of
ARR3 correlates with an increased level of arsenical resistance, serial dilution spotting assays were performed on
rich media supplemented with increasing concentrations
of sodium arsenite (0–20 mM) (fig. 5D). While the reference
strain H99 could not grow in the presence of 5 mM arsenite, isolates with 3–5 copies of the repeat unit were generally able to grow in the presence of 5 mM arsenite, isolates
with 9–11 copies grew in the presence of 10 mM arsenite and
isolates with 13–19 copies grew in the presence of 15 mM
arsenite. No strains were able to grow in the presence of
20 mM arsenite. Copy number of the ARR3 amplicon is thus
positively correlated with the level of arsenical resistance.
To determine if further amplification of the ARR3 repeat
was possible, we next performed an experimental evolution
assay in an attempt to increase amplicon copy number by
repeated selection of colonies displaying increased arsenite
resistance. The 14 copy-containing strain M8A, which could
grow in concentrations as high as 15 mM arsenite, was subcultured onto media containing increasing concentrations
of arsenite in a stepwise fashion. Spontaneous colonies with
high resistance were easily identified as the strain was exposed to progressively higher concentrations of arsenite.
Using this approach, we isolated derivatives of strain
M8A that could withstand up to 30 mM arsenite. Southern
blot analysis of the subcultured strains revealed a massive
amplification of the ARR3 amplicon in the experimentally
evolved strains (supplementary fig. S2, Supplementary Material online).
The Presence of ARR3 in High Copy Number Does
Not Contribute to the Success of Infection of an
Animal Host
It has been postulated that the virulence factors C. neoformans employs during host infection have been co-opted
from features that promote survival in the environmental
niche (Buchanan and Murphy 1998). To determine if ARR3
has been co-opted to play a role during infection, virulence
of the arr3n mutant was assessed using the murine inhalation model of cryptococcosis. One hundred percent of
mortality was reached in 21 days in BALB/c mice infected
with the arr3n strain compared with 23 days for control
strain H99 and 19 days for arr3DþARR3 the complemented strain (fig. 7A). The survival rate of mice infected
with the arr3n strain was not significantly different to the
survival rates of the mice infected with the wild-type or
Massive Gene Amplification in C. neoformans · doi:10.1093/molbev/mss066
MBE
complemented strains, suggesting that Arr3 does not play
a significant role during infection.
To ascertain if the amplification of ARR3 could contribute to virulence, two experimentally evolved strains M8A13 (30 copies of the amplicon) and M8A-11 (80 copies)
were compared with the original strain M8A in a second
virulence assay. Mice infected with the control strain M8A
succumbed within 21 days, whereas mice infected with the
evolved strains of M8A-13 and M8A-11 did not survive past
22 days and 24 days, respectively, and were not significantly
different (fig. 7B). Strains containing increased copy number of the ARR3 amplicon were thus unaffected for virulence (fig. 7A and B).
The lack of a detectable role in virulence for Arr3 does
not preclude this protein from having a function during
infection; as an efflux pump, Arr3 could potentially have
substrate specificity that extends beyond arsenite alone,
perhaps into the efflux of antifungal agents. Treatment of
cryptococcal meningoencephalitis traditionally involves the
antifungal agents amphotericin B, flucytosine, and azoles such
as fluconazole. To investigate if the arsenite efflux transporter
has been co-opted to play a role in multidrug resistance, we
tested the sensitivity of M8A and the experimentally evolved
strains (M8A-11 and M8A-13), and the H99 arr3n deletion
strain to the antifungal drugs fluconazole, itraconazole, amphotericin B, and 5-fluorocytosine using a MIC assay. The
MIC50 and MIC90 of the experimentally evolved strains did
not differ from the MICs of the wild-type strain (table 3). Similarly, the arr3D mutant displayed identical sensitivity to all
antifungals tested. This suggests that massive amplification of
the arsenite efflux transporter gene does not contribute to
antifungal resistance.
Discussion
FIG. 5. Presence of ARR3 amplification correlates with increased
arsenite resistance Cryptococcus neoformans var. grubii VNI laboratory
reference strain H99 was used as a negative control and a strain with
ARR3 gene amplification was used as a positive control. (A) Schematic
diagram showing the cut sites of restriction enzyme XhoI and the
location of the ARR3 amplicon–specific primers. The primers are designed such that a product (1.3 kb in size) is amplified if the amplicon
units were tandemly arrayed. The amplicon unit (highlighted by the
gray box) is 3,177 bp in size and consists of the promoter and terminal
regions of the ARR3 gene and a fragment of the adjacent predicted
MFS sugar phosphate permease gene (SEO1). (B) Strains M8A, G0, and
NIH 38 showed a 1.3 kb amplified product, indicating that the strains
contained that ARR3 amplification. (C) Amplicon copy number was
determined based on the size of the Xho1 cleavage band. The number
of tandem repeats in the isolates was found to be unstable; copy
number varied from 3 to 19 copies. (D) Arsenical resistance spot assay
results showed a general correlation between copy number and arsenical resistance. Strains were grown on YPD media.
In S. cerevisiae and Homo sapiens, subtelomeres are regions
near the telomeres that are repeat rich and gene poor
(Pryde et al. 1997). These regions have been extensively
studied in S. cerevisiae in particular, where despite being
gene depleted, there is an enrichment of genes with known
functions in transport and fermentation (Winzeler et al.
2003; Brown et al. 2010). A recent study estimated that
these regions are approximately 33 kb in length in the Saccharomycetales (Brown et al. 2010); we sought to determine the subtelomeric length in C. neoformans using the
same gene density-based approach. We found that unlike
S. cerevisiae, the subtelomeres in Cryptococcus appear to
possess a gene density that is equivalent to the rest of
the genome. The subtelomeres are therefore yet another
genomic feature of C. neoformans that differ in their fundamental structure compared with S. cerevisiae. For instance,
the centromeres of S. cerevisiae all possess a defined consensus sequence, whereas the centromeres of C. neoformans are
large transposon-dense structures (Hegemann and Fleig
1993; Fleig et al. 1995; Loftus et al. 2005). In S. cerevisiae,
MAT is small and has a cassette-based switching system,
whereas in C. neoformans, MAT is large and does not change
(Fraser et al. 2003; Fraser, Giles, et al. 2005; Morrow and
1995
Chow et al. · doi:10.1093/molbev/mss066
FIG. 6. Arr3 function is conserved across multiple fungal phyla.
Deletion of the ARR3 resulted in arsenite sensitivity in the mutant
and complementation with the wild-type gene restored arsenite
resistance. Complementation of the Saccharomyces cerevisiae arr3n
mutant with the protein-coding ORF of the Cryptococcus ARR3
gene restored arsenite resistance strongly indicates that the
Cryptococcus ARR3 gene is an ortholog of the ARR3 gene in S.
cerevisiae. Serial dilution spotting assay of the strains were grown on
YPD media.
Fraser 2009). The subtelomeres now join this list as being
distinct from yeast, instead sharing more similarity to species
like Magnaporthe oryzae, where the gene density within the
subtelomeric regions is also similar to that in the rest of the
genome (Farman and Leong 1995; Mizuno et al. 2006; Farman
2007).
Our synteny analyses revealed that the ;40-kb regions
near the ends of the chromosomes of Cryptococcus nevertheless still possess unique characteristics, exhibiting a higher
rate of rearrangement and an enrichment of certain gene
types. These observations are consistent with the known
potential for rapid evolution of genes that reside in the subtelomeric regions that has been particularly well exploited
by a number of fungi and protists, many of which have ac-
MBE
cumulated families of genes involved in niche adaptation
(Adams et al. 1992; De Bruin et al. 1994; De Las Penas et al.
2003). Such genes tend to be nonessential but provide
the capability to respond to specific ecological challenges.
We found that the subtelomeric regions in C. neoformans
are enriched in hexose transporters in a similar fashion to
S. cerevisiae (Winzeler et al. 2003). We therefore defined the
subtelomeric regions in C. neoformans as ;40 kb, based on
our synteny analysis and the enrichment of hexose transporter genes in these regions. Studies by Xue et al. (2010)
previously described a myo-inositol transporter gene family, with 5 of 11 members lying within 30 kb of the chromosome end. Since C. neoformans can utilize inositol as
a sole carbon source and since inositol has been demonstrated to be involved in virulence in multiple fungal species, it is likely that this enrichment arose as an adaptive
mechanism in response to the environment (Chen et al.
2008; Bethea et al. 2010; Xue et al. 2010; Wang et al.
2011). We postulate that the enrichment of hexose transporter genes is likely to also play a role in virulence, as acquisition of carbon sources is of critical importance during
infection (Perfect 2005; Price et al. 2011).
To expand on our analysis of C. neoformans subtelomeres,
we focused on one subtelomere in particular, the right subtelomere of chromosome 3, as this region displayed the least
synteny between var. grubii and var. neoformans in our synteny analysis. This focus enabled us to identify the presence
of a tandemly arrayed amplification involving the ARR3
gene. Studies in a range of both eukaryotes and prokaryotes
suggest that tandem gene arrays originate from an initial
gene duplication event due to a double-stranded DNA break
(Romero and Palacios 1997; Haber and Debatisse 2006; Hastings 2007), representing a possible mechanism for the origin
of the ARR3 amplification. The initial duplication may have
arisen from a double-stranded break from the stalling or collapse of a replication fork followed by repair through breakinduced replication (Bosco and Haber 1998; Haber et al.
2004; Watanabe and Horiuchi 2005; McEachern and Haber
2006). In such a scenario, the duplication would be in direct
FIG. 7. The deletion or amplification of ARR3 does not affect virulence in a murine model. (A) Deletion of ARR3 gene does not affect virulence
in a mouse model (log-rank [Mantel–Cox] test P value 5 0.85). H99 median survival 5 16 days; arr3D median survival 5 17 days; and arr3D þ
ARR3 median survival 5 16 days. (B) Amplification of the ARR3 gene does not confer an obvious advantage during infection (log-rank [MantelCox] test P value 5 0.47). M8A (14 copies) median survival 5 19 days; M8A-11 (80 copies) median survival 5 19.5 days; and M8A-13 (30
copies) median survival 5 18 days.
1996
Massive Gene Amplification in C. neoformans · doi:10.1093/molbev/mss066
Table 3. Minimal Inhibitory Concentrations of Cryptococcus
neoformans Strains Tested.
MIC (mg/ml) reading at 72 h
AmB
Strain
name
MIC90 MIC50
H99
1
0.25
narr3
1
0.25
narr3 1
CnARR3
1
0.25
M8A
1
0.25
M8A-11
1
0.25
M8A-13
1
0.25
5-FC
Flu
Itr
MIC90 MIC50 MIC90 MIC50 MIC90 MIC50
0.25
0.125 6.25 3.125
1
0.5
0.25
0.125 6.25 3.125
1
0.5
0.25
0.125 6.25
0.125 <0.125 12.5
0.125 <0.125 12.5
0.125 <0.125 12.5
3.125
3.125
3.125
3.125
1
2
2
2
0.5
0.5
0.5
0.5
NOTE.—AmB, amphotericin B; 5-FC, 5-fluorocytosine; Flu, Fluconazole; and Itr,
Itraconazole.
orientation, consistent with the observed rearrangement of
the ARR3 amplicon.
Break-induced replication occurs regularly near telomeres of S. cerevisiae and can result in gene amplifications
from mismatched templates as replication proceeds to the
chromosome end (Haber et al. 2004; Koszul et al. 2004;
Reams and Neidle 2004; Schildkraut et al. 2005). After
the initial duplication event, subsequent expansion can occur by unequal crossing-over between sister chromatids,
rolling-circle replication, double rolling-circle replication,
or extrachromosomal amplification and reintegration
(Watanabe and Horiuchi 2005). Tandem genomic duplications make further rearrangements, such as those we have
observed conferring arsenic hypertolerance, particularly
facile through ectopic recombination, as the region is prone
to misalignment. Alternatively, unequal crossing-over yielding a change in DNA copy number could occur as a result of
replication initiation at an origin of replication and subsequent replication fork breakage, which has been demonstrated during ribosomal DNA amplification (Ganley et al.
2009). While it is not known if the ARR3 gene is located near
a replication origin, it is likely subsequent amplification
events involving ARR3 in C. neoformans occurred as a consequence of unequal sister chromatid exchange.
Our genetic studies confirmed ARR3 encodes an arsenite
efflux transporter that appears similar in function to its ortholog in S. cerevisiae. As arsenic is a ubiquitous contaminant of soil and groundwater that is regularly encountered
by many organisms, we postulate that the founder strain of
subclade A5 may have originated from an environment
with high levels of arsenite contamination. High levels of
arsenite are genotoxic and cytotoxic, adversely affecting cell
signaling and proliferation, and impairing cellular respiration and ATP production (Hosiner et al. 2009). Accordingly,
arsenical detoxification mechanisms are ubiquitous in nature, and resistance has been characterized in many organisms ranging from bacterial to mammalian cells (Dabbs and
Sole 1988; Broer et al. 1993; Rosen 1995; Wysocki et al. 1997).
In S. cerevisiae, three independent pathways for arsenical
detoxification have been identified. The first and best studied of these involves the arsenical resistance genes ARR1,
ARR2, and ARR3, which are found in a cluster 7.5 kb from
the end of chromosome XVI (Rosen 1995, 2002; Wysocki
MBE
et al. 1997; Mukhopadhyay and Rosen 1998). The transcription factor Arr1p regulates production of arsenate reductase
Arr2p required for reducing arsenate As(V) to arsenite As(III)
and the plasma membrane arsenite efflux transporter Arr3p.
The second pathway involves the sequestration of arsenite in
vacuoles as glutathione conjugates via the ATP-binding cassette transporter Ycf1p (Wysocki et al. 1997; Ghosh et al.
1999). In the third mechanism, the extrusion of arsenite
is mediated by the bidirectional aquaglyceroporin Fps1p,
which is also involved in arsenite uptake (Wysocki et al.
2001; Maciaszczyk-Dziubinska et al. 2010).
Tolerance assays showed a positive correlation between
copy number and level of arsenical resistance in C. neoformans. This mechanism is analogous to increased copper
and cadmium resistance observed in S. cerevisiae when the
metallothionein gene CUP1 is amplified (Wysocki et al.
1997; Ghosh et al. 1999). To our knowledge, this is the first
example of a naturally occurring gene amplification leading
to high-level arsenite tolerance in a fungus. Although triplication of S. cerevisiae ARR3 has been reported in sake yeast,
this involves a large region spanning nine genes, with selection for the aquaporin-encoding gene AQY1 proposed to be
the likely reason for the amplification (Ogihara et al. 2008). In
this instance, the AQY1-ARR3 region in chromosome XVI was
translocated onto chromosomes IV and XIII in a nonreciprocal manner. The presence of ARR3 in the amplicon region is
probably coincidental as S. cerevisiae is unlikely to encounter
arsenite during sake brewing.
Of all fungi, why would C. neoformans in particular carry
an amplification of an arsenite transporter gene? Insight
again comes from elegant studies in S. cerevisiae that have
revealed that arsenite uptake can be mediated via hexose
transporters (Liu et al. 2004). Interestingly, the genes of
both arsenite and hexose transporters are associated with
the subtelomeric region in C. neoformans as well as in the
sugar-loving S. cerevisiae. Aside from the metabolic requirements for hexose sugars, the polysaccharide capsule of
C. neoformans is composed primarily of the complex sugars glucuronoxylomannan and galactoxylomannan, which
contain high concentrations of the hexose sugars xylose,
mannose, and galactose, plus glucuronic acid, which is derived from glucose. The hexose transporter genes likely
represent a key gene family that is important in acquiring
resources for capsule biosynthesis and expansion could
therefore potentially be related to synthesis of the capsule, as disruption to hexose transporter activity reduces
capsule formation (Chikamori and Fukushima 2005). Increased hexose transporter activity characteristic of the
sugar-coated killer C. neoformans could unintentionally
lead to increased arsenite uptake in contaminated niches;
an isolated gene duplication event involving a subtelomeric
arsenite efflux gene may have therefore been rapidly selected for and subsequently amplified in response to environmental arsenite levels. In support of this model, we
demonstrated that further ARR3 amplification could be
selected for through passage on increasing concentrations
of arsenite. Expansion of amplicon units did not adversely
affect normal growth but mediated the degree of arsenite
1997
Chow et al. · doi:10.1093/molbev/mss066
resistance when the cells were placed under this particular
selective pressure.
Finally, although the ARR3 gene in C. neoformans does
not appear to play a role in virulence or pathogenesis, the
occurrence of the amplification, as well as the enrichment
of hexose transporter genes in the subtelomeric regions, is
evidence that the subtelomeric regions of this fungus are
likely important in niche adaptation. Future studies may
therefore reveal further gene duplication or amplification
events taking place in the subtelomeres of C. neoformans
during infection of the human host, providing further insight
into mechanisms of virulence in this dangerous pathogen.
Supplementary Material
Supplementary tables S1 and S2 and figures S1 and S2 are
available at Molecular Biology and Evolution online (http://
www.mbe.oxfordjournals.org/).
Acknowledgments
We would like to thank Drs Joseph Heitman and Anastasia
P. Litvintseva (Duke University Medical Centre) for kindly
providing strains used in the study. We thank Dr Austen R.
D. Ganley (Institute of Natural Sciences, Massey University,
New Zealand) for helpful comments on the manuscript.
This study was funded by the National Health and Medical
Research Council of Australia (Project Grant 455980 and
CDA 569673).
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