Microevolution of Cryptococcus neoformans Driven by ...

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

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Abstract

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Chow et al. · doi:10.1093/molbev/mss066

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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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.

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Massive Gene Amplification in C. neoformans · doi:10.1093/molbev/mss066 Table 1. Fungal Strains Used in the Study. 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 This study 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

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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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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)

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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

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

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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

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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.

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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

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).

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  10
7), 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

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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).

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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

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.

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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  10
7). 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.

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Massive Gene Amplification in C. neoformans · doi:10.1093/molbev/mss066

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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

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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.

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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

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

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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.

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Massive Gene Amplification in C. neoformans · doi:10.1093/molbev/mss066

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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

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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.

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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-

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.

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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.

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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