Modulation of gene expression in drug resistant Leishmania is associated with gene amplification, gene deletion and chromosome aneuploidy

Gene expression and DNA copy number analyses using full genome oligonucleotide microarrays of Leishmania reveal molecular mechanisms of methotrexate resistance.


Background
The protozoan parasite Leishmania is distributed worldwide and is responsible for a wide spectrum of diseases, including cutaneous, mucocutaneous and visceral leishmaniasis. No vaccines are presently available against Leishmania infections [1] and treatments rely primarily on chemotherapy. The chemotherapeutic arsenal is limited and resistance to the mainstay of pentavalent antimonials has reached epidemic proportions in parts of India [2]. Several studies dealing with drug resistance in Leishmania have highlighted the plasticity of the Leishmania genome [3,4]. The antifolate methotrexate (MTX) has been one of the first and most widely used drugs for understanding drug-induced plasticity and resistance mechanisms [5][6][7][8]. While Leishmania is sensitive to MTX, the drug is not used clinically to treat leishmaniasis. However, Leishmania is a folic acid auxotroph and studies of MTX resistance mechanisms have highlighted several novel aspects of folate metabolism in this parasite that could be exploited for drug interventions [9,10]. Indeed, the development of novel antifolate molecules for Leishmania and related parasites has been ongoing in several laboratories [11][12][13].
Leishmania resists MTX by a number of mechanisms. Leishmania has the capacity to transport folic acid, but this activity is often impaired in MTX resistant cells [8,[14][15][16][17]. The main Leishmania folate transporter FT1 has been isolated [18,19] and is part of a large family of folate biopterin transporter (FBT) proteins with 14 members in Leishmania (AA Ouameur et al., unpublished data). Rearrangements of FBT genes are correlated with MTX resistance [19][20][21]. A frequent mechanism of drug resistance in Leishmania is gene amplification [3]. Small chromosomal regions of 20-70 kb that are part of one of the 36 Leishmania chromosomes are amplified as part of extrachromosomal elements [3]. These elements are usually formed by recombination between repeated homologous sequences [22][23][24]. Amplification of the gene coding for the target dihydrofolate reductase-thymidylate synthase (DHFR-TS) has been described in MTX resistant parasites [5,6,[25][26][27][28][29]. Work on MTX resistance also led to the characterization of the pteridine reductase PTR1, whose main function is to reduce pterins. However, when overexpressed it can also reduce folic acid and lead to MTX resistance by by-passing DHFR-TS activity [30][31][32][33]. The PTR1 gene is frequently amplified as part of extrachromosomal circular or linear amplicons [6,16,22,[34][35][36][37][38]. In addition to these three main mechanisms of resistance, perturbation in folate metabolism [39,40], in one carbon metabolism [41] or in DNA metabolism [42] have also been associated with MTX resistance. Several of these mutations can co-exist in the same cell, demonstrating that resistance can be a complex multi-gene phenomenon. Genome wide expression profiling scans represent a useful tool for understanding complex resistance mechanisms and may lead either to the discovery of novel resistance mechanisms and/or could provide clues about mechanisms of gene rearrangements.
Indeed, DNA microarrays have been useful for investigating the mode of action of drugs [43] and mechanisms of resistance (reviewed in [44][45][46]). DNA microarrays for Leishmania have evolved from random genomic DNA clones [47][48][49][50], cDNA clones [51,52], targeted PCR fragments [29], selected 70-mer oligonucleotides [53,54] to full genome microarrays [55,56]. Targeted microarrays have been used previously for the study of drug resistance in Leishmania [29,52,54,57]. We present here the generation of full genome DNA microarrays for both L. major and L. infantum and their use in the study of one L. major and one L. infantum MTX resistant mutant. These genome wide expression profiling experiments illustrate the complexity of resistance mechanisms present in the same cell. They allowed the definition of the precise mechanisms leading to the formation of extrachromosomal circular and linear amplicons, the definition of gene deletion events and revealed the involvement of aneuploidy in the complex genotype of MTX resistance.

RNA expression profiling in methotrexate resistant Leishmania cells
Completion of the L. major genome has allowed the generation of arrays containing 60-mer oligonucleotide probes designed by NimbleGen Systems [55,56] and in this work, we present the generation of a full genome DNA microarray composed of 70-mer oligonucleotide probes suitable for both L. major and L. infantum analysis (see Materials and methods for a full description of the arrays). These full genome arrays were used for deciphering how Leishmania resists the antifolate model drug MTX. Two MTX resistant mutants, L. major MTX60.4, which has previously been studied with small targeted arrays [29], and L. infantum MTX20.5, were studied using the full-genome microarrays. Mutants of both species are highly resistant to MTX (Figure 1a), and since they were selected in a stepwise fashion, it is likely that multiple resistance mechanisms may exist in these mutants and could thus be uncovered by these arrays. The resistant cells had a similar generation time as the wild-type parent cells.
The DNA microarrays were first validated by hybridizing fluorescently labeled digested DNA of wild-type L. major and L. infantum cells. The arrays were found to yield uniform and reproducible results (not shown) and were deemed appropriate for RNA expression profiling experiments. Total RNAs were thus purified for both wild-type and mutant strains, used to synthesize fluorescent probes, and hybridized to the microarrays as described in Materials and methods. Scanning and normalization led to expression data that were first represented as scatter plots. As evident from these plots (inserts in Figure 2a,b), most genes in both species are equally expressed between the sensitive and resistant strains. Indeed, the bulk of expression (RNA level) ratios between sensitive and resistant strains were close to 1. Nonetheless, there were notable differences. First, the RNA levels of a total of 61 genes were found to be modulated (cut-off of 2, p < 0.05) in the L. infantum MTX20.5 mutant compared to the wild-type strain ( Figure 2a; Table S1 in Additional data file 1) and the expression levels of 75 genes were changed significantly (cut-off of 2, p < 0.05) in the L. major MTX60.4 mutant compared to the wild-type strain (Figure 2b; Table S1 in Additional data file 1). Secondly, a majority of genes whose expression was modulated by more than two-fold had increased expression levels in L. infantum MTX20.5 but the majority of another set of genes had decreased expression levels in L. major MTX60.4 (inserts of Figure 2; Table S1 in Additional data file 1). If the expression modulation cut-off was changed from 2 to 1.5 (p < 0.05), we found 251 and 372 genes that were differentially expressed in L. infantum MTX20.5 and L. major MTX60.4, respectively ( Figure 2). Surprisingly, few differentially expressed genes were found to be modulated similarly in both mutants ( Figure 3; Table S1 in Additional data file 1). One notable exception is a region of chromosome 6 that corre-sponds to a six gene locus including the DHFR-TS gene. DHFR-TS is the main target for MTX and its gene was frequently found amplified in L. major MTX resistant mutants as part of extrachromosomal circles (reviewed in [3,4]).
The DNA microarray data were supported by selected quantitative real-time reverse transcription PCR (qRT-PCR) assays in both the L. major and L. infantum mutants (Figure 3). In only two cases we found a discrepancy between the two techniques. LmjF04.0160 and its orthologue LinJ04_V3.0160 were found down-regulated in both mutants using DNA microarrays, but this was confirmed only in the L. major mutant by qRT-PCR ( Figure 3). The other discrepancy between microarray and qRT-PCR data was for FT1, but this is explained by a gene deletion event (see below). The only other gene that was modulated similarly in the two mutants was the ABC protein gene ABCA2 and this was confirmed by qRT-PCR ( Figure 3). Other genes were modulated in both mutants but in different ways. While LmjF31.0720 was downregulated in L. major MTX60.4, its orthologue LinJ31_V3.0750 in L. infantum MTX20.5 was overexpressed ( Figure 3). Otherwise, genes differentially expressed were specific to individual mutants.
The differential gene expression of the MTX resistant mutants was also represented in a chromosome by chromosome fashion ( Figure 2). This has permitted us to visualize regions that are differently expressed (red/orange, corresponding to overexpressed genes in the mutants). Two regions were clearly overexpressed in the L. infantum MTX20.5 mutant. One region was on chromosome 6 (DHFR-TS loci) and the second was in the left portion of chromosome 23 ( Figure 2a). For the L. major MTX60.4 mutant, we also saw an increase in expression of selected genes present on chromosome 6 (DHFR-TS loci), but we also observed a number of whole chromosomes (for example, chromosome 22; colored predominantly red in Figure 2b).

Extrachromosomal circular amplification of DHFR-TS
DHFR-TS is present on chromosome 6 and by close examination of the expression data derived from the arrays we were able to precisely define the genes with increased expression in both the L. major and L. infantum mutants. In L. infantum, the genomic region overexpressed is delimited by genes LinJ06_V3.0860 and LinJ06_V3.0910 (Figure 4a). Most interestingly, the same region is overexpressed in L. major MTX60.4 ( Figure 4a). As Leishmania is devoid of control for the initiation of transcription (no pol II promoter has yet been isolated in this parasite [58]), it is possible that the amplification of a small genomic region containing the DHFR-TS gene is responsible for the increased gene expression as determined by DNA microarrays. This was tested by hybridization of a blotted pulsed-field gel electrophoresis (PFGE) gel with a DHFR probe. Wild-type cells gave rise to two hybridizing bands, suggesting that the two homologous chromosomes 6 have different sizes (Figure 4b, lanes 1 and 3), a well established phenomenon in Leishmania [59]. The two mutants had an extra band hybridizing to the DHFR probe, which with its hybridizing smear is characteristic of extrachromosomal circles (Figure 4b, lanes 2 and 4). The genesis of circular DNA in Leishmania has been studied and is often due to homologous recombination between direct repeats bordering the regions amplified [22][23][24]. Close examination of the sequences flanking the regions amplified indeed pointed to the presence of repeated sequences (Figure 4a). The repeated sequences were highly similar between L. major (575 bp) and L. infantum (837 bp) ( Figure S1 in Additional data file 2). To provide evidence that the DHFR-TS containing circles were generated through homologous recombination between these direct repeated sequences, we used two primers (6a and 6b in Figure 4a,c) that should give rise to a PCR amplification product only when an extrachromosomal circle is formed (Figure 4c). Indeed, when using this primer pair, PCR fragments of the expected size were observed in L. infantum MTX20.5 and L major MTX60.4 (Figure 4d, lanes 2 and 4) while no amplification was observed in the wild-type cells (Figure 4d, lanes 1 and 3). The difference in size of the PCR fragments between L. major and L. infantum is due to the difference in size of the repeats in the two species ( Figure  S1 in Additional data file 2). Sequencing of the PCR generated amplicon derived from L. major MTX60.4 [Gen-Bank:EU346088] confirmed the scenario of homologous recombination between the repeated sequences ( Figure S1d in Additional data file 2).

Linear amplification of PTR1
In mutant L. infantum MTX20.5 we observed a region of chromosome 23 that was overexpressed (increased RNA levels; Figure 2a). This region contains the gene for pteridine reductase 1 (PTR1), a well established MTX resistance gene whose product can reduce folic acid, hence by-passing the need for DHFR-TS [30,31]. Similarly to the DHFR-TS loci, the microarray expression data have allowed the precise determination of the region that was overexpressed, which started at the telomeric end and extended 120 kb up to gene LinJ23_V3.0380 (Figure 5a). The putative presence of telomeric sequences would suggest a linear amplification instead of a circular amplification. Hybridization of a chromosome PFGE blot has shown that PTR1 hybridized to the approximately 800 kb chromosome in both wild-type and resistant cells but also to a smaller linear amplicon of approximately 230 kb in L. infantum MTX20.5 ( Figure 5b). This amplicon also hybridized to a telomere probe (Figure 5b). The size of the amplicon suggests that the amplified region was

Chromosomes Chromosomes
Base pairs Base pairs 1,000,000 2,000,000 1,000,000 2,000,000 duplicated. The LinJ23_V3.0390 gene is clearly not overexpressed and thus not part of the amplicon (Figure 5a). Three genes, LinJ23_V3.0360, LinJ23_V3-0370 and Lin23_V3.0380, were less overexpressed than the other genes that are part of the amplicon (Figure 5a). Examination of the sequences where expression changed enabled the detection of inverted homologous repeats of 578 bp ( Figure  S2 in Additional data file 2) between LinJ23_V3.0350 and Lin23_V3.0360, and between LinJ23_V3.0380 and Lin23_V3.0390 ( Figure 5a). Interestingly, similar repeats of 574 bp with 91% identity were found at the same position in the L. major genome [60]. The presence of these inverted repeats and the microarray expression data would suggest the formation of a linear amplicon with large inverted duplications that was formed by annealing of the identical 578 bp inverted repeats (Figure 5c). To obtain support for this scenario, we used PCR primer pairs (23a and 23b, or 23c and 23d) that would lead to a PCR product only if the rearrangement had occurred at the level of the inverted repeats (as, for example, during a block in DNA replication). Indeed, we obtained a product of the expected size with these pairs of primers in L. infantum MTX20.5 but no product was obtained from DNA derived from wild-type cells (Figure 5d). The nucleotide sequence of the PCR amplicon obtained with primer pair 23a/23b [GenBank:EU346089] is entirely consistent with the model shown in Figure 5c ( Figure S2 in Additional data file 2).

Decrease in gene expression due to deletion of folate transporter genes
Leishmania spp. have a large gene family of conserved folate transporters with 14 FBT members (AA Ouameur et al., unpublished data). Part of this family located on chromosome 10 is shown in Figure 6a. Microarray expression data indicated that FT1, coding for the main Leishmania folate transporter [18,19], is down-regulated in L. major MTX60.4 but not in L. infantum MTX20.5 ( Figure 3). The level of conservation of the various FBTs precluded that the 70-mer  oligonucleotides spotted on the arrays would discriminate several of these closely related genes. The use of qRT-PCR to confirm the microarray data indicated that FT1 may be absent ( Figure 3). This was suggestive of a gene deletion event and indeed a Southern blot of L. major MTX60.4 DNA hybridized with a probe recognizing the majority of FBT genes confirmed this extensive gene rearrangement ( Figure 6b) and bands corresponding to LmjF10.0380, LmjF10.0385 (FT1) and LmjF10.0390 were either lacking or rearranged. Using PCR primers (labeled F and R in Figure 6a,c), we were able to demonstrate that FT1 (LmjF10.0385) was deleted following an event of homologous recombination between conserved sequences between LmjF10.0380 and LmjF10.0390 ( Figure   6c). Indeed, primers F and R gave rise to a PCR fragment of 2. the majority of genes present on chromosomes 11 and 12 appeared down-regulated while the expression of genes located on chromosomes 7, 22, 28 and 32 seemed up-regulated ( Figure 2). Chromosome 6 of L. infantum MTX20.5 also appears to be in more than two copies. This chromosomewide uniform modulation of expression was represented more thoroughly for selected chromosomes by plotting the fold modulation in gene expression along the chromosome (Figure 7). The normalized microarray data indicated that genes of chromosomes 22 and 28 were overexpressed 1.7-and 1.5-fold, respectively, in the resistant strain L. major MTX60.4 compared to the wild-type strain. The expression of genes on chromosomes 11 and 12 seemed, in general, to be 50% underexpressed in the mutant strain compared to wildtype cells (Figure 7).
A number of hypotheses can explain this whole chromosomespecific gene regulation and we tested whether the copy number of specific chromosomes changed upon MTX selection in L. major MTX60.4. Quantitative Southern blot analyses with two distinct probes derived from chromosome 22 revealed that if the wild-type cells contain two homologous Linear amplification of PTR1 as a large inverted duplication  (Figure 7c,d). Aneuploidy of specific chromosomes and drug resistance has been described in cancer cells (reviewed in [61]) and fungi [62,63]. To test this possibility, we generated a revertant line of L. major MTX60.4 by successive passages in the absence of MTX; under these conditions, resistance to the drug decreased (Figure 1b). Revertant cells were not as sensitive as wild-type cells to MTX but this is expected as a deletion of FT1 ( Figure 6) will lead to resistant parasites [19]. The aneuploidy of chromosomes 22 and 28 regressed to diploidy (similar to wild-type diploidy) after 30 passages, thus circumstantially linking resistance levels ( Figure 1b) and copy number of these chromosomes (Figure 7a,b, lanes 2-6). With the cells now diploid, additional passages (for example, passage 42) did not decrease resistance further.

Comparative genomic hybridization
Since several of the changes in RNA levels were correlated with gene amplification or gene deletion, we undertook a comparative genomic hybridization (CGH) study using the full genome array. The DNA of mutant L. major MTX60.4 was labeled and changes in copy number in comparison to sensitive wild-type cells were measured using CGH. The CGH data are represented in a chromosome by chromosome fashion in Figure S4 in Additional data file 3. A qualitative correlation was observed between CGH and RNA-based hybridization ( Figure 8). Indeed, amplification of the DHFR-TS locus, derived from chromosome 6, was easily detected by both techniques and quantification of the DNA amplification was compared to RNA levels ( Figure 4). The deletion of FT1 was also detected by CGH and the latter technique was found to be quantitative. Indeed, the 70-mers recognizing FT1 recognized three conserved FT genes. In the MTX60.4 mutant two of these genes are deleted, hence explaining the ratio of 0.33 obtained by CGH ( Figure 6). Polyploidy was also easily detected by CGH ( Figure 8). Indeed, a similar qualitative pattern of hybridization intensities was obtained for both RNA expression profiling and CGH (Figure 8). Interestingly, while RNA expression profiling showed that chromosome 11 was down-regulated, quantitative Southern blots indicated that the copy number of the chromosome remained unchanged (Figure 7). This was also confirmed by CGH (Figure 8). There are some differences, however, between RNA expression profiling and CGH. For example, the latter technique showed that chromosome 2 is polyploid ( Figure S4 in Additional data file 3) but this is likely due to the dynamic process of cell culture and parasite evolution, as DNA and RNA were prepared 1.5 years apart, rather than a difference in the techniques.
Chromosome aneuploidy in L. major selected for MTX resistance

Discussion
The use of DNA microarrays is now useful to understand both the mode of action of drugs and the mechanisms of drug resistance (reviewed in [44][45][46]). Since Leishmania has no control at the level of transcription initiation [58], it is unlikely that drug response profiling using microarrays will be helpful to understand the mode of action of drugs in Leishmania. Results using MTX as a lead drug and qRT-PCR to monitor key genes, such as DHFR-TS, PTR1, and FT1, appeared to confirm this lack of RNA modulation of target genes upon drug exposure (unpublished observations). This is unfortunate, as the mode of action of most anti-Leishmania drugs is unknown. Nonetheless, microarrays are likely to be useful for studying resistance in Leishmania since it is often mediated by gene amplification [3,4] and we show here that DNA arrays hybridized to cDNAs were most valuable for detecting gene amplification events (Figures 2, 4, and 5).
Since resistance is mostly correlated with gene amplification, we also used CGH and found a good qualitative correlation between RNA expression profiling and CGH (Figure 8). The technique of CGH was found to be technically simpler, Genome Biology 2008, 9:R115 3, and Figure 7 for chromosomes 11 and 12), hybridization with cDNAs is likely to be more comprehensive. Nonetheless, modulation in RNA levels without changes in copy number of a gene is an infrequent event in drug resistant Leishmania. The use of both L. infantum and L. major MTX resistant mutants validated the design of our multi-species array but has also illustrated that the cellular resistance genotype can be complex and differ considerably between different mutants selected for resistance to the same drug. The modulation in expression of a few genes was common to both mutants, and only ABCA2 and DHFR-TS could be confirmed by qRT-PCR (Figure 3). Down-regulation of the ABC protein gene ABCA2 has never been described in MTX resistant Leishmania cells and additional investigations would be required to test whether it has any role in MTX resistance.
DHFR-TS was the first amplified gene studied in a protozoan parasite [5] but its exact mechanism of amplification has never been reported. In addition to detecting gene amplification events, microarray data, whether derived from RNA expression profiling or CGH, were also useful in mapping the exact regions that were amplified. We show that DHFR-TS is amplified in L. major MTX60.4 as an extrachromosomal circle through homologous recombination between non-coding repeated sequences (Figure 4). This is consistent with other loci that were also found to be amplified by homologous recombination between relatively long repeated sequences [22][23][24]. Blast searches have shown that these exact repeated sequences are found only on chromosome 6. Remarkably, the same similar repeated sequences (albeit with different sizes) have also been conserved in L. infantum ( Figure S1 in Additional data file 2). The same observation was made for the inverted repeats close to PTR1 that were conserved between L. major and L. infantum. L. major and L. infantum are thought to have diverged 0.5 million years ago [67] and it thus seems that there is considerable selective pressure to keep these repeated sequences intact. Since folates and pterins are important for Leishmania growth, it is possible that the presence of these repeats may allow a strategy to rapidly increase DHFR-TS or PTR1 levels in conditions of limited substrates. With its lack of transcription initiation control, Leishmania may utilize this alternative strategy of flanking key metabolic genes by repeated sequences to amplify these genes when required. Consistent with this proposal, DNA amplification has been observed in Leishmania cells subjected to nutrient shocks [68].
PTR1 is a well established MTX resistance gene product [30,31] and the amplification of its gene was first reported as part of extrachromosomal circles [6,[34][35][36]. Linear amplification of PTR1 with inverted duplications was described later [16,24,37] and linear amplicons could be precursors of circular amplicons [38]. Linear amplicons derived from other loci than the PTR1 region with inverted duplications have also been described in Leishmania [69][70][71][72][73]. The microarray hybridization data have enabled the elaboration of a plausible model for the generation of a linear amplicon that contained large inverted duplications formed at the site of inverted repeats ( Figure 5). This is consistent with other models of gene amplification in Leishmania [16,37] where inverted repeats seem to be a major pathway to generate amplified large DNA palindromes (inverted duplications), as described in Tetrahymena [74], yeast [75] and mammalian cancer cells [76,77]. One of the large inverted duplications extends from the inverted repeats, where rearrangement has occurred, to the telomeric sequences ( Figure 5). These data exclude the necessity of chromosomal breaks/rearrangements at two independent positions, but it remains to be determined whether a double-stranded break, a single-stranded break or blocks in replication are facilitating inverted repeat annealing.
Gene deletions were thought to be associated with MTX resistance in Leishmania [19,20] but had not yet been characterized at the molecular level. The microarray data, either derived from RNA expression profiling or CGH, has led to the observation that a diploid non-conservative deletion occurred by homologous recombination between two members of the large FBT gene family ( Figure 6). The mechanism of gene deletion thus resembles the mechanism of amplification. Usually, amplification in Leishmania is conservative, and only a few instances of non-conservative amplification (loss of one allele) have been described in it [3,22,23]. In the L. major MTX60.4 mutant, we observed a diploid deletion of the FT1 gene ( Figure 6). It is not known whether the second allele is deleted by homologous recombination or by a gene conversion event such as a loss of heterozygosity, but there is a strong selection pressure to delete FT1, the main folate (and MTX) transporter in Leishmania. Without FT1, cells can become resistant to MTX but folates or related molecules will still need to be transported. It will be of interest to determine whether the fusion FBT protein produced by the recombination event ( Figure 6) is active or not.
The microarray approach has shown that modulation of gene expression could (rarely) be due to differential RNA expression without changes in copy number (Figure 3) [29]; it could be more frequently due to gene amplification (Figures 4 and  5) and, as determined now, to gene deletion ( Figure 6). Two novel strategies were highlighted through the use of microarrays. In the L. major MTX60.4 mutant, the entire set of genes of chromosomes 11 and 12 is down-regulated while all the genes present on chromosomes 22 and 28 and possibly a few other chromosomes are overexpressed. The mechanism underlying an upregulation in gene expression results from a change in chromosome ploidy (Figure 7). Changes in ploidy have been observed when attempting to inactivate essential genes in Leishmania [78], but not in resistant parasites. We recently observed a similar phenomenon with other resistant Leishmania cells (P Leprohon et al., unpublished data), suggesting that chromosome aneuploidy is part of the Leishmania arsenal for responding to drug pressure. There was a good correlation between resistance levels and the copy number of these supernumerary chromosomes (Figures 1 and  7), linking this genetic event to the resistance phenotype. Obviously, additional studies will be required to determine which gene(s) is (are) responsible for resistance. A putative mechanism for increasing the levels of a gene product in Leishmania would thus be to generate supernumerary chromosomes. This may occur when direct or inverted repeats are absent in the vicinity of a gene conferring a selective advantage. While this is plausible, especially for an organism lacking control at the level of transcription initiation, this drug induced aneuploidy has been well documented in cells with transcriptional control, such as cancer cells (reviewed in [61]) or fungi [62,63]. The mechanism of down-regulation of whole chromosome expression does not seem to involve a change in chromosome number (Figures 7 and 8) and may involve epigenetic factors that will need to be investigated.

Conclusion
The microarray approach was useful in highlighting several mechanisms used by resistant cells to modulate the copy number of genes by: gene deletion or extrachromosomal circular or linear amplicons; through supernumerary chromosomes; and by decreasing the expression of whole chromosomes by a mechanism that remains to be identified. In the case of the first two events, the rearrangements have occurred at the site of repeated (direct or inverted) sequences. It is possible that these repeats are not randomly distributed to allow the amplification of specific chromosomal regions. Using DNA microarrays it was shown that inverted duplications are frequent in cancer cells; these are not randomly distributed, and a subset are associated with gene amplification [79]. The availability of DNA microarrays for Leishmania has highlighted the role of repeated sequences and of chromosome ploidy in responding to environmental changes. Aneuploidy has been suggested as an important cause of cancer specific drug resistance [61] and further work should reveal the potential importance of this phenomenon in drug resistance in Leishmania.

Cell culture
The wild-type strain L. major LV39 and the mutants L. major MTX60.4 have been described previously [65]. The L. infantum strain (MHOM/MA/67/ITMAP-263) was selected in vitro in a stepwise fashion starting with its EC 50 (0.5 μM) with doubling concentrations of MTX when cells were adapted to yield L. infantum MTX20.5 growing at 20 μM of MTX. All cells were grown in M199 medium supplemented with 10% heat-inactivated fetal bovine serum and 5 μg/ml hemin at 25°C.

DNA manipulation
Chromosomes in agarose blocks were prepared and separated by PFGE as described previously [38]. For Southern blot and PCR, genomic DNA was isolated using the DNAzol technique (Invitrogen, Carlsbad, CA, USA) as recommended by the manufacturer. Southern blots, hybridization, and washing conditions were done following standard protocols [80]. For chromosome copy number investigation, Southern spots were quantified using ImageQuant 5.2 (GE Healthcare, Upsala, Sweden) and the reference gene α-tubulin was used for normalization.

L. infantum and L. major DNA oligonucleotides full genome microarray design
The recent completion of the sequence of the L. major [81] and L. infantum [82] genomes, allowed the generation of multispecies high-density oligonucleotide microarrays. Our analysis of open reading frame sequence conservation between L. major and L. infantum revealed that these two species share 91-96% nucleotide identity, suggesting that interspecies microarray probes can be designed. Therefore, 70-mer oligonucleotides were designed for each open reading frame of L. infantum and L. major using automated bioinformatic procedures. The genomes of both species were first compared using BLAST and homologous genes were grouped together. Probes were designed with consistent thermodynamic properties. Probes were initially designed for L. infantum with the added requirement that the region targeted by the probes had perfect homology between both species. For common probes, up to 2 mismatches (out of 70 nucleotides) were tolerated. In the case that more than two mismatches were present in a given gene between L. infantum and L. major, a new probe was designed specifically for L. major (956 probes). The microarray included a total of 8,978 70-mer probes that recognized with no mismatches all L. infantum genes (8,184, GeneDB version 3) and also all L. major genes (8,370, GeneDB version 5.1) with a small percentage of the probes having at most 2 mismatches. Also, 372 control probes were included in the microarray for assessing synthesis variability, and location of the probe within a given open reading frame and of mismatches on hybridization. The probes were synthesized in 384-well plates by Invitrogen. The microarrays were printed on SuperChip (Erie Scientific, Portsmouth, NH, USA) using a BioRobotics MicroGrid (Genomic solutions Inc, Ann Arbor, MI, USA). Each probe was printed in duplicate. Our microarray platform is described in the Gene Expression Omnibus (GEO) with accession number GPL6855.

Total RNA preparation and labeling
Total RNA was isolated from 10 8 Leishmania cells during the mid-log phase using RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany). The RNA preparation was treated with TURBO DNase (Ambion, Austin, TX, USA) to avoid any genomic contamination. The purity, integrity and quantity of the RNA were assessed on the Agilent 2100 bioanalyzer with the RNA 6000 Nano LabChip reagent set (Agilent Technologies, Santa Clara, CA, USA). For each probe, 10 μg of RNA were converted to aminoallyl-dUTP incorporated cDNA using random hexamers (Roche, Basel, Switzerland) and the SuperScript III RNase H Reverse Transcriptase (Invitrogen). Probes were thereafter coupled to the fluorescent dye Alexa Fluor555 or Alexa Fluor647 (Invitrogen) following the manufacturer's recommendations. Fluorescent probes were then purified with MinElute Spin Columns (QIAGEN) and quantified spectrophotometrically.

Genomic DNA preparation and labeling
Genomic DNA from 10 8 cells was isolated using the DNAzol technique (Invitrogen) as recommended by the manufacturer. Total DNA was then fragmented by successive passages through 22G1" and 27G 1/2" needles (Becton Dickinson Franklin Lakes, NJ, USA). Fragmented DNA was then double digested with PvuII and MseI restriction enzymes. Digested DNA was purified by phenol-chloroform, followed by an ethanol precipitation. For each probe, 4 μg of purified fragmented and digested genomic DNA were converted to fluorescently labeled DNA using Cy5-or Cy3-dCTP (Amersham, Piscataway, NJ, USA), random hexamers (Roche) and the exo -Klenow DNA polymerase (NEB, Ipswich, MA, USA). Fluorescent probes were then purified with ArrayIt columns (TeleChem International, Sunnyvale, CA, USA) and quantified spectrophotometrically.

Fluorescence detection, data processing and statistical analysis
The Perkin Elmer ScanArray 4000XL Scanner was used for image acquisition (Perkin Elmer, Waltham, MA, USA). Gene-Pix Pro 6.0 image analysis software (Axon Instruments, Union City, CA, USA) was used to quantify the fluorescence signal intensities of the array features. Four different RNA preparations of each mutant and their respective wild-type strain were analyzed, including dye-swaps. Raw data from GenePix were imported in R 2.2.1 for normalization and statistical analyses were performed using the LIMMA (version 2.7.3) package [83][84][85]. Before processing, probes were flagged according to the hybridization signal quality [86]. Weights were assigned to each array in order to give less weight to arrays of lesser quality [87]. Data were corrected using background subtraction based on convolution of normal and exponential distributions [88]. Intra-array normalization was carried out using the 'print-tip loess' statistical method and inter-array normalization was done by using the 'quantiles of A' method for each array [89]. Statistical analysis was done using linear model fitting and standard errors were moderated using a simple empirical Bayes [83]. Multiple testing corrections were done using the FDR method with a threshold p-value of 0.05. Only genes statistically significant with an absolute log ratio greater than 0.58 (log 2 1.5) were considered as differentially expressed. Species comparison was performed only on probes that had less than two mismatches when hybridized to either Leishmania species. GeneSpring GX 3.1 was used for the generation of scatter plots and for chromosome by chromosome analysis. The entire data set has been deposited in GEO under the accession number series GSE9949. The comparative genomic hybridization data are deposited under reference number GSE11623.

qRT-PCR
Three independent RNA preparations were conducted for each condition. First-strand cDNA was synthesized from 2 μg of total RNA using the Superscript III RNase H Reverse Transcriptase enzyme and random hexamers (Roche) according to the manufacturer's instructions. The resulting cDNA samples were stored at -20°C until use. Control PCR amplification was carried out using primers from different internal controls (GAPDH and actin) to evaluate the uniformity of cDNA synthesis in different samples. Primers, TaqMan probes, experimental procedures and quantification for qRT-PCR of the folate transporter genes was as described (AA Ouameur et al., unpublished data) using the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) for normalization. For all other genes, equal amounts of cDNA were run in triplicate and amplified in a 15 μl reaction containing 7.5 μl of 2× Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 10 nM of Z-tailed forward primer, 100 nM of reverse primer, 250 nM of Amplifluor Uniprimer probe (Chemicon Int., Temecula, CA, USA), and 1 μl of cDNA target. Reactions were performed at the Gene Quantification core laboratory of the Centre de Génomique de Québec using the Applied Biosystems Prism 7900 Sequence Detector [90]. Amplification was normalized to two genes showing a highly stable expression in wild-type and resistant strains: LinJ18_V3.0630/ LmjF18.0620 encoding a putative 60S ribosomal protein L10a, and LinJ36_V3.0850/LmjF36.2500 encoding a chromatin assembly factor 1 subunit b-like protein.