Genome plasticity and its role in leishmania adaptation and drug resistance

Aneuploidy in Leishmania

Aneuploidy is defined as the abnormal number of chromosomes within a cell. Aneuploidy is typically detrimental to the fitness of an organism, for example, trisomy of chromosome 21 is the causative agent for Down syndrome.^[8] However, microorganisms, including; Candida, Trypanosoma Cruzi, and Leishmania, have been observed to display aneuploidy.^[8,9,11,12] Fluorescence in situ hybridization (FISH) and whole genome sequencing (WGS) utilizing Illumina high throughput sequencing followed by Read Depth coverage (RDC) have revealed great variation in chromosome copy number (CCN), both within and among, species, strains, and population of Leishmania.^[10,13] FISH and WGS demonstrate that the chromosomes of Leishmania exist in varying states of ploidy among monosomic, disomic, trisomic and tetrasomic.^[13] Such heterogeneity in ploidy expression has been termed as aneuploidy mosaicism.^[8-10,13]

FISH indicates that the genome of L.major has been shown to be predominantly disomic, with chromosomes 1, 5, and 27 displaying trisomy, and chromosome 2 displaying mainly monosomy.^[10] However, CCN did vary among cells of this L.major population.^[10] Chromosomes 12, 27, and 36, appeared stable in somy expression at a population-level, whereas ploidy in chromosomes 1, 2, 5, and 17, fluctuated between cells of the population.^[10] For example; chromosome 1 was observed to be trisomic in 62% of cells and disomic in 37% of cells.^[10] As illustrated in Figure 1.

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Figure 1:: Variable ploidy for seven chromosomes in L.major (Amended from Sterkers., et al., 2010). Figure 1 illustrates aneuploidy and mosaicism in an L.major strain. Numerous chromosomes within the Leishmania genome display varying levels of aneuploidy, evidencing the high incidence and importance of this genome plasticity feature to the parasite. Variable ploidy expression, in seven chromosomes studied, between cells of an L.major strain. Percentage of monodi- and tri-somic chromosomes in L.major is demonstrated by the y-axis. Each bar represents one of the seven chromosomes studied. Yellow indicates monosomic, blue indicates disomic, and red indicates trisomic.

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Several methodological strengths increase the validity of the results generated by FISH analysis. To avoid the influence of biases due to artefact in the counting of chromosomes, two color labeling of the same chromosome was performed. Furthermore, all counts were performed blinded and by two independent counters. The results were additionally verified through the study of two other L.major strains obtained from both human and dog. Thus, overall, it appears that FISH evidence for aneuploidy did not result from artifactual genetic drift due to long-term laboratory cultivation. The results generated from this study are in accordance with those derived from a study utilizing a WGS approach to study CCN variation.^[13] However, whilst both studies determined the L.major genome to be predominantly disomic, the WGS study did not detect high levels of monosomy or trisomy in chromosomes 2, and 1, 5 and 17, respectively. These differences may be accounted for by the differing methodologies. FISH is the only technique capable of discerning the number of aneuploidies within each individual cell in a given population,^[10] whilst WGS yields a clearer population wide view of aneuploidy as it analyses many millions of cells.^[13] In addition, the WGS study found that, for L.infantum, nine chromosomes displayed trisomy. Moreover, in one L.mexicana isolate three chromosomes displayed trisomy, whereas in a different L.mexicana isolate, only one chromosome was shown to display trisomy, suggesting variation in chromosomes affected. Interestingly, a variable number of chromosomes within L.mexicana were of intermediate read-depth, being neither disomic nor trisomic, indicating a mixture of individual cells within the population displaying di- and tri-somy. This phenomenon was also observed for L.braziliensis. One conserved finding between all strains and species of Leishmania was chromosome 31 always being supernumerary. Results from WGS and RDC, were further confirmed via allele frequency analysis and real time polymerase chain reaction.(RT-PCR) Such reproducibility within the results confers greater reliability to the findings. Nonetheless, there were some exceptions to the general concordance. For instance, chromosomes 5 and 16 in L.mexicana were predicted to be disomic by allele frequency analysis, but supernumerary by RDC data. One possible explanation for this discrepancy in data may relate to the decrease in accuracy observed in RDC when detecting multiple copy genes.^[13] Alternatively, this discrepancy may be explained as a result of the variable ploidy expressed by cells of the same strains of Leishmania species, thus ploidy levels may differ between different isolates of the same strain, resulting in contrasting data when different test isolates are used. Overall, data collated from numerous studies employing different experimental methodologies, evidences the existence of widespread aneuploidy in different Leishmania species.

Aneuploidy as a survival strategy

Mechanism of aneuploidy generation

Mosaic aneuploidy, as observed in Leishmania, is thought to result from a lack of control regarding chromosomal replication, thus resulting in over or under replication of the chromosome, and, consequently, gain or loss of the chromosome copy, respectively.^[10] Post-translationally modified histones may modify auto replicating sequences via epigenetic mechanisms, resulting in the defective chromosome replication control.^[15] Alternatively, mosaic aneuploidy may arise following chromosome mis-segregation.^[7] Asymmetric segregation of chromosomes can be observed during mitosis, with dividing cells displaying asymmetric allotment of chromosome homologues into the two daughter cells in either a 1+2 or a 2+3 pattern.^[14] Such that the total number of chromosomes is always odd.

Gene copy number variants

Leishmania is possessive of the ability to both amplify and delete specific DNA sequences.^[22] One mechanism by which Leishmania is able to modify gene expression is through induction of gene CNVs.^[8] DNA amplification can result from homologous recombination (HR).^[23,24] DNA amplifications can be observed as the formation of extrachromosomal regions of the Leishmania genome, as either circular elements or linear minichromosomes, formed through rearrangements of direct or inverted homologous repeated sequences, respectively.^[22,17] Alternatively, HR can promote intrachromosomal tandem duplication.^[22] Variation in gene copy number frequently occurs in response to parasite environment and selection pressure, such as that exerted by drug administration.^[22,17]

Gene copy number variation and drug resistance

Genome plasticity, in the form of variations in gene copy number, has been implicated in the development of drug resistance, as evidenced by significant differences in gene expression between MTX-drug-resistant L.major and L.infantum species and wild-type strains,^[17] as demonstrated in Figure 2.

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Figure 2:: DNA microarray data highlighting differential gene expression in MTX-resistant and wild-type L.infantum [amended from Ubeda et al., 2008]. The figure illustrates numerous genes which are differently expressed between the wild-type L.infantum and the MTX-resistant L.infantum, indicating the possibility that copy number variation, and consequent up- or down- regulation of genes, contributes to the development of MTX resistance. The red line on the scatter plot indicates a 1.5 fold increase in gene transcription, with the blue line indicating a 1.5 fold decrease in gene transcription, between the wild-type L.infantum and the MTX-resistant L.infantum, the y-axis represents the expression ratio between mutant and wild type cells, and the x-axis represents signal intensity. One of the genes demonstrating a 1.5 fold increase in expression between MTX-resistant L.infantum and wild-type L.infantum, was identified as DHFR-TS.

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Utilization of a comparative wild-type group in the study, affords greater validity to this conclusion. Gel electrophoresis data exemplified gene amplification in the DHFR-TS gene, present on chromosome 6, in MTX-drug-resistant mutants.^[17] This finding was further confirmed via PCR.^[17] Such reproducibility of these findings improves the reliability of the data. DHFR-TS was amplified on circular extrachromosomal DNA regions through the process of HR between repeated sequences. DHFR-TS upregulation results in increased production of DHFR, a key enzyme in parasite DNA biosynthesis.^[25] The same study also reported linear amplification of the PTRI gene in MTX-resistant mutants.^[17] The PTRI gene encodes the protein PTRI, whose enzymatic activity is similar to that of DHFR, and thus, is an important determinant to parasite survival.^[26] Both the DHFR-TS and the PTRI genes are flanked by numerous repeated sequences, thus providing a mechanism from rapid amplification of these metabolic genes in situations of limited nutrients. DNA deletion events can also promote drug resistance. For example, the FT1 gene, which encodes folate transporters, is downregulated in MTX-resistant L.major and L.infantum mutants.^[17] MTX gains entry to cells via folate transporters, thus FT1 downregulation results in decreased uptake of MTX.^[27] Similarly, cosmid-sequencing demonstrated a significant difference in gene CNVs between wild-type and MTX-drug-resistant Leishmania strains.^[22] One limitation of this methodology, however, is the inability to detect loss-of-function mutations, and therefore, cannot be utilized to confirm the finding of FT1 downregulation. Furthermore, linear amplification has also been shown for the ABC transporter encoding gene MRPA, in L.major and L.infantum strains resistant to antimonials.^[17] Amplifications promoting drug resistance confer a survival advantage to the parasite, and thus, are positively selected for. Illustrated by increased presence of amplifications in L. major and L.infantum isolates exposed to drugs, when compared to unstressed controls.^[17] Contrastingly to gene amplifications occurring within extrachromosomal regions, gene amplification can also occur in intrachromosomal regions of the Leishmania genome and can confer drug resistance to the parasite.^[28] Next generation sequencing (NGS) revealed DNA amplification on chromosome 19 to occur on intrachromosomal, as opposed to extrachromosomal, regions.^[28] This finding was further validated by RT- PCR.^[27] Similar to previous studies, NGS demonstrated that gene deletions also result in drug resistance.^[25] DNA deletion on chromosome 31 in the aquaporin 1 (AQP1) encoding region has been linked to Leishmania resistance to antimony. The association between AQP1 downregulation and Leishmania antimony resistance has been found in both laboratory cultured Leishmania strains and field isolates, conferring greater external validity to this finding. AQP1 provides a route of entry for antimony compounds in Leishmania, thus providing an explanation for the resistance acquired in the absence of this transporter protein.

Overall, evidence drawn from multiple studies demonstrates a role for CNV in the upregulation of genes conferring drug resistant capabilities to the parasite.

Gene copy number variation and adaptation to environment

Numerous studies have reported gene CNVs in the DNA regions encoding proteins, including; amastins, GP63, and peptidases.^[8,29,30] Genomic amplification in these encoding regions, and the consequent upregulation of the encoded proteins, promotes parasite survival within the host. Amastins are cell surface expressed proteins that aid in parasite adhesion to host cells.^[8,29] The amastin repertoire has been reported to vary between Leishmania species, which may reflect parasitic adaptation to the variety of host environments each Leishmania species infects.^[8] GP63 is another parasite protein involved in cell adhesion. Within the sand fly host, GP63 functions in midgut attachment. Whereas within the vertebrate host, upregulation of this protein is associated with inhibition of the complement pathway via complement mediated cleavage of C3, and inhibition of antiparasitic activity via interaction with JAK kinases.^[8] Similar to the amastin protein family, GP63 expression also varies between Leishmania species, with limited activity of GP63 reported for L.tarentolae in comparison with the L.vianna species, which is reflected by the lack of intracellular life cycles stages for L.tarentolae and the different hosts each species infects. Peptidase expression prevents parasite clearance, by down regulating the host T-helper-1-cell immune response.^[8] One common methodological limitation present in studies investigating gene CNV, is the focus upon Leishmania strains from a specific, singular geographical location, as variation may occur both inter- and intra-species based upon geographical location. Therefore, studies focusing on a singular geographic location may lack both external validity and generalizability. The amplification of differing genetic regions promoting parasite continual survival between Leishmania species and intra-species life cycle stages, suggests gene amplification acts as an adaptive survival strategy to differing host environments.

Leishmania transcriptional control

Leishmania parasites exert unique transcriptional programs. For the majority of eukaryotes, each protein is encoded by a single transcription unit, with its own promoter and terminator. In contrast, kinetoplastid, including Leishmania, genes are transcribed collectively as part of a polycistronic transcription unit (PTU), and each PTU has the ability to transcribe up to hundreds of genes.^[31] Leishmania possesses unique DNA replication initiation pathways, as the origin of replication sites are detected in a single region within each chromosome, as evidenced by marker-frequency-analysis.^[35] This is in direct contrast to eukaryotic genomes, in which multiple origins of replication sites are located across the linear chromosome.^[35] Whilst a multitude of studies evidence unique DNA replication programs existing within the Leishmania genome, evidence is currently lacking to explain the fitness advantage posed by the possession of such unique replication mechanisms. The ability to exert a unique mechanism of DNA replication and control, and the resultant genomic plasticity, may provide the parasite with the ability to modulate its genome to respond to environmental stresses, such as the selection pressure exerted via drug administration. Therefore, favoring parasite resistance, consequent survival, and establishment of infection. However, studies evidencing this hypothesis are required, for instance studies comparing parasite response to drug pressure between Leishmania species expressing such unique DNA replication pathways, and biologically similar parasite species who lack such unique DNA replication pathways.

Homologous recombination in Leishmania

HR is a mechanism whose activity ensures completion of DNA replication. In addition, HR also functions in DNA repair.^[31] HR is regulated by the enzyme Rad51 recombinase. HR factors such as Rad51 have been proven to function in episome formation, which in turn has been linked to the acquisition of drug resistance.^[31] MER11 is another factor proven essential in the process of DNA repair, MER11 action is essential to the functionality of Rad51^[35,36,37]. MRE11 is responsible for resection of the ends of DNA double-strand breaks.^[31] Similar to Rad51, MRE11 promotes the formation of episomes, specifically extrachromosomal linear amplicons, in response to drug pressure.^[31] In contrast, Rad51 favors circular amplicon formation.^[31] Linear amplification of the PTR1 gene, mediated by MRE11, and circular amplification of the DHFR-TS gene, mediated by Rad51, has been shown to lead to MTX resistance in Leishmania.^[31] The majority of studies investigating HR in Leishmania utilize a common experimental model.^[23,31-40] Such standardization in methodology imparts greater reproducibility and reliability to the results generated from such studies. Consequently, the conclusions are afforded greater internal validity.