Investigating aneuploidy and mosaicism as a mechanism for survival and adaptation of Leishmania

Leishmania is a protozoan parasite causing a neglected tropical disease known as leishmaniasis, which is fatal in visceral infections if untreated. Few drugs are available for treatment and the emergence of drug resistance poses a threat to current elimination efforts. Leishmania has a plastic genome characterized by dynamic modulation of chromosomes copy number (aneuploidy), which leads to different karyotypes co-existing in clonal populations (mosaic aneuploidy). It is hypothesized that Leishmania exploits mosaic aneuploidy as a strategy for early adaptation to environmental stresses, in particular to drug pressure. In this thesis, we aimed to investigate the extent and dynamics of mosaic aneuploidy during adaptation to standard in vitro conditions as well as to drugs. We first applied a high throughput single-cell genome sequencing (SCGS) to reveal for the first time the complete karyotype of thousands of individual Leishmania parasites in two distinct clonal populations in standard in vitro culture. We observed that drastic changes in karyotypes quickly emerge in a population stemming from an almost euploid cell, with mosaic aneuploidy further increasing by moderate and gradual karyotypic alterations. We also found that all chromosomes are prone to somy changes, but only some polysomies can achieve high frequencies in the population, suggesting that natural selection determines which karyotypes emerge and propagate. In a second set of experiments, we investigated the dynamics of mosaic aneuploidy during adaptation to high drug pressure (SbIII and miltefosine) in vitro. By combining SCGS with lineage tracing using cellular barcodes and longitudinal genome characterization, we revealed that early aneuploidy changes observed under SbIII pressure result from the polyclonal selection of pre-existing karyotypes, complemented by rapid cumulative karyotypic alterations which culminates in similar aneuploidy modifications, pointing to a process of convergent evolution. In the case of miltefosine, early parasite adaptation was associated with independent pre-existing point mutations in a miltefosine transporter gene and aneuploidy changes only emerged later, upon exposure to increased concentration of the drug. Different mutants of the miltefosine transporter gene were identified, also pointing out the polyclonal origin of miltefosine resistance. In summary, the present thesis demonstrates that polyclonality and genome plasticity are hallmarks of Leishmania adaptation, with the scenario of aneuploidy dynamics being dependent on the nature and strength of the environmental stress as well as on the existence of other pre-adaptive mechanisms.