Authors

Discussion

Aneuploidy is a ubiquitous feature of Leishmania and represents an adaptive mechanism, modulating gene expression and possibly impacting phenotypes. Aneuploidy may vary within single parasites in clonal populations, a phenomenon termed mosaic aneuploidy (MA), with important evolutionary and functional implications which remains under-explored. In previous work (1), we applied and validated a high throughput single-cell genome sequencing (SCGS) method to study for the first time the extent and dynamics of whole karyotype heterogeneity in two Leishmania donovani clonal populations representing different stages of MA evolution in vitro. We found that drastic changes in karyotypes quickly emerge in a population stemming from an almost euploid (except for chr 31) founder cell -here further called euploid-. This possibly involves polyploidization/hybridization at an early stage of population expansion, followed by assorted ploidy reduction. During further stages of expansion, MA increases by moderate and gradual karyotypic alterations, affecting a defined subset of chromosomes. To gain insights into the first molecular events during MA emergence and identify potential drivers, we set up new series of experiments (unpublished): we derived 20 subclones (euploid or highly aneuploid) from one L. donovani strain and characterized the independent evolution of 8 of those clones over 10 passages in vitro (~70 generations). With longitudinal bulk genome sequencing, we revealed a process of convergent evolution where subclones starting with different founder karyotypes developed similar aneuploidy profiles in short time periods (4-5 passages). Moreover, a longitudinal SCGS monitoring of one of these euploid subclones revealed that mosaic aneuploidy is already detectable – to a minor extent – at stages as early as 21 generations after cloning, with ~21 karyotypes identified between 720 cells. At this stage, 95% of the sequenced parasites still had the euploid karyotype. However, at generation 36, different sub-populations were identified with distinct highly aneuploidy karyotypes, with one of these subpopulations further outgrowing the group of parasites with the euploid profile at generation 46, suggesting a fitness gain in this highly aneuploidy group. The longitudinal SCGS also revealed that the rate at chromosome copy number change increase exponentially over time, suggesting that maintenance in culture is accompanied by increase in genome instability, promoting higher karyotype heterogeneity in fewer generations. Lastly, a longitudinal single-cell RNA sequencing of this same clone revealed candidate genes that might be involved in this increased genome instability. These results indicate that the mechanisms governing mosaic aneuploidy in Leishmania can be differentially modulated to increase or constrain the diversification of karyotypes and provide a first glance at which molecular factors can act in this regulation.

Reference 1. Negreira GH, Mon