High throughput single-cell genome sequencing gives insights into the generation and evolution of mosaic aneuploidy inLeishmania donovani

Abstract

Leishmania, a unicellular eukaryotic parasite, is a unique model for aneuploidy and cellular heterogeneity, along with their potential role in adaptation to environmental stresses. Somy variation within clonal populations was previously explored in a small subset of chromosomes using fluorescence hybridization methods. This phenomenon, termed mosaic aneuploidy (MA), might have important evolutionary and functional implications but remains under-explored due to technological limitations. Here, we applied and validated a high throughput single-cell genome sequencing method to study for the first time the extent and dynamics of whole karyotype heterogeneity in two clonal populations of Leishmania promastigotes 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 founder cell. 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. Our data provide the first complete characterization of MA in Leishmania and pave the way for further functional studies.

Life in plastic, it’s fantastic! How Leishmania exploit genome instability to shape gene expression

“…Disomy (i.e. two chromosome copies) likely predominates in Leishmania , however mosaic aneuploidy (variable aneuploidy states) is common in vitro and within natural populations, suggesting it is a constitutive feature of their genome ( Rogers et al., 2011 ; Sterkers et al., 2011 ; Negreira et al., 2022 ). Why aneuploidy is frequent in Leishmania is unclear but like extrachromosomal amplification, varying chromosome number may provide an additional method of mRNA regulation by increasing DNA copies.…”

“…Over the last decade, single cell sequencing (SCS) technologies ( Imamura et al., 2020 ; Louradour et al., 2020 ; Bussotti et al., 2021 ; Negreira et al., 2022 ), novel screening strategies ( Baker et al., 2021 ), improved genetic engineering using CRISPR/Cas9 ( Zhang and Matlashewski, 2015 ; Espada et al., 2021 ; Beneke et al., 2022 ) and inducible gene deletion ( Duncan et al., 2016 ; Damasceno et al., 2020b ; Yagoubat et al., 2020 ) have seen the Kinetoplastid parasite Leishmania , a single-celled eukaryote, emerge as a strong model of adaptive genome plasticity due to its surprising tolerance for extensive genomic alterations ( Rogers et al., 2011 ; Sterkers et al., 2011 ; Lachaud et al., 2014 ; Ubeda et al., 2014 ). Over 20 species of Leishmania cause the vector-borne, neglected tropical disease (NTD) leishmaniasis in humans and animals.…”

Experimental Hybridization in Leishmania: Tools for the Study of Genetic Exchange

“…Minicircle diversity is most likely not uniform in the cultured Leishmania promastigote population, as MSCs are expected to change after successive rounds of mitosis in a stochastic manner. For the study of nuclear genome heterogeneity, single-cell WGS (scWGS) has been used to describe the rise of different nuclear karyotypes within a clonal L. donovani culture [ 35 ]. Similarly, analysis of single-cell kDNA or single-kinetoplast composition is important for a comprehensive understanding of minicircle heterogeneity, MSC cooperative functioning, and maxicircle–minicircle combinations in Leishmania populations.…”

Paving the Way: Contributions of Big Data to Apicomplexan and Kinetoplastid Research

“…These examples of species-specific innovations are most frequent amongst the genes necessary for the coating and/or decoration of the parasite’s cell surface, and are likely to determine key pathways for parasite survival and adaptation in different hosts and environments. Recently, the field has used whole genome amplification of single cells and single-cell sequencing as means to detect aneuploidy mosaicism and reveal the specifics of its generation and evolution ( Imamura et al., 2020 ; Negreira et al., 2022 ).…”

“…Comparative genomics has been key to understanding the microevolution of parasite lineages, as a high-throughput method of population genetics. As the field progresses to single-cell genomics ( Poran et al., 2017 ; Negreira et al., 2022 ) ( Figure 3 ), long-read sequencing, and “post-genomic” tools (e.g. SNP barcoding panels ( Daniels et al., 2008 ; Preston et al., 2014 ; Baniecki et al., 2015 )), we will gain greater resolution into the dynamics of gene gain and loss, chromosomal reassortment, haplotype diversity and de novo mutations that may affect parasite fitness.…”