Aneuploidy and mosaicism: a strategy for early adaptation to drug pressure in Leishmania

Maintenance of stable ploidy over continuous mitotic events is a paradigm for most higher eukaryotes. Defects in chromosome partitioning and/or under and over replication of chromosomes can lead to aneuploidy, a deleterious condition for most organisms. Surprisingly, in Leishmania, a Protozoan parasite, aneuploidy is a constitutive feature, where modulations of chromosome copy number (CCN) represent a mechanism of gene expression adaptation, possibly impacting phenotypes. Moreover, even in clonal populations, individual CCN varies significantly between single cells, a phenomenon named mosaic aneuploidy. At populational level, this can act as an important adaptation potential in early response to environmental stresses, such as hosts immune system activity and drug pressure. Resistance to drug treatment has increasingly become one of the hindrances to the elimination of Visceral Leishmaniasis (VL), a lethal disease caused by Leishmania donovani. Antimonials, the first class of drugs used to treat VL, lost their efficacy in the Indian sub-continent (ISC) due to the emergence of drug resistant parasites, and there are reports of resistance against Miltefosine, the drug that replaced antimonials in ISC. The mechanisms by which these parasites can rapidly develop resistance to different drugs is still poorly understood. We hypothesize that mosaic aneuploidy could represent a key mechanism driving early adaptation to drug pressure due to intercellular genotypic and phenotypic diversity. In this project, we will address this hypothesis by employing Single Cell Genomic Sequencing (SCGC) and lineage tracking with DNA barcodes. A barcoded clonal population of L. donovani will be generated by inserting in the genome of each cell a unique random DNA sequence, termed a DNA barcode. The evolution of individual cell lineages during early adaptation to drug pressure and the emergence of drug tolerant lineages will be tracked by quantitative sequencing of the DNA barcodes in different timepoints during in vitro and in vivo drug exposure. Parallelly, SCGS will be used to estimate the ploidy profile of individual cells before and after drug treatment. Thus, we expect to assess which lineages survive drug pressure and develop resistance, how karyotypes change over treatment, and if genomic changes are pre-existing or de novo adaptations. Additionally, SCGS and lineage tracking will be employed to parasites under standard culture conditions to address how quickly mosaicism arises and evolves over time in clonal populations. Together, these results will provide important insights to fundamental questions of Leishmania genomic plasticity and its role in early adaptation to environmental changes, including drug pressure.

Keywords:B780-tropical-medicine

Project type:PhD project