PhD defence Gabriel Heringer Negreira

Supervisors

• Prof. dr. Jean-Claude Dujardin (ITM/University of Antwerp)

• Dr. Malgorzata Domagalska (ITM)

Abstract

Leishmania is a genus of protozoan parasites causing leishmaniasis, a neglected tropical disease with a wide range of clinical manifestations ranging from self-healing but stigmatizing skin lesions to a visceral syndrome, fatal 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 rapid and dynamic modulation of chromosomes copy number (aneuploidy), which leads to different karyotypes co-existing even among sister parasites in clonal populations (mosaic aneuploidy). It is hypothesized that Leishmania exploits aneuploidy and its mosaicism 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 drug pressure.

We first applied a high throughput single-cell genome sequencing (SCGS) technology to investigate the extent of mosaic aneuploidy in two distinct clonal Leishmania donovani populations, in standard in vitro culture. We revealed for the first time the complete karyotype of thousands of individual Leishmania parasites, identifying hundreds of karyotypes co-existing in each population. 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 selection dictates 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. In the case of SbIII, we consistently observed drastic changes in aneuploidy emerging in a short period of ~3 weeks and affecting multiple chromosomes, with the reproducible dosage increase of a subset of chromosomes including chromosome 23, which bears the MRPA gene, a known driver of SbIII tolerance. By combining SCGS with lineage tracing using cellular barcodes and longitudinal genome characterization, we revealed that early aneuploidy changes generated under SbIII pressure result from the polyclonal selection of pre-existing karyotypes, complemented by rapid de novo alterations in chromosome copy number 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.