Biology subjects

Negreira, G. H.

Publications and source records attributed to Negreira, G. H..

4 recordsLinked to original sources

bioRxiv2025

A novel high-throughput single-cell DNA sequencing method reveals hidden genomic heterogeneity in the unicellular eukaryote Leishmania

Genome instability is considered a major driver of adaptation in eukaryotic microorganisms, but its study is hampered by the limited availability of genomic technologies with single-cell resolution. Here we present a novel high throughput method to reconstruct both structural and nucleotide information at single-cell level in Leishmania, a protozoan parasite with a remarkable genome plasticity characterized by frequent gene copy number variations (CNVs) and high aneuploidy mosaicism. By combining the use of semi-permeable capsules with primary template-directed amplification, we could determine the karyotypes of hundreds of Leishmania parasites, detect distinct CNVs between different cell populations, and identify sub population of cells harboring distinct nucleotide variants, including in genes associated with drug resistance. This approach provides a powerful new framework to uncover hidden evolutionary potential in complex microbial populations, with application in studying adaptation, drug resistance, and genome evolution in Leishmania and other pathogens.

molecular biology↗

bioRxiv2022

The role of aneuploidy and polyclonality in the adaptation of the Protozoan parasite Leishmania to high drug pressure

Aneuploidy is generally considered harmful, but in some microorganisms, it can act as an adaptive mechanism against environmental stresses. Here, we used Leishmania - a protozoan parasite with a remarkable genome plasticity - to study the early evolution of aneuploidy under high drug pressure (antimony or miltefosine) as stressor model. By combining single-cell genomics, lineage tracing with cellular barcodes and longitudinal genome characterization, we revealed that antimony-induced aneuploidy changes result from the polyclonal selection of pre-existing karyotypes, complemented by further and rapid de novo alterations in chromosome copy number along 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. Thus, polyclonality and genome plasticity are hallmarks of parasite adaptation, but the scenario of aneuploidy dynamics is dependent on the nature and strength of the environmental stress as well as on the existence of other pre-adaptive mechanisms.

molecular biology↗

bioRxiv2021

High throughput single-cell genome sequencing gives insights into the generation andevolution of mosaic aneuploidy in Leishmania donovani

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 Leishmania 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 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. MA usually affected a defined subset of chromosomes, of which some display an enrichment in snoRNA genes which could represent an adaptative benefit to the amplification of these chromosomes. Our data provide the first complete characterization of MA in Leishmania and pave the way for further functional studies. Note to the BioRxiv communityThe present preprint is a revision of an older preprint posted on 06th March 2020 on BioRxiv (https://www.biorxiv.org/content/10.1101/2020.03.05.976233v1). Here we included two extra samples in our single-cell genome sequencing (SCGS) analysis - the BPK081 cl8 clone (a nearly euploid strain) and a population consisting of a mixture of four L. donovani strains which was used as control for high levels of mosaicism in aneuploidy and for estimation of doublets. We also upgraded the bioinformatics pipeline to determine single-cell karyotypes and performed new fluorescence in situ hybridization (FISH) analysis. The new findings observed especially in the BPK081 cl8 led to a reformulation of the text, a new hypothesis for the evolution of mosaicism and a general restructuring of the article. Therefore, the older preprint is obsolete.

genomics↗

bioRxiv2020

Exploring the evolution and adaptive role of mosaic aneuploidy in a clonal Leishmania donovani population using high throughput single-cell genome sequencing

Maintenance of stable ploidy over continuous mitotic events is a paradigm for most higher eukaryotes. Defects in chromosome segregation and/or replication can lead to aneuploidy, a condition often considered deleterious. However, in Leishmania, a Protozoan parasite, aneuploidy is a constitutive feature, where variations of somies represent a mechanism of gene expression adaptation, possibly impacting phenotypes. Strikingly, clonal Leishmania populations display cell-to-cell somy variation, a phenomenon named mosaic aneuploidy (MA). However, until recently, no method was available for the determination of the complete karyotype of single Leishmania parasites. To overcome this limitation, we used here for the first time a high-throughput single-cell genomic sequencing (SCGS) method to estimate individual karyotypes of 1560 promastigote cells in a clonal population of Leishmania donovani. We identified 128 different karyotypes, of which 4 were dominant. A network analysis revealed that most karyotypes are linked to each other by changes in copy number of a single chromosome and allowed us to propose a hypothesis of MA evolution. Moreover, aneuploidy patterns that were previously described by Bulk Genome Sequencing as emerging during first contact of promastigotes populations with different drugs are already pre-existing in single karyotypes in the SCGS data, suggesting a (pre-)adaptive role of MA. Additionally, the degree of somy variation was chromosome-specific. The SCGS also revealed a small fraction of cells where one or more chromosomes were nullisomic. Together, these results demonstrate the power of SCGS to resolve sub-clonal karyotype heterogeneity in Leishmania and pave the way for understanding the role of MA in these parasites adaptability. Update: 25th May 2021A revision of the present preprint was released in BioRxiv on 11th May 2021 (https://www.biorxiv.org/content/10.1101/2021.05.11.443577v2). In the new version, we included two extra samples in our single-cell genome sequencing (SCGS) analysis - the BPK081 cl8 clone (a nearly euploid strain), and a population consisting of a mixture of four L. donovani strains which was used as control for high levels of mosaicism in aneuploidy and for estimation of doublets. We also upgraded the bioinformatics pipeline to determine single-cell karyotypes and performed new fluorescence in situ hybridization (FISH) analysis. The new findings observed especially in the BPK081 cl8 led to a reformulation of the text, a new hypothesis for the evolution of mosaicism and a general restructuring of the article. Therefore, the present preprint is obsolete. Please refer to the new preprint entitled "High throughput single cell genome sequencing gives insights in the generation and evolution of mosaic aneuploidy in Leishmania donovani" for more information.

genomics↗