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Monsieurs, P.

Publications and source records attributed to Monsieurs, P..

13 recordsLinked to original sources
bioRxiv2026

Trypanosoma congolense Variant Surface Glycoprotein gene expression occurs in the absence of monoallelic transcription control

Antigenic variation is a very widespread process for pathogen evasion of mammalian adaptive immunity, involving the continuous change of exposed antigens. In many single-celled pathogens, antigen expression during antigenic variation is monoallelic: just a single gene from a large family is expressed in one cell at a time. In African trypanosomes, antigenic variation relies on expression of Variant Surface Glycoprotein (VSG), and in Trypanosoma brucei there is detailed understanding of the machinery that dictates that only one of approximately 15 VSG expression sites is actively transcribed at a time. In the closely related African trypanosome, T. congolense, which remains a significant blight on agriculture productivity in sub-Sharan Africa, we have no such understanding of VSG gene expression control or dynamics. Here, we have investigated the mechanics of antigenic variation in T. congolense, first examining the patterns of VSG expression at the transcript and protein level in small parasite populations in vitro. Surprisingly, this analysis revealed much greater VSG diversity than seen in T. brucei, with such expression diversity unaltered in mutants that impair homologous recombination. Using single cell transcriptomics, we explain this diversity, since we find no evidence for monoallelic transcription of T. congolense VSGs, but show instead that each parasite can dynamically express up to [~]40 different VSGs in a single cell both in vitro and in vivo. VSG co-expression occurs from VSG genes distributed across the genome, indicating the lack of a dedicated locus for VSG transcription. Thus, comparing two trypanosome species that rely on the same class of surface antigen for immune evasion has revealed highly distinct mechanisms for controlling antigen gene expression, challenging the assumed common operation of antigenic variation across African trypanosome species.

microbiology↗
bioRxiv2026

A Plasmodium knowlesi A1-H.1 transcriptome time course focusing on the late asexual blood stages

BackgroundThe zoonotic parasite Plasmodium knowlesi is closely related to Plasmodium vivax, the second leading cause of human malaria. P. knowlesi line A1-H.1 can be maintained in human erythrocytes and is an experimental model for P. vivax biology. We present the first transcriptome time-course of the intraerythrocytic developmental cycle (IDC) of P. knowlesi A1-H.1 grown in human normocytes. ResultsBulk RNA-sequencing was performed at five IDC stages using tightly synchronised cultures, enabling identification of constitutively expressed genes and stage-specific biomarkers, and investigation of temporal expression patterns of multigene families. Comparative analysis with P. vivax orthologues revealed strong genome-wide conservation of temporal expression. Analysis of invasion-associated genes and ApiAP2 transcription factors identified genes with conserved expression patterns for which P. knowlesi is likely a good model to study the P. vivax gene. To support comparative analyses, we developed an open-access interactive webtool to explore and visualise P. knowlesi - P. vivax orthologue expression across the IDC. ConclusionsThis time-course dataset provides a reference transcriptome framework for P. knowlesi A1-H.1 and a resource for comparative Plasmodium biology. The interactive webtool facilitates rapid identification of candidate genes with conserved temporal expression, streamlining functional studies in which P. knowlesi A1-H.1 serves as a model for P. vivax.

genomics↗
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↗
bioRxiv2025

Direct genome sequencing of Leishmania tropica in tissues of Moroccan patients with cutaneous leishmaniasis reveals micro-focal transmission underlain by clonal and sexual reproduction modes

Leishmania tropica is causing cutaneous leishmaniasis (CL) from North Africa to India and in Ethiopia and is reported to be transmitted from humans to humans through sand fly bites. While this species is characterized by a high genetic diversity all over the area of endemicity, there is very little information on diversity at micro-epidemiological scale. Here, we zoomed on an epidemic Moroccan focus of CL - restricted in space and time - and studied transmission patterns by comparative genomics of parasites in human patients. We used a culture-independent method of genome sequencing based on Leishmania genome capture (SureSelect-sequencing, SuSL-seq), applied directly on dermal scrapings. We first compared the genome of paired samples: i.e. parasites in host tissues analyzed by SuSL-seq and derived isolates shortly maintained in culture and analyzed by whole genome sequencing (WGS). Despite the low passage number of isolates, significant differences were observed between karyotypes of 4/7 paired samples, highlighting the clinical and epidemiological relevance of direct genome sequencing. Secondly, we identified 7 groups of nearly identical genotypes, characteristic of clonal propagation as well as parasites with mixed ancestry, a signature of genetic exchange. Our results reveal a micro-focal transmission among humans, underlain by clonal and sexual reproductive modes. This study demonstrates the power of direct genome sequencing for evolutionary genetics at a micro-epidemiological scale.

genomics↗
bioRxiv2025

Multi-drug tolerance in Leishmania persister-like cells

Leishmaniasis is caused by parasitic protozoa of the genus Leishmania and is found widely across the tropics and sub-tropics, afflicting hundreds of thousands of people. The disease is notoriously difficult to treat. Here, we present evidence of the existence of persister-like cells in cultured Leishmania populations, induced upon exposure to normally lethal doses of antimony, a widely used anti-leishmanial. Persisters are a small fraction of non-proliferative cells with reduced metabolism that are adapted to withstand a variety of environmental assaults, including lethal doses of antimicrobials. We show that Leishmania persister-like cells survive lethal doses of antimonials by adopting a quiescence phenotype characterised by reduced proliferation, constrained metabolism, and diminished mitochondrial membrane potential. What is more, these cells demonstrate cross-tolerance to other anti-leishmanial drugs. Wild-type persister-like cells reverted to similar levels of drug susceptibility once the antimony-induced pressure was removed. Surprisingly, cells which had previously been selected for genetic changes causing resistance to antimony acquired a level of hyper-resistance after transient passage through the quiescent state, without further genetic change. Our results demonstrate the extreme versatility of this eukaryotic pathogen in adaptation to drug pressure and highlight the need for the development of new anti-leishmanials targeting non-proliferative forms.

microbiology↗
bioRxiv2024

Genomic characterization of a unique population of Leishmania tropica in a new emerging focus of cutaneous leishmaniasis in Somali region of Ethiopia

We sequenced Leishmania tropica genomes directly from eight human skin samples collected in a newly emerging focus of cutaneous leishmaniasis in Somali region, Ethiopia. We found a new variant with unique genomic signatures of drug resistance. Genomic homogeneity in the sampled population demonstrates the occurrence of a L. tropica outbreak.

microbiology↗
bioRxiv2024

Novel single-cell preservation and RNA sequencing technology unlocks field studies for Plasmodium natural infections

Single-cell RNA sequencing (scRNA-seq) is a powerful technology used to investigate cellular heterogeneity. When applied to unicellular eukaryotes such as Plasmodium parasites, scRNA-seq provides a single-cell resolution particularly valuable to study complex infections which are often comprised of mixed life stages and clones. Until now, the application of scRNA-seq has been mainly limited to in vitro and animal malaria models, despite known transcriptional differences as compared to circulating parasite populations. This is primarily due to the challenges of working with Plasmodium natural infections in endemic settings. We validated sample preparation methods and a novel single-cell RNA sequencing technology for the first time in P. knowlesi parasites which can be effectively implemented to analyze natural infections in low-resource settings. We recovered 22,345 P. knowlesi single-cell transcriptomes containing all asexual blood stages from 6 in vitro culture samples, with conditions mimicking natural infections, and generated the most extensive P. knowlesi single-cell dataset to date. All 6 samples produced reproducible circular UMAP projections with consistent cluster localization and high gene expression correlation, regardless of the sample preparation methods used. Biomarker expression and life stage annotation using the Malaria Cell Atlas P. knowlesi reference dataset further confirmed these results. In conclusion, the combination of adaptable sample preparation methods with novel preservation and scRNA-seq technology has the potential to fundamentally transform the study of natural infections. This approach unlocks the use of scRNA-seq in field studies which will lead to new insights into Plasmodium parasite biology. ImportanceSequencing unicellular organisms, such as malaria parasites, at the single-cell level is important to understand the diversity present in cell populations. Until now, single-cell sequencing of malaria has been primarily limited to laboratory models. While these models are key to understanding biological processes, there are known differences between lab models and parasite populations circulating in natural human infections. This study presents sample preparation methods and a new single-cell RNA sequencing technology that enables sample collection from natural infections in low-resource settings. Using a mock natural infection, we validated this new single-cell RNA sequencing technology using marker genes with known expression patterns and a reference dataset from the Malaria Cell Atlas. We demonstrate that high-quality single-cell transcriptomes with consistent expression patterns can be recovered using various sample preparation methods, thereby unlocking single-cell sequencing for field studies and leading to additional insights into parasite biology in the future.

microbiology↗
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↗
bioRxiv2021

Genomic and phenotypic characterization of experimentally selected resistant Leishmania donovani reveals a role for dynamin-1 like protein in the mechanism of resistance to a novel anti-leishmanial compound

The implementation of prospective drug resistance (DR) studies in the R&D pipelines is a common practice for many infectious diseases, but not for Neglected Tropical Diseases. Here, we explored and demonstrated the importance of this approach, using as paradigms Leishmania donovani, the etiological agent of Visceral Leishmaniasis (VL), and TCMDC-143345, a promising compound of the GSK Leishbox to treat VL. We experimentally selected resistance to TCMDC-143345 in vitro and characterized resistant parasites at genomic and phenotypic levels. We found that it took more time to develop resistance to TCMDC-143345 than to other drugs in clinical use and that there was no cross resistance to these drugs, suggesting a new and unique mechanism. By whole genome sequencing, we found two mutations in the gene encoding the L. donovani dynamin-1-like protein (LdoDLP1) that were fixed at highest drug pressure. Through phylogenetic analysis, we identified LdoDLP1 as a family member of the dynamin-related proteins, a group of proteins that impacts the shapes of biological membranes by mediating fusion and fission events, with a putative role in mitochondrial fission. We found that L. donovani lines genetically engineered to harbor the two identified LdoDLP1 mutations were resistant to TCMDC-143345 and displayed altered mitochondrial properties. By homology modeling, we showed how the two LdoDLP1 mutations may influence protein structure and function. Taken together, our data reveal a clear involvement of LdoDLP1 in the adaptation/resistance of L. donovani to TCMDC-143345. ImportanceHumans and their pathogens are continuously locked in a molecular arms race during which the eventual emergence of pathogen drug resistance (DR) seems inevitable. For neglected tropical diseases (NTDs), DR is generally studied retrospectively, once it has already been established in clinical settings. We previously recommended to keep one step ahead in the host-pathogen arms race and implement prospective DR studies in the R&D pipeline, a common practice for many infectious diseases, but not for NTDs. Here, using Leishmania donovani, the etiological agent of Visceral Leishmaniasis (VL), and TCMDC-143345, a promising compound of the GSK Leishbox to treat VL, as paradigms, we experimentally selected resistance to the compound and proceeded to genomic and phenotypic characterization of DR parasites. The results gathered in the present study suggest a new DR mechanism involving the L. donovani dynamin-1 like protein (LdoDLP1) and demonstrate the practical relevance of prospective DR studies.

microbiology↗
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↗
bioRxiv2020

Evaluation of whole genome amplification and bioinformatic methods for the characterization of Leishmania genomes at a single cell level

Here, we report a pilot study paving the way for further single cell genomics studies in Leishmania. First, the performances of two commercially available kits for Whole Genome Amplification (WGA), PicoPlex and RepliG was compared on small amounts of Leishmania donovani DNA, testing their ability to preserve specific genetic variations, including aneuploidy levels and SNPs. We show here that the choice of WGA method should be determined by the planned downstream genetic analysis, Picoplex and RepliG performing better for aneuploidy and SNP calling, respectively. This comparison allowed us to evaluate and optimize corresponding bio-informatic methods. As PicoPlex was shown to be the preferred method for studying single cell aneuploidy, this method was applied in a second step, on single cells of L. braziliensis, which were sorted by fluorescence activated cell sorting (FACS). Even sequencing depth was achieved in 28 single cells, allowing accurate somy estimation. A dominant karyotype with three aneuploid chromosomes was observed in 25 cells, while two different minor karyotypes were observed in the other cells. Our method thus allowed the detection of aneuploidy mosaicism, and provides a solid basis which can be further refined to concur with higher-throughput single cell genomic methods.

microbiology↗