Biology subjects

Sterkers, Y.

Publications and source records attributed to Sterkers, Y..

7 recordsLinked to original sources

bioRxiv2026

The Nucleoporin MLP1 of the nuclear basket is essential for nuclear integrity and ploidy maintenance in Trypanosoma brucei

Trypanosoma brucei is a divergent eukaryote parasite responsible for neglected tropical diseases in human and animals, sleeping sickness or human African trypanosomiasis and nagana respectively. Besides scientific interest, understanding the specific features of its biology has medical and economical relevance. Nuclear pore complexes (NPCs) are large multiprotein channels embedded in the nuclear envelope that regulate nucleo-cytoplasmic transport. Beyond this role, NPCs also participate in essential nuclear processes such as chromosome segregation, transcription, and cytokinesis. Here, we showed that Myosin-like protein-1 (MLP1) localized to the nuclear basket of NPCs in T. brucei. Silencing of TbMLP1 by RNA interference in T. brucei procyclic cells caused marked defects in parasite growth, severe impairment of messenger RNA export, disorganization of nuclear structure, and pronounced genomic instability. Flow cytometry and FISH analyses revealed abnormal DNA content and a sharp reduction in disomic cells, accompanied by increased numbers of monosomic, trisomic, and polysomic cells, indicating an untolerated aneuploidy. These findings reveal that TbMLP1 links NPC function to multiple key cellular pathways. We provide new insights into the mechanisms that preserve nuclear architecture, nuclear envelope morphology, genome stability, and faithful chromosome segregation, including effects on kinetochore distribution and organization of the mitotic spindle. Author SummaryTrypanosoma brucei is the microscopic parasite responsible for sleeping sickness in humans and nagana in cattle; learning more about its basic biology may help identify weak points that could one day be targeted in new treatments. In this work, we studied a protein called MLP1, which sits at the nuclear pores--the gateways that control what enters and leaves the nucleus. Our findings revealed that MLP1 is essential for ensuring proper movement of messenger RNA, keeping the nucleus stable, and allowing chromosomes to be shared equally when cells divided. In total, we gained new insight into the basic biology of a parasite that remains a major health concern in many regions of the world, showing how a single structural protein can influence many vital processes within it.

microbiology↗

bioRxiv2025

A novel Leishmania infantum reference strain for gene editing and the study of visceral leishmaniasis

Parasites of the Leishmania donovani complex are responsible for visceral leishmaniasis, a vector-borne disease transmitted through the bite of female phlebotomine sand flies. As well as the human hosts, these parasites infect many mammals which can serve as reservoirs. Dogs are particularly important reservoirs in Europe. Transmission is widespread across Asia, Africa, the Americas, and the Mediterranean basin, including South of France. Visceral leishmaniasis poses a fatal threat if left untreated. Research into the pathophysiology of this neglected disease is of prime importance, as is the development of new drugs. In this study, we evaluated the growth, differentiation, and macrophage infectivity of four L. donovani complex strains and identified L. infantum S9F1 (MHOM/MA/67/ITMAP263, clone S9F1) as a well-adapted strain for genetic engineering studies. We present here the genome sequence and annotation of L infantum S9F1 T7 Cas9, providing the scientific community with easy access to its genomic information. The data has been integrated into the LeishGEdit online resource to support primer design for CRISPR-Cas9 experiments. We now aim to make this strain widely available to foster pathogenesis studies of visceral leishmaniasis. AUTHOR SUMMARYVisceral leishmaniasis is a disease caused by parasites of the Leishmania donovani complex. These parasites are spread to humans and animals through the bites of sand flies, and this disease affects millions of people worldwide, particularly in regions such as the Americas, Asia, Africa, and the Mediterranean basin. If left untreated, it can be fatal. Researchers need to study the biology of the parasite that causes the disease to better understand how it develops and progresses. In this study, we identified a L. infantum strain that is amenable to genetic modification in the laboratory and may serve as a representative model of the causative agent of visceral leishmaniasis. We tested two CRISPR-Cas9 strategies on this strain, re-sequenced and annotated its genome, and made the data available on the LeishGEdit website. By sharing this strain with the research community, we aim to support further studies on the pathogenesis of visceral leishmaniasis.

microbiology↗

bioRxiv2024

Stranded short nascent strand sequencing reveals the topology of eukaryotic DNA replication origins in Trypanosoma brucei.

The universal features that define genomic regions acting as replication origins remain unclear. In this study, we mapped a set of origins in Trypanosoma brucei using stranded short nascent strand sequencing method. Our results showed that DNA replication predominantly initiates in intergenic regions between poly(dA)- and poly(dT)-enriched sequences. G4 structures were detected in the vicinity of some origins and were embedded in poly(dA)-enriched sequences in a strand-specific manner: G4s on the plus strand were located upstream, while those on the minus strand were located downstream of the centre. The origins centres were found to be areas of low nucleosome occupancy, surrounded by regions of high nucleosome occupancy. Furthermore, our results demonstrate that 90% of replication origins overlap with a minor proportion of the previously reported RNA: DNA hybrids. These findings shed new light on the sequence and structural features that define the topology of replication origins in T. brucei. To further characterize replication dynamics at the single-molecule level, we employed DNA combing analysis.

genomics↗

bioRxiv2024

Apoptotic proteins in Leishmania donovani: In silico screening, modelling, and validation by knock-out and gene expression analysis

Visceral leishmaniasis (VL), a life-threatening vector-borne illness that disproportionately affects children and elderly immunocompromised people, is a primary tropical neglected disease. No apoptotic partner proteins in L. donovani have been reported yet, which might contribute to the knowledge of parasite cell death and the establishment of alternative therapeutics. We used the Orthologues algorithm to search for the mammalian Bcl-2 family proteins orthologs, one anti-apoptotic and two pro-apoptotic, in L. donovani. We also included a pro-death aquaporin (AQP) protein due to its characteristic BH3 domain, which is known to interact with pro-apoptotic proteins in mammals. Molecular docking and molecular dynamics simulation studies were conducted to assess the protein-protein interaction between the identified apoptotic proteins and mimic mammalian intrinsic apoptotic pathways. The results showed that the pro-apoptotic protein interacted with the hydrophobic pocket of the anti-apoptotic ortholog, forming a stable complex, which may represent a critical event in the apoptotic pathways of leishmaniasis. To further establish an apoptotic pathway in L. donovani, we used several CRISPR-Cas9 approaches to target the identified proteins. The pure knocked population mutants, and episomal over-expressing mutant cells were exposed to apoptotic stimuli. TUNEL assay and quantitative expression profiling suggested that these proteins are needed during the parasites apoptosis and could play a role in the parasites survival. Author SummaryVisceral leishmaniasis, a fatal systemic infection affecting internal organs, is one of three types of leishmaniasis in mammals alongside cutaneous and mucocutaneous leishmaniasis. It predominantly occurs in tropical and subtropical climatic zones, Leishmania donovani predominant in the Indian subcontinent and Leishmania infantum in the Mediterranean basin, the Middle East, Central Asia, South America, and Central America. This disease primarily affects children, immunocompromised adults, and the elderly. L donovani, transmitted by the infected sandflies complete its life cycle in humans, serving as reservoir. During its life cycle, at a particular stage, the parasite undergoes apoptotic-like events, yet underlying proteins or key factors remain unidentified. Using computational methods, we screened the L. donovani genome for potential candidate genes of the Bcl-2 family apoptotic proteins. We biologically/experimentally validated our in-silico findings using molecular editing tools like CRISPR-Cas9, advancing our understanding of the parasites apoptotic pathway. Targeting this pathway could lead to more effective therapeutics against visceral leishmaniasis.

microbiology↗

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↗

bioRxiv2019

Original research. Inhibition of Polymerase Chain Reaction: Pathogen-Specific Controls are better than Human Gene Amplification

PCR inhibition is frequent in medical microbiology routine practice and may lead to false-negative results; however there is no consensus on how to detect it. Pathogen-specific and human gene amplifications are widely used to detect PCR inhibition. We aimed at comparing the value of PCR inhibitor detection using these two methods. We analysed Cp shifts ({Delta}Cp) obtained from qPCRs targeting either the albumin gene or the pathogen-specific sequence used in two laboratory-developed microbiological qPCR assays. 3152 samples including various matrixes were included. Pathogen-specific amplification and albumin qPCR identified 62/3152 samples (2.0 %), and 409/3152 (13.0%) samples, respectively, as inhibited. Only 16 samples were detected using both methods. In addition, the use of the Youdens index failed to determine adequate Cp thresholds for albumin qPCR, even when we distinguished among the different sample matrixes. qPCR targeting the albumin gene therefore appears not adequate to identify the presence of PCR inhibitors in microbiological PCR assays. Our data may be extrapolated to other heterologous targets and should discourage their use to assess the presence of PCR inhibition in microbiological PCR assays.

microbiology↗