Analyse fonctionnelle des nucléoporines dans le maintien de la ploïdie chez les trypanosomatidés

Trypanosoma brucei et Leishmania sp sont des parasites eucaryotes divergents appartenant à l’ordre des kinetoplastidae. Outre l’intérêt scientifique, la compréhension de leur biologie présente un intérêt médical et économique. Ces parasites proches d’un point de vue phylogénétique présentent des caractéristiques moléculaires et cellulaires originales communes qui restent mal connues, telles que l'organisation génomique en grandes unités transcriptionnelles polycistroniques, l'absence de régulation transcriptionnelle et les modifications post-transcriptionnelles de l'ARNm. Parmi les caractéristiques spécifiques de ces parasites, nous nous sommes intéressés au comportement des pores nucléaires et à leur importance, en particulier pendant la mitose. Les trypanosomatidés présentent une mitose dite fermée durant laquelle l'enveloppe nucléaire reste intacte. Les complexes de pores nucléaires (CPN) sont de grands canaux multiprotéiques intégrés dans l'enveloppe nucléaire et constitués de nucléoporines (NUP). La fonction principale des CPN est de réguler le transport nucléo-cytoplasmique. Cependant, les NUP apparaissent également comme d'importants acteurs dans d'autres processus fondamentaux, notamment dans la ségrégation chromosomique, la transcription et la cytokinèse. En utilisant la technique du CRISPR-Cas9, nous avons marqué la nucléoporine MLP1 et montré qu’elle était située dans le panier nucléaire des CPN chez T. brucei et L. mexicana. Le knockdown par ARN interférence de MLP1 chez les formes procycliques de T. brucei entraîne un défaut de croissance, une désorganisation nucléaire et une grave instabilité génomique. L'analyse par FISH et par cytométrie en flux a montré une forte réduction des cellules disomiques qui a été compensée par une augmentation des cellules monosomiques et trisomiques. Ainsi, le knockdown de TbMLP1 est associé à une aneuploïdie mosaïque mal tolérée chez T. brucei. La voie ARNi étant absente chez Leishmania, nous avons dû mettre au point et utiliser un système CRISPR-Cas9 inductible pour étudier LmxM_MLP1. Le knockout inductible de MLP1 a entrainé une forte réduction de la croissance, une réduction importante du pourcentage de cellules diploïdes et un défaut dans la progression du cycle cellulaire associé à une perturbation générale de l'architecture nucléaire. Le marquage de protéines du kinétochore et du fuseau mitotique a montré l’importance de MLP1 dans le processus de ségrégation des chromosomes. La colocalisation avec d'autres nucléoporines a permis d’observer l’architecture de l'enveloppe nucléaireAinsi, nos résultats indiquent que MLP1 est une protéine essentielle, impliquée dans le maintien de l’intégrité de la ploïdie et de l'organisation nucléaire chez T. brucei et L. mexicana. De plus, le séquençage des brins naissants a montré son implication potentielle, à confirmer, dans la réplication de l'ADN.Trypanosoma brucei and Leishmania are divergent eukaryotes belonging to the kinetoplastidae. Understanding their biology, besides its scientific interest, has medical and economical relevance. These 'sister' parasites exhibit common original molecular and cellular features which remain poorly known such as the genomic organization into large polycistronic transcriptional units, the absence of transcriptional regulation and the messenger RNA (mRNA) processing. Among the specific features of these parasites we got interested into the nuclear pore behaviors and importance particularly during mitosis. Trypanosomatids display a closed mitosis where the nuclear envelop remains intact. Nuclear pore complexes (NPCs) are large multiprotein channels embedded in the nuclear envelope (NE) and made of nucleoporins (NUPs). The primary function of NPCs is to regulate nucleo-cytoplasmic transport; however, NUPs are also emerging as important actors in other core processes including chromosomal segregation, transcription, and cytokinesis. Here, using CRISPR-Cas9, we showed that the GFP-tagged nucleoporin MLP1 is located in the nuclear basket of the NPCs in T. brucei and Leishmania. TbMLP1 RNA interference knockdown in T. brucei PCFs led to a growth defect, nuclear disorganization and severe genomic instability. FISH and flow cytometry analysis demonstrated a large reduction in disomic cells, which was compensated by an increase in monosomic and trisomic cells, unsurprisingly undetectable by bulk WGS. Thus, TbMLP1 knockdown is associated with an un-tolerated mosaic aneuploidy in T. brucei. The RNA interference (RNAi) pathway being absent in Leishmania, we successfully developed an inducible CRISPR-Cas9 system to study essential genes in L. mexicana, among which LmxM_MLP1. The inducible knockout (iKO) of MLP1 led to a severe growth defect, a reduction in diploid cells and a defect in cell cycle progression associated with a general disturbance of the nuclear architecture. Taken together, our results indicate that MLP1 is required for the maintenance of genome and nuclear organization in T. brucei and L. mexicana. With a potential implication in: DNA replication showed using Small Nascent Strand sequencing (SNS-seq), nuclear envelop morphology using colocalization analysis of other nucleoporins and in segregation process using mitotic spindle and kinetoplastid kinetochore (KKT) proteins distribution. Our data already provide novel insights into the molecular mechanisms regulating genome stability and its interesting connection to the NPCs in the Trypanosomatids

Gene Editing in Trypanosomatids: Tips and Tricks in the CRISPR-Cas9 Era

International audienceGene editing in trypanosomatids has long been proven difficult. The development of CRISPR-Cas9 has improved this issue, opening the way to a better understanding of biological processes and drug-resistance mechanisms, and screening of drug targets. Different strategies have now been developed: either PCR- or plasmid-based, differing mainly in the nature of the donor DNA and the single guide RNA transcription. Here we review the main genetic tools available for Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei for gene tagging, single-base editing, and deletion of nonessential and essential genes. We discuss the main advantages and challenges of different strategies and how to choose 'the right cut' depending on the importance of untranslated regions. These considerations allow selection of the most accurate gene editing approach for a given functional analysis

Universal highly efficient conditional knockout system in Leishmania , with a focus on untranscribed region preservation

International audienceTrypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features that remain poorly understood; particularly, Leishmania is known for its high degree of genomic plasticity that makes genomic manipulation challenging. CRISPR-Cas9 has been applied successfully to these parasites providing a robust tool to study non-essential gene functions. Here, we have developed a versatile inducible system combining Di-Cre recombinase and CRISPR-Cas9 advantages. Cas9 is used to integrate the LoxP sequences, and the Cre-recombinase catalyses the recombination between LoxP sites, thereby excising the target gene. We used a Leishmania mexicana cell line expressing Di-Cre, Cas9, and T7 polymerase and then transfected donor DNAs and single guide RNAs as polymerase chain reaction (PCR) products. Because the location of LoxP sequences in the genomic DNA can interfere with the function and localisation of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed "universal" template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR-based (molecular cloning-free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania

Genetic characterisation of carbapenem-resistant Gram-negative bacteria isolated from the University Hospital Mohamed Boudiaf in Ouargla, southern Algeria

International audienceObjectives: The aim of this study was to investigate the molecular epidemiology and the genetic support of carbapenem-resistant Enterobacteriaceae and Acinetobacter spp. isolates collected in the University Hospital of Ouargla, southern Algeria. Methods: A total of 99 Gram-negative bacteria (GNB) were collected from stool samples of colonised patients and from inanimate surfaces in the hospital environment between December 2014 and August 2015. Selected Enterobacteriaceae and Acinetobacter spp. isolates with reduced susceptibility to carbapenems were subjected to phenotypic study, including antibiotic susceptibility testing according to CA-SFM-EUCAST 2015 guidelines and modified Carba NP test. Genes encoding carbapenemases, extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases were screened by PCR and sequencing. Clonal relatedness was determined by multilocus sequencing typing (MLST). Results: Of the 99 GNB isolates, 10 (10.1%) showed reduced susceptibility to carbapenems were studied further, including 7 Acinetobacter baumannii, 1 Acinetobacter nosocomialis, 1 Escherichia coli and 1 Klebsiella pneumoniae. PCR and sequencing showed that four A. baumannii isolates and the single A. nosocomialis isolate harboured blaNDM-1. In addition, blaOXA-23 was observed in three A. baumannii isolates, and blaOXA-48 was detected in the two Enterobacteriaceae isolates. MLST assigned the K. pneumoniae to ST999 and the E. coli to ST38. The seven A. baumannii isolates belonged to ST85 and ST2. Conclusions: This study describes the epidemiology of carbapenemases produced by Enterobacteriaceae and Acinetobacter spp. in southern Algeria and reports the first description of metallo-beta-lactamase NDM1-producing A. nosocomialis in Algeria. (C) 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved

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

International audienceAbstract 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 clonal populations of Leishmania promastigotes 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, affecting a defined subset of chromosomes. Our data provide the first complete characterization of MA in Leishmania and pave the way for further functional studies