Caractérisation structurale et régulation de l'activité de deux Polo-like kinases de Schistosoma mansoni (SmPlk1 et SmSak)

La schistosomiase est l'infection helminthique humaine la plus importante en termes de morbidité et de mortalité dans de nombreux pays. L'émergence récente de cas de résistances à la seule drogue efficace contre le schistosome, le praziquantel (PZQ) fait de cette maladie une priorité pour l'Organisation Mondiale de la Santé (OMS) de rechercher de nouvelles cibles thérapeutiques. La ponte massive des oeufs (jusqu'à 300 oeufs par jour par femelle) est responsable de la transmission de la maladie par des parasites complexes trématodes mais aussi de toute la pathologie de la schistosomiase. Mon travail de Doctorat vise avant tout à déchiffrer les mécanismes complexes de la reproduction des schistosomes et à élucider les voies de signalisation qui régulent cette hyperfécondité, dont les acteurs moléculaires peuvent être des cibles évidentes pour le contrôle de la schistosomiase. Les kinases Polo sont des membres conservés de la famille des Polo-like kinases (Plks) qui sont impliquées dans la progression du cycle cellulaire. Actuellement, les Plks humaines constituent une cible importante dans les traitements anticancéreux de par leur expression aberrante dans les tumeurs et signent d ailleurs un mauvais pronostic. Sur la base de recherches in silico, nous avons caractérisé SmPlk1 (Schistosoma mansoni Plk1), homologue à la Plk1 humaine. SmPlk1 était principalement transcrit dans des stades de multiplication cellulaire intense et préférentiellement dans les organes reproducteurs des schistosomes adultes suggérant un rôle potentiel de SmPlk1 dans les processus de division. Nous avons montré que SmPlk1 possédait un rôle conservé dans la transition G2/M dans le modèle d ovocyte de Xénope. L inhibition dose-dépendante de l'ovogénèse et la spermatogénèse par le BI 2536, un inhibiteur spécifique des Plks, indiquait un rôle de SmPlk1 dans la multiplication et la différenciation des gamètes chez le parasite et suggérait que cette kinase pourrait être une nouvelle cible dans le traitement anti-schistosome. Parallèlement à ces travaux, une seconde Plk, SmSak, a été identifiée dont les résultats préliminaires ont montré une structure et des fonctions différentes de celles de SmPlk1 suggérant un rôle distinct de cette dernière. De plus, nous avons identifié un activateur potentiel de Plk, SmSLK (S. mansoni Ste20-like kinase) capable d'activer spécifiquement SmPlk1 dans des conditions d'activation particulières dépendant de deux mécanismes originaux, l'un dépendant des caspases et l'autre dépendant d'ARN anti-sens.Schistosomiasis is the most important helminthic infection in term of morbidity and mortality in many developing countries and represents the second parasitic disease to malaria. The evidence for praziquantel (PZQ) resistance, the only drug currently used to treat the disease, led the World Health Organization (WHO) to consider as a priority the finding of novel therapeutic targets. Egg production is responsible for disease transmission by complex trematodes parasites but also for the pathology of schistosomiasis. My PhD work contributes to a better understanding of the complex mechanisms that regulate schistosome reproduction. One particularity of schistosomes is that the sexual development of females requires a permanent contact with the male, allowing a level of fecundity exceptionally high. Therefore, the molecular mechanisms implied in this hyperfecundity are obvious targets for the control of schistosomiasis. Polo kinases are serine/threonine kinases, belonging to the Polo-like kinase family (Plks) whose members are conserved from yeast to mammals. During last years, human Plks have been extensively studied and considered as major targets for cancer because of their dramatic overexpression in proliferating cells and many tumors. In silico researches have led us to the characterization in S. mansoni, SmPlk1, homologous to human Plk1. SmPlk1 was abundantly transcribed in parasite stages containing germinal cells expected to undergo frequent cell divisions, and particularly in the reproductive organs of adult worms suggesting a potential role of SmPlk1 in division processes. We have shown that SmPlk1 induced resumption of meiosis in oocytes of Xenopus. Moreover, the specific Plk1- inhibitor BI 2536 used in clinical trials, induces morphological aberrations in reproductive organs and inhibits oogenesis and spermatogenesis in paired worms, indicating a role of SmPlk1 in gamete multiplication and differentiation in S. mansoni parasites and so the possibility that this kinase could be a novel potential target for schistosomiasis treatment. In parallel to this work, we recently identified a second Plk in S. mansoni, SmSak different for its structure and its functions, and notably its role in the centriole duplication. Moreover, we identified a potential activator of Plk, SmSLK (S. mansoni Ste-20 like kinase) able to activate specifically SmPlk1 in particular conditions. Indeed, two original mechanisms, one dependent on caspases and the other one dependent on antisense RNA, could regulate the kinase activity of SmSLK and therefore, the activity of SmPlk1.LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Schistosoma mansoni polo-like kinases and their function in control of mitosis and parasite reproduction

Polo-like kinases are important regulators of cell cycle progression and mitosis. They constitute a family of conserved serine/threonine kinases which are highly related in their catalytic domains and contain polo boxes involved in protein-protein interactions and subcellular localization. In mammals, five Plks (Plk 1-5) encompass diverse roles in centrosome dynamics, spindle formation, intra S-phase and G2/M checkpoints and DNA damage response. Plk1 is a key positive regulator of mitosis and is overexpressed in various types of cancers. Plk4 is a divergent member of the Plk family, with essential functions in centriole duplication. Homozygous disruption of Plk1 or Plk4 in mice is lethal in embryos. Two Plk members SmPlk1 and SmSak, homologous to Plk1 and Plk4 respectively, are present in the parasitic platyhelminth Schistosoma mansoni. Structural and functional analyses of SmPlk1 have demonstrated its conserved function in the regulation of cell cycle G2/M transition in Xenopus oocytes. The anti-cancer drug BI 2536 (the most potent and selective Plk1 inhibitor) inhibits specifically the catalytic activity of SmPlk1 and induced profound alterations in schistosome gonads, indicating a role of SmPlk1 in parasite gametogenesis and its potential as a novel chemotherapeutic target against schistosomiasis. Functions of SmSak in cell cycle regulation and schistosome gonad development are currently investigate

Droplet digital PCR as a tool to detect resistant isolates of Dirofilaria immitis

Prevention of canine heartworm disease, caused by Dirofilaria immitis, relies on macrocyclic lactones for which drug resistance is now a concern. Although genetic polymorphisms have been associated with resistance in D. immitis populations, the mechanism is still not well understood. The lack of reliable in vitro assays to detect resistance is a limitation for confirming resistance. Ten single nucleotide polymorphisms (SNPs) were previously clinically validated in D. immitis resistant isolates, using the MiSeq platform. This technique although useful for research studies is expensive and does not facilitate rapid detection of these markers in small numbers of clinical samples. We developed a droplet digital PCR protocol for detecting SNPs correlating with ML resistance. Specific primers and hydrolysis probes encompassing the wildtype and mutant alleles were designed to amplify the SNP targets from genomic DNA of different D. immitis isolates. Allele frequencies were determined and the suitability of the ddPCR assay was assessed and compared with MiSeq data. The ddPCR assay accurately detected and quantified alternate nucleotides in two isolates of reference, the ML-susceptible Missouri (MO) and ML-resistant JYD-34, at the previously identified SNP positions. The presence of the SNPs was also determined in additional isolates with known or putative susceptible or resistant phenotypes. We observed SNP1 and SNP2 are more predictive markers and appear suitable for rapid detection and monitoring of drug resistance. Our results suggested that ddPCR could be employed to distinguish infection due to actual genetic resistance from infection with susceptible parasites and also for rapid detection of isolates not only with ML susceptible and resistant genotypes but also mixed genotypes that correspond to heterogeneous isolates containing a mixed population of ML susceptible and resistant parasites. DdPCR may be a useful tool for conducting surveys, or assessments of individual isolates, for genetic evidence of resistance or developing resistance