Evidence for Integrin – Venus Kinase Receptor 1 Alliance in the Ovary of Schistosoma mansoni Females Controlling Cell Survival

Parasites of the genus Schistosoma cause schistosomiasis, a life-threatening infectious disease for humans and animals worldwide. Among the remarkable biological features of schistosomes is the differentiation of the female gonads which is controlled by pairing with the male and a prerequisite for egg production. Eggs, however, are not only important for the maintenance of the life-cycle; they also cause the pathological consequences of schistosomiasis. Part of the eggs gets trapped in host tissues such as liver and spleen and trigger inflammatory processes, finally leading to liver cirrhosis. Research activities of the last decade have indicated that different families of cellular and receptor-type kinases but also integrins contribute to the control of mitogenic activity and differentiation the female goands. In this context an unusual class of receptor tyrosine kinases (RTKs) has been identified, the venus kinase receptors (SmVKRs). By biochemical and molecular approaches we demonstrate that SmVKR1 activation can be achieved by cooperation with a signaling complex consisting of the beta integrin receptor Smß-Int1 and the bridging molecules SmILK, SmPINCH, SmNck2. Besides unravelling a novel way of SmVKR1 activation, we provide evidence that this complex controls the differentiation status of oocytes by regulating cell death-associated processes

A New Family of Receptor Tyrosine Kinases with a Venus Flytrap Binding Domain in Insects and Other Invertebrates Activated by Aminoacids

Background: Tyrosine kinase receptors (RTKs) comprise a large family of membrane receptors that regulate various cellular processes in cell biology of diverse organisms. We previously described an atypical RTK in the platyhelminth parasite Schistosoma mansoni, composed of an extracellular Venus flytrap module (VFT) linked through a single transmembrane domain to an intracellular tyrosine kinase domain similar to that of the insulin receptor. Methods and Findings: Here we show that this receptor is a member of a new family of RTKs found in invertebrates, and particularly in insects. Sixteen new members of this family, named Venus Kinase Receptor (VKR), were identified in many insects. Structural and phylogenetic studies performed on VFT and TK domains showed that VKR sequences formed monophyletic groups, the VFT group being close to that of GABA receptors and the TK one being close to that of insulin receptors. We show that a recombinant VKR is able to autophosphorylate on tyrosine residues, and report that it can be activated by L-arginine. This is in agreement with the high degree of conservation of the alpha amino acid binding residues found in many amino acid binding VFTs. The presence of high levels of vkr transcripts in larval forms and in female gonads indicates a putative function of VKR in reproduction and/or development. Conclusion: The identification of RTKs specific for parasites and insect vectors raises new perspectives for the control of human parasitic and infectious diseases

Transcriptome analyses of inhibitor-treated Schistosome females provide evidence for cooperating Src-kinase and TGFbeta receptor pathways controlling mitosis and egshell formation

Schistosome parasites cause schistosomiasis, one of the most prevalent parasitemias worldwide affecting humans and animals. Constant pairing of schistosomes is essential for female sexual maturation and egg production, which causes pathogenesis. Female maturation involves signaling pathways controlling mitosis and differentiation within the gonads. In vitro studies had shown before that a Src-specific inhibitor, Herbimycin A (Herb A), and a TGFb receptor (TbR) inhibitor TRIKI) have physiological effects such as suppressed mitoses and egg production in paired females. As one Herb A target, the gonad-specifically expressed Src kinase SmTK3 was identified. Here, we comparatively analyzed the transcriptome profiles of Herb A- and TRIKI-treated females identifying transcriptional targets of Src-kinase and TbRI pathways. After demonstrating that TRIKI inhibits the schistosome TGFbreceptor SmTbRI by kinase assays in Xenopus oocytes, couples were treated with Herb A, TRIKI, or both inhibitors simultaneously in vitro. RNA was isolated from females for microarray hybridizations and transcription analyses. The obtained data were evaluated by Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA), but also by manual classification and intersection analyses. Finally, extensive qPCR experiments were done to verify differential transcription of candidate genes under inhibitor influence but also to functionally reinforce specific physiological effects. A number of genes found to be differentially regulated are associated with mitosis and differentiation. Among these were calcium-associated genes and eggshell-forming genes. In situ hybridization confirmed transcription of genes coding for the calcium sensor hippocalcin, the calcium transporter ORAI-1, and the calcium-binding protein calmodulin-4 in the reproductive system pointing to a role of calcium in parasite reproduction. Functional qPCR results confirmed an inhibitor-influenced, varying dependence of the transcriptional activities of Smp14, Smp48, fs800, a predicted eggshell precursor protein and SmTYR1. The results show that eggshell-formation is regulated by at least two pathways cooperatively operating in a balanced manner to control egg production

The Formin-Homology Protein SmDia Interacts with the Src Kinase SmTK and the GTPase SmRho1 in the Gonads of Schistosoma mansoni

BACKGROUND:Schistosomiasis (bilharzia) is a parasitic disease of worldwide significance affecting human and animals. As schistosome eggs are responsible for pathogenesis, the understanding of processes controlling gonad development might open new perspectives for intervention. The Src-like tyrosine-kinase SmTK3 of Schistosoma mansoni is expressed in the gonads, and its pharmacological inhibition reduces mitogenic activity and egg production in paired females in vitro. Since Src kinases are important signal transduction proteins it is of interest to unravel the signaling cascades SmTK3 is involved in to understand its cellular role in the gonads. METHODOLOGY AND RESULTS:Towards this end we established and screened a yeast two-hybrid (Y2H) cDNA library of adult S. mansoni with a bait construct encoding the SH3 (src homology) domain and unique site of SmTK3. Among the binding partners found was a diaphanous homolog (SmDia), which was characterized further. SmDia is a single-copy gene transcribed throughout development with a bias towards male transcription. Its deduced amino acid sequence reveals all diaphanous-characteristic functional domains. Binding studies with truncated SmDia clones identified SmTK3 interaction sites demonstrating that maximal binding efficiency depends on the N-terminal part of the FH1 (formin homology) domain and the inter-domain region of SmDia located upstream of FH1 in combination with the unique site and the SH3 domain of SmTK3, respectively. SmDia also directly interacted with the GTPase SmRho1 of S. mansoni. In situ hybridization experiments finally demonstrated that SmDia, SmRho1, and SmTK3 are transcribed in the gonads of both genders. CONCLUSION:These data provide first evidence for the existence of two cooperating pathways involving Rho and Src that bridge at SmDia probably organizing cytoskeletal events in the reproductive organs of a parasite, and beyond that in gonads of eukaryotes. Furthermore, the FH1 and inter domain region of SmDia have been discovered as binding sites for the SH3 and unique site domains of SmTK3, respectively

Involvement of the Cytokine MIF in the Snail Host Immune Response to the Parasite Schistosoma mansoni

We have identified and characterized a Macrophage Migration Inhibitory Factor (MIF) family member in the Lophotrochozoan invertebrate, Biomphalaria glabrata, the snail intermediate host of the human blood fluke Schistosoma mansoni. In mammals, MIF is a widely expressed pleiotropic cytokine with potent pro-inflammatory properties that controls cell functions such as gene expression, proliferation or apoptosis. Here we show that the MIF protein from B. glabrata (BgMIF) is expressed in circulating immune defense cells (hemocytes) of the snail as well as in the B. glabrata embryonic (Bge) cell line that has hemocyte-like features. Recombinant BgMIF (rBgMIF) induced cell proliferation and inhibited NO-dependent p53-mediated apoptosis in Bge cells. Moreover, knock-down of BgMIF expression in Bge cells interfered with the in vitro encapsulation of S. mansoni sporocysts. Furthermore, the in vivo knock-down of BgMIF prevented the changes in circulating hemocyte populations that occur in response to an infection by S. mansoni miracidia and led to a significant increase in the parasite burden of the snails. These results provide the first functional evidence that a MIF ortholog is involved in an invertebrate immune response towards a parasitic infection and highlight the importance of cytokines in invertebrate-parasite interactions

The Syk Kinase SmTK4 of Schistosoma mansoni Is Involved in the Regulation of Spermatogenesis and Oogenesis

The signal transduction protein SmTK4 from Schistosoma mansoni belongs to the family of Syk kinases. In vertebrates, Syk kinases are known to play specialized roles in signaling pathways in cells of the hematopoietic system. Although Syk kinases were identified in some invertebrates, their role in this group of animals has not yet been elucidated. Since SmTK4 is the first Syk kinase from a parasitic helminth, shown to be predominantly expressed in the testes and ovary of adult worms, we investigated its function. To unravel signaling cascades in which SmTK4 is involved, yeast two-/three-hybrid library screenings were performed with either the tandem SH2-domain, or with the linker region including the tyrosine kinase domain of SmTK4. Besides the Src kinase SmTK3 we identified a new Src kinase (SmTK6) acting upstream of SmTK4 and a MAPK-activating protein, as well as mapmodulin acting downstream. Their identities and colocalization studies pointed to a role of SmTK4 in a signaling cascade regulating the proliferation and/or differentiation of cells in the gonads of schistosomes. To confirm this decisive role we performed biochemical and molecular approaches to knock down SmTK4 combined with a novel protocol for confocal laser scanning microscopy for morphological analyses. Using the Syk kinase-specific inhibitor Piceatannol or by RNAi treatment of adult schistosomes in vitro, corresponding phenotypes were detected in the testes and ovary. In the Xenopus oocyte system it was finally confirmed that Piceatannol suppressed the activity of the catalytic kinase domain of SmTK4. Our findings demonstrate a pivotal role of SmTK4 in gametogenesis, a new function for Syk kinases in eukaryotes

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

Les récepteurs Tyrosine kinases de Schistosoma mansoni : à la recherche de nouvelles cibles thérapeutiques

Malgré les efforts entrepris pour contrôler sa transmission, la schistosomose reste la seconde endémie parasitaire mondiale et représente toujours un véritable problème de santé publique ainsi qu’un handicap pour la croissance économique de bon nombre de pays en voie de développement. Dans le cas de l’infection à Schistosoma mansoni, l’apparition de cas de résistance au praziquantel, drogue de choix actuelle pour le traitement de la maladie, représente une menace importante pour la santé des populations et motive la poursuite de recherches visant à la découverte de nouvelles molécules. Les récents succès obtenus en chimiothérapie anticancéreuse avec l’utilisation d’inhibiteurs spécifiques de récepteurs tyrosine kinases (RTK), tel que le récepteur EGF, nous ont conduits à considérer que, d’une manière analogue, les récepteurs aux facteurs de croissance du parasite pourraient être des cibles thérapeutiques potentielles. Trois RTK ont été identifiés chez S. mansoni : un récepteur de l’EGF, un récepteur de l’insuline et un récepteur complètement original appartenant probablement à une classe de RTK qui n’avait pas été encore identifiée. Les études structurales et fonctionnelles ont démontré la conservation et les divergences de ces RTK parasitaires et en font des candidats privilégiés en tant que nouvelles cibles chimiothérapeutiques

Etude des Récepteurs Tyrosine Kinase du parasite helminthe Schistosoma mansoni (découverte des Récepteurs Venus Kinase, une nouvelle famille de RTK)

La schistosomiase constitue un problème majeur de santé publique dans de nombreux pays émergents d'Afrique, d'Amérique Latine et d'Asie du Sud Est, causant près de 300 000 décès par an. Cette maladie est due au schistosome qui est un ver parasite possédant un cycle de vie complexe. A ce jour une seule drogue est utilisée en monothérapie, le praziquantel ou PZQ, pour lutter contre cette maladie. En raison d'apparitions de résistances au PZQ, il devient nécessaire de rechercher de nouvelles cibles thérapeutiques contre le ver. Au cours de ma thèse je me suis penché sur l'utilisation potentielle des Récepteurs Tyrosine Kinase (RTK) de schistosomes comme cibles thérapeutiques contre le parasite. En effet, les kinases du schistosome semblent présenter un fort degré de spécificité, ce qui les rend attractives pour le l'élaboration d'inhibiteurs potentiels. Dans ce cadre, nous avons poursuivi l'étude de SmIR-1, un récepteur de l'insuline de Schistosoma mansoni découvert au laboratoire, et montré qu'il pouvait être impliqué dans la prise de glucose chez le parasite. Dans un second temps, à partir d'un RTK totalement atypique décrit au laboratoire chez S. mansoni, nous avons découvert une nouvelle famille de RTK nommés les VKR pour Venus Kinase Recepteur, qui semblerait étendue à l'ensemble des invertébrés et dont nous avons entrepris l'étude fonctionnelleLILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Digestion des protéines de l'hôte par le réseau de protéases intestinales du parasite helminthe schistosoma mansoni

En utilisant les inhibiteurs protéasiques et la technique d'interférence à l'ARN, nous avons décomposé le réseau d'endopeptidases impliquées dans la digestion intestinale de Schistosoa mansoni. L'initiation de la dégradation des protéines de l'hôte, hémoglobine et albumine, avec les protéases à cystéine ou à acide aspartique du parasite, s'est révélée ordonnée, ocasionnellement redondante et spécifique au substrat. Alors que l'inhibition de la cathepsine D a un effet majeur surla dégradation de l'hemoglibine, les protéases à cystéine semblent prédominer dans la dégradation. Une asparaginyl endopeptidase synergise avec les cathepsines B et L soit en activant les zymogènes, soit en facilitant le clivage du substrat. Ces protéases fonctionnent de façon optimale dans des compartiments acidiques de la lumière intestinale ou du gastroderme. Définir le rôle de ces enzymes permet de mieux comprendre l'intéraction des protéases au sein d'un réseau complexe, conservé au cours de l'évolution des invertébrés, des nématodes aux insectes. Cette étude fournit également des éléments intéressants quant à la détermination de protéases pouvant être utilisées comme cilbles thérapeutiques potentielles contre la schistosomiase, un problème de santé mondial.Utilizing protease inhibitors and Rna interference, we deconvulated the network of endopeptidases functioning in intestinal protein digestion in Schistosoa mansoni. We showed that initial degradation of hemoglobin and albumin by parasite cysteine and aspartic rpoteases, is ordered, occasionally redundant, and substrate specific. While inhibition of cathepsin D had a greater effect on primary cleavage of hemoglobin, inhibition of cysteine proteases predominated in albumin degradation. Nevertheless, in both cases inhibitor combinations were synergistic. An asparaginyl endopeptidase synergized with cathepsin B and L by zymogen activation or facilitating substrate cleavage ; these proteases operate optimally in two distinct acidic compartments : one formed by lamellar fusion in the gut lumen and the second in the gastrodermis. Definig the role of these enzymes provides a clearer understanding of the function of a complex protease network found throughout invertebrate evolution from nematodes to insects. It also provides insights into which of these protease are logical chemotherapeutic targets for schistosomiasis, a major global health problem.LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF