Svenja Beckmann

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.

Schistosoma mansoni: signal transduction processes during the development of the reproductive organs.

Among the topics of considerable interest concerning our understanding of the unusual biology of schistosomes is the sexual maturation of the female. The identification of genes coding for signal transduction proteins controlling essential steps of the pairing-dependent differentiation of the reproductive organs, vitellarium and ovary will help to substantiate our knowledge about this unique parasite. Furthermore, such signalling proteins could be potential targets to interfere with the development of this parasite to combat schistosomiasis since its pathology is caused by the eggs. This review summarises first post-genomic steps to elucidate the function of gonad-specific signalling molecules which were identified by homology-based cloning strategies, by in silico identification or by yeast two-hybrid interaction analyses, using a combination of novel techniques. These include the in vitro culture of adult schistosomes, their treatment with chemical inhibitors to block enzyme activity, the use of RNAi to silence gene function post-transcriptionally, and confocal laser scanning microscopy to study the morphological consequences of these experimental approaches. Finally, we propose a first model of protein networks that are active in the ovary regulating mitogenic activity and differentiation. Some of these molecules are also active in the testes of males, probably fulfilling similar roles as in the ovary.

Imatinib has a fatal impact on morphology, pairing stability and survival of adult Schistosoma mansoni in vitro

Schistosomes cause bilharzia (schistosomiasis), one of the most prevalent parasitic diseases for human and animals worldwide. Praziquantel (PZQ) is the only widely used drug for treatment and control of this parasitemia. Since a vaccine is not yet available, and in light of emerging resistance against PZQ, the search for alternatives has high priority. Here we present that Imatinib, a compound used in human cancer therapy (Gleevec; STI-571), significantly affected schistosome morphology and physiology in vitro. Besides its negative effect on gonad development and pairing stability, Imatinib led to pathological alterations of the gastrodermis, which finally caused the death of the parasite.

Schistosoma mansoni Polo-like kinase 1: A mitotic kinase with key functions in parasite reproduction

Polo-like kinases (Plks) are conserved regulators of mitosis. In mammals, Plk1 is over-expressed in a wide range of tumour cells and constitutes a valuable target for anti-cancer therapy. This work presents the characterisation of the Plk1 homologue (SmPlk1) of Schistosoma mansoni, a trematode responsible for schistosomiasis, one of the most important parasitic diseases, second only to malaria. The intense levels of disease transmission and the severity of pathologies are the consequences of the exceptional reproductive activity of schistosomes, in which Plks may play a decisive role. Structural and functional analyses of SmPlk1 have demonstrated its homology with other Plk1 members and its conserved function in mitotic processes. Activation of SmPlk1 was shown to be dependent on phosphorylation of its conserved threonine residue (T(182)) and the ability of active SmPlk1 to induce mitosis was demonstrated in the Xenopus oocyte model. SmPlk1 transcripts were detected abundantly in parasite stages containing a high amount of germinal cells. A potential role of SmPlk1 in mitosis and/or meiosis in schistosomes was supported by the in situ detection of SmPlk1 transcripts in female vitelline cells and oocytes as well as in male spermatocytes. Several Plk inhibitors were shown to inhibit SmPlk1 activity in Xenopus oocytes, and BI 2536 (the first-in-class prototype Plk1 inhibitor) induced in vitro dramatic alterations in schistosome gonads, which affected oogenesis and spermatogenesis. These results indicate a major role for SmPlk1 in parasite reproduction and suggest its importance as a potential new target against schistosomiasis.

Molecular characterisation of kappa-5, a major antigenic glycoprotein from Schistosoma mansoni eggs

The major immunopathological consequences of infection with Schistosoma mansoni, a T helper type 2 response and granuloma formation leading to fibrotic tissue damage, are caused by the egg stage of the parasite. Three antigens of S. mansoni eggs, termed IPSE/alpha-1, omega-1 and kappa-5, have been found to be the primary targets of the egg-directed antibody response of the host. Here, we report on the isolation, cloning and characterisation of kappa-5. Apart from an uncharacterised mRNA sequence in S. japonicum, no significant similarities of kappa-5 to known sequences from other species were found. In contrast to IPSE/alpha-1 and omega-1, which have been found only in eggs, kappa-5 was present in miracidia as well as in eggs at the mRNA and protein levels. In eggs, isoforms of kappa-5 were observed with both three and four fully occupied N-glycosylation sites, while in miracidia only one isoform with four N-glycans could be detected. Interestingly, in Western blots sera from S. mansoni-infected Africans were reactive against kappa-5 with IgE and IgG isotype antibodies, but against IPSE/alpha-1 and omega-1 only with IgG antibodies. The further characterisation of kappa-5 as one of the three major egg antigens should help to better understand the immunology and immunopathology of schistosomiasis.

Characterization of the SRC/ABl hybrid-kinase SMTK6 of Schistosoma mansoni

Background: SmTK6 was identified as interaction partner of SmTK4. Results: SmTK6 is a Src/Abl hybrid kinase and interacts also with the uncommon SmVKR1 and SmTK3. Conclusion: SmTK6 is suggested to be part of a complex of receptors, Syk and Src kinases, which are involved in gonad development. Significance: SmTK6 represents an Abl kinase progenitor, for which a function in reproduction could be assigned. Cellular protein-tyrosine kinases play key roles in signal transduction processes in eukaryotes. SmTK4 was the first Syk kinase identified in a parasite and found to be tissue-specifically transcribed in the gonads of adult Schistosoma mansoni. Functional analyses confirmed its role in oogenesis and spermatogenesis. As an SmTK4 upstream binding partner, the cellular protein-tyrosine kinase SmTK6 was isolated from a yeast two-hybrid library. Phylogenetic analyses performed in this study confirmed the first suggestions of a hybrid character of SmTK6. Biochemical studies made in Xenopus oocytes using inhibitors against Src (herbimycin A) and Abl (imatinib) kinases exhibited a biochemical inhibition profile of SmTK6, which was intermediate of Src and Abl kinases. As SmTK6 upstream interaction partners, we identified among others the known Src kinase SmTK3 and the Venus kinase receptor SmVKR1 of S. mansoni by yeast two-hybrid analyses, all of which co-localized in the gonads. Co-immunoprecipitation experiments confirmed interactions between SmTK6 and SmTK3 or SmVKR1. In Xenopus oocytes, it was finally shown that SmVKR1 but also SmTK3 were able to activate SmTK6 enzymatic activity indicating its functions in a receptor tyrosine kinase signal transduction cascade. These results not only demonstrate an intermediate but Src-biased profile of the unusual kinase SmTK6. They also strongly substantiate previous indications for a kinase complex, consisting of a receptor tyrosine kinase, Syk and Src kinases, which has been hypothesized to be involved in proliferation and differentiation processes in the gonads of schistosomes.

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.

Protein Kinases as Potential Targets for Novel Anti-Schistosomal Strategies

Schistosome parasites are the causative pathogens of schistosomiasis (bilharzia), a disease of worldwide significance. In terms of patient numbers, schistosomiasis ranks second to malaria as a parasitosis affecting more than 200 million people of the tropics and subtropics. Since the 1970s Praziquantel (PZQ) is the drug of choice and nearly exclusively used for treatment. However, drug resistance is an increasing threat, particularly with respect to large-scale PZQ administration programs. Last decades research indicated that resistance against PZQ can be induced under laboratory conditions, and field studies provided first indications for the possibility of reduced PZQ efficacy. Furthermore, clear evidence for the molecular armamentarium of schistosomes with multidrug transporters was found, one of which was responding to PZQ challenge. Also the development of a vaccine still represents an elusive goal, although effort and time have been invested in this subject. In light of these facts it is commonly accepted that new drugs are urgently needed. Research on signal transduction processes in Schistosoma mansoni has provided an unexpected and novel perspective towards this end. Molecular, biochemical, and physiological studies elucidating principles of schistosome development have demonstrated the essential role of protein kinases (PKs). In humans, PKs are known to be involved in cancer development. Since a variety of approved anticancer drugs targeting PKs exist, first studies have been performed to investigate whether these drugs are able to also inhibit schistosome PKs. Indeed, promising results have been obtained indicating the potential of PKs as privileged targets for new concepts in fighting schistosomes.

Venus kinase receptors control reproduction in the platyhelminth parasite Schistosoma mansoni.

The Venus Kinase Receptor (VKR) is a single transmembrane molecule composed of an intracellular tyrosine kinase domain close to that of insulin receptor and an extracellular Venus Flytrap (VFT) structure similar to the ligand binding domain of many class C G Protein Coupled Receptors. This receptor tyrosine kinase (RTK) was first discovered in the platyhelminth parasite Schistosoma mansoni, then in a large variety of invertebrates. A single vkr gene is found in most genomes, except in S. mansoni in which two genes Smvkr1 and Smvkr2 exist. VKRs form a unique family of RTKs present only in invertebrates and their biological functions are still to be discovered. In this work, we show that SmVKRs are expressed in the reproductive organs of S. mansoni, particularly in the ovaries of female worms. By transcriptional analyses evidence was obtained that both SmVKRs fulfill different roles during oocyte maturation. Suppression of Smvkr expression by RNA interference induced spectacular morphological changes in female worms with a strong disorganization of the ovary, which was dominated by the presence of primary oocytes, and a defect of egg formation. Following expression in Xenopus oocytes, SmVKR1 and SmVKR2 receptors were shown to be activated by distinct ligands which are L-Arginine and calcium ions, respectively. Signalling analysis in Xenopus oocytes revealed the capacity of SmVKRs to activate the PI3K/Akt/p70S6K and Erk MAPK pathways involved in cellular growth and proliferation. Additionally, SmVKR1 induced phosphorylation of JNK (c-Jun N-terminal kinase). Activation of JNK by SmVKR1 was supported by the results of yeast two-hybrid experiments identifying several components of the JNK pathway as specific interacting partners of SmVKR1. In conclusion, these results demonstrate the functions of SmVKR in gametogenesis, and particularly in oogenesis and egg formation. By eliciting signalling pathways potentially involved in oocyte proliferation, growth and migration, these receptors control parasite reproduction and can therefore be considered as potential targets for anti-schistosome therapies.

Transcriptome Analyses of Inhibitor-treated Schistosome Females Provide Evidence for Cooperating Src-kinase and TGFβ Receptor Pathways Controlling Mitosis and Eggshell 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 TGFβ receptor (TβR) 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 TβRI pathways. After demonstrating that TRIKI inhibits the schistosome TGFβreceptor SmTβRI 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.