Schistosoma mansoni: Molecular genetic effects following inhibitor treatment; functional characterization of differentially expressed genes potentially involved in regulating mitoses and egg-formation processes
Zusammenfassung der Projektergebnisse
Microarray studies of our lab with RNA of inhibitor-treated male and female S. mansoni resulted in the identification of differentially expressed genes (DEGs) potentially involved in regulating egg production. The inhibitors comprised the Src-kinase inhibitor Herbimycin A (Herb A) and the TGFβ-RI kinase inhibitor (TRIKI). Bioinformatics and molecular analyses showed that Src-kinase and TGFβ-RI kinase pathways cooperate to regulate reproduction-associated processes in S. mansoni including mitosis. A stronger influence of the TGFβ/TGFβ-RI was found while the Src-kinase containing pathway seemed to modulate the TGFβ/TGFβ-RI pathway effects. To obtain more insight in processes downstream of these cooperating signaling cascades, we started to characterize selected DEGs. Their annotation indicated functions in mitotic processes as supposed for a c-Mycbinding-related gene, p53, and dynactin, or a participation in calcium (Ca2+) metabolism-associated processes as supposed for hippocalcin, calmodulin-4, and the Ca2+-channel protein ORAI-1. The genes coding for c-Myc-binding protein (Smp_125290.1) and p53 (Smp_ 139530.1) were found to be pairingdependently transcribed in males and in the ovary of females according to our RNA-Seq data that were obtained in parallel to this project. Due to the relatively high expression in ovaries of unpaired females (sF), which mainly contain stem cell-like oogonia, we hypothesize that Smp_125290.1 may contribute to stem cell-associated cell division processes. First in-vitro culture experiments with a c-Myc inhibitor revealed a negative effect on the egg production of paired worms whose egg production seemed unaffected by sub-lethal inhibitor concentrations. Although a c-Myc gene has not yet been identified in S. mansoni, it may be worth following its trace based the finding made. Since mitosis and apoptosis regulation belong to the processes controlled by p53 and cMyc, the identified bias of transcript levels of both genes towards unpaired females suggests that apoptosis may play a dominating role in sF. In vitro, the p53 inhibitor Pifithrin-α induced separation of couples and the death of worms. Sublethal concentrations negatively affected egg production, and by confocal microscopy (CLSM) a perforated tissue structure was observed in the vitellarium of paired females, which represents a target organ for p53-mediated and apoptosis-related processes. The transcript levels of hippocalcin (Smp_085650.1), which was found to be transcribed in the ovary, the vitellarium, and the Mehlis´ glands of paired females, and dynactin (Smp_062630.1) were in part influenced by pairing in the female and mainly in its ovary or vitellarium, respectively, according to the RNA-Seq data. Attempts to localize dynactin by in-situ hybridization have failed so far for unknown reasons, as well as first RNAi experiments for hippocalcin and dynactin. Calmodulin-4 (Smp_032990.1) transcripts were mainly localized in the female vitelloduct and around the ootype by in situhybridization, and RNA-Seq analysis showed a high transcript level in sF. Full-length cloning confirmed the existence of two EF hands, and closest similarity of this gene to orthologs in S. japonicum and Clonorchis sinensis was found by in silico analyses. Yeast two-hybrid (Y2H) library screening identified a binding partner that is still annotated as hypothetical protein (Smp_073450). According to the RNA-Seq data Smp_073450 is also pairing-dependently transcribed with a bias towards unpaired females. In vitro, the inhibitor Calmidazolium affected worm vitality and tissue destruction as revealed CLSM. In a parallel approach we identified the epidermal growth factor substrate protein SmEps8 by Y3H analyses as an upstream interaction partner of the Src kinase SmTK3, the target of Herb A. Interaction analyses revealed a contribution of the SH2 and SH3 domains of SmTK3 to binding. RT-PCR analyses and in situ hybridization experiments demonstrated colocalization of SmTK3 and SmEps8 in the gonads, and first evidence was obtained for SmEps8 interaction and colocalization with SER, one of the epidermal growth factor receptors discovered in S. mansoni. These results provided first evidence for a SER-SmEps8- SmTK3-SmDia (diaphanous) signal transduction pathway controlling differentiation processes in the gonads of S. mansoni. The Ca2+-channel protein SmORAI-1 (Smp_076650.1/2) was localized in the ovary and vitellarium of the female and testes of the male by in-situ hybridization and organ-specific RT-PCR analyses. RNA-Seq provided evidence for a pairing-influenced transcription in females and their ovaries, which was partly confirmed by qRT-PCR. Using the ORAI inhibitor 2-APB in vitro with adults showed that egg production and quality were negatively affected in paired females, which was paralleled by a perforated structure of the vitellarium and a mislocalization of mature oocytes as detected by confocal microscopy. Phenotype analyses following siRNA- and/or dsRNA-mediated RNAi confirmed the deleterious effect in the vitellarium. In contrast to the female gonads, an additional phenotype was found in the testes of males. EdU-incorporation assays demonstrated a reduction of labeled cells in the testes indicating a negative effect on spermatogenesis affecting gonadal stem cells. Finally, Ca2+-flux was investigated at the physiological level by a newly established Ca2+-imaging protocol of our lab. Using this method we obtained first evidence that SmORAI-1 is indeed a Ca2+ transporter in the gonads, whose function can be influenced by 2-APB and RNAi. With respect to understanding the unusual biology of schistosomes, the pairing-dependent sexual maturation of the female is of high interest. The discovery of the prominent roles of kinases, TGFβ-signalling and Ca2+ metabolism-associated processes controlling gonad differentiation in the female gonads will help to unravel the science puzzle of schistosome reproduction and in parallel to identify potential targets for new strategies fighting schistosomiasis, whose pathology is caused by the eggs. The results obtained during this project thus contribute to both aims opening further research options.
Projektbezogene Publikationen (Auswahl)
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(2010) Schistosoma mansoni: Signal transduction processes during the development of the reproductive organs. Parasitology 137, 497-520
Beckmann, S., Quack, T., Burmeister, C., Buro, C., Long, T., Dissous, C., Grevelding C.G.
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(2012) Protein kinases as potential targets for novel anti-schistosomal strategies. Curr. Pharm. Des. 18, 3579-3594
Beckmann, S., Leutner, S., Gouignard, N., Dissous, C., Grevelding, C.G.
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(2012) SmSak, the second Polo-like kinase of the helminth parasite Schistosoma mansoni: conserved and unexpected roles in meiosis. PLoS One 7(6), e40045
Long T, Vanderstraete M, Cailliau K, Morel M, Lescuyer A, Gouignard N, Grevelding CG, Browaeys E, Dissous C
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(2013) Transcriptome analyses of inhibitor-treated schistosome females provide evidence for cooperating Src-kinase and TGFβ receptor pathways controlling mitosis and eggshell formation. PLoS Pathogens 9(6), e1003448
Buro, C., Oliveira, K.C., Leutner, S., Beckmann, S., Lu, Z., Dissous, C., Cailliau, K., Verjovski-Almeida, S., Grevelding, C.G.
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(2013) Whole-organ isolation approach as a basis for tissue-specific analyses in Schistosoma mansoni. PLoS Neglected Tropical Diseases 7(7), e2336
Hahnel, S., Lu, Z., Wilson, A., Grevelding, C.G., Quack, T.
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(2014) Receptor tyrosine kinases and schistosome reproduction: new targets for chemotherapy. Frontiers in Genetics 5: 238
Morel, M., Vanderstraete, M., Hahnel, S., Grevelding, C.G., Dissous, C.
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(2014) Serum albumin and α-1 acid glycoprotein impede the impact of Imatinib on adult and larval stages of Schistosoma mansoni. International Journal for Parasitology: Drugs and Drug Resistance 4(3): 287-295
Beckmann, S., Long, T., Scheld, C., Geyer, R., Caffrey, C., Grevelding, C.G.
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(2014) Venus kinase receptors control reproduction in the platyhelminth parasite Schistosoma mansoni. PLoS Pathogens 10(5): e1004138
Vanderstraete, M., Gouignard, N., Cailliau, K., Morel, M., Hahnel, S., Leutner, S., Beckmann, S., Grevelding, C.G., Dissous, C.
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(2015) Isolation, enrichment and primary characterization of vitelline cells from Schistosoma mansoni obtained by the organ-isolation method. International Journal for Parasitology 45(9-10), 663-672
Lu, Z., Quack, T., Hahnel, S., Gelmedin, V., Pouokam, E., Diener, M., Hardt, M., Michel, G., Baal, N., Hackstein, H., Grevelding CG.
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(2015) Re-positioning protein kinase inhibitors against schistosomiasis. Future Medical Chemistry 7(6), 737-752
Gelmedin, V., Dissous, C., Grevelding, C.G.
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(2017) Identification and first characterization of SmEps8, a potential interaction partner of SmTK3 and SER transcribed in the gonads of Schistosoma mansoni. Experimental Parasitology 180, 55-63
Buro, C., Burmeister, C., Quack, T., Grevelding, CG.
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(2017) Ova and oogenesis in Schistosoma. In: Schistosoma, Biology, Pathology and Control. Edited by Barrie G. M. Jamieson; Taylor & Francis Group, CRC Press, Boca Raton; p. 320- 332 [Print ISBN: 978-1-4987-4425-6; eBook ISBN: 978-1-4987-4426-3]
Grevelding, C.G., Hahnel, S., Lu, Z.
