Date Published: June 13, 2013
Publisher: Public Library of Science
Author(s): Christin Buro, Katia C. Oliveira, Zhigang Lu, Silke Leutner, Svenja Beckmann, Colette Dissous, Katia Cailliau, Sergio Verjovski-Almeida, Christoph G. Grevelding, David L. Williams.
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.
Blood-dwelling endoparasites of the genus Schistosoma are the only trematodes that have evolved a gender dimorphism , . These parasites cause schistosomiasis, which is of worldwide significance for humans and animals in tropical and sub-tropical areas . About 780 million people live in endemic areas being at risk of schistosomiasis, of which 200 million are infected generating annual losses of 1.7 to 4.5 million disability adjusted life years (DALYs) of humans as determined by the Global Burden of Disease Programme , . Living in the abdominal veins of their vertebrate hosts, adult paired females produce up to 300 eggs per day. Half of these eggs penetrates the epithelia and reach the gut lumen (e.g. S. mansoni) or the bladder (S. haematobium) to be transported to the environment for continuing the life cycle. The remaining eggs migrate via the blood stream to different organs such as spleen and liver, where they get trapped causing granuloma formation and liver cirrhosis , .
Inhibitor experiments in previous studies and in this study have indicated that Src kinase- and TβRI-containing pathways influence mitotic activity and egg production in paired schistosome females , , and that SmTK3 ,  and SmTβRI are targets of Herb A or TRIKI, respectively. Furthermore, a yeast-two-hybrid (Y2H) cDNA-library screening and subsequent qualitative and quantitative analyses identifying and characterizing binding partners acting “downstream” of SmTK3 detected besides others a homolog of the BAF60 subunit of the SWI/SNF complex (SmBAF60) and a diaphanous homolog (SmDia) as the strongest interacting partners . The SWI/SNF complex is involved in chromatin-remodeling activities, DNA-damage responses, transcriptional activation, sliding of nucleosomes, and alteration of histone-DNA contacts . Diaphanous proteins belong to the big group of formin-homology proteins known to play roles in actin-mediated processes controlling cell and tissue architecture, cell-cell interactions, gastrulation, and cytokinesis .