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

Combinatory microarray and SuperSAGE analyses identify pairing-dependently transcribed genes in Schistosoma mansoni males, including Follistatin

Background: Schistosomiasis is a disease of world-wide importance and is caused by parasitic flatworms of the genus Schistosoma. These parasites exhibit a unique reproduction biology as the female’s sexual maturation depends on a constant pairing-contact to the male. Pairing leads to gonad differentiation in the female, and even gene expression of some gonad-associated genes is controlled by pairing. In contrast, no morphological changes have been observed in males, although first data indicated an effect of pairing also on gene transcription in males. Methodology/Principal Findings: To investigate the influence of pairing on males, we performed a combinatory approach applying SuperSAGE and microarray hybridization, generating the most comprehensive data-set on differential transcription available to date. Of 6,326 sense transcripts detected by both analyses, 29 were significantly differentially transcribed. Besides mutual confirmation, the two methods complemented each other as shown by data comparison and real-time PCR, which revealed a number of genes with consistent regulation across all methods. One of the candidate genes, follistatin of S. mansoni (SmFst) was characterized in more detail by in situ hybridization and yeast two-hybrid (Y2H) interaction analyses with potential binding partners. Conclusions/Significance: Beyond confirming previously hypothesized differences in metabolic processes between pairingexperienced (EM) and pairing-unexperienced males (UM), our data indicate that neuronal processes are involved in malefemale interaction but also TGFb-signaling. One candidate revealing significant down-regulation in EM was the TGFbpathway controlling molecule follistatin (SmFst). First functional analyses demonstrated SmFst interaction with the S. mansoni TGFb-receptor agonists inhibin/activin (SmInAct) and bone morphogenic protein (SmBMP), and all molecules colocalized in the testes. This indicates a yet unknown role of the TGFb-pathway for schistosome biology leading to male competence and a possible influence of pairing on the male gonad

Arylmethylamino steroids as antiparasitic agents

In search of antiparasitic agents, we here identify arylmethylamino steroids as potent compounds and characterize more than 60 derivatives. The lead compound 1o is fast acting and highly active against intraerythrocytic stages of chloroquine-sensitive and resistant Plasmodium falciparum parasites (IC50 1–5?nM) as well as against gametocytes. In P. berghei-infected mice, oral administration of 1o drastically reduces parasitaemia and cures the animals. Furthermore, 1o efficiently blocks parasite transmission from mice to mosquitoes. The steroid compounds show low cytotoxicity in mammalian cells and do not induce acute toxicity symptoms in mice. Moreover, 1o has a remarkable activity against the blood-feeding trematode parasite Schistosoma mansoni. The steroid and the hydroxyarylmethylamino moieties are essential for antimalarial activity supporting a chelate-based quinone methide mechanism involving metal or haem bioactivation. This study identifies chemical scaffolds that are rapidly internalized into blood-feeding parasites

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

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

Serum albumin and α-1 acid glycoprotein impede the killing of Schistosoma mansoni by the tyrosine kinase inhibitor Imatinib

In the search for new drugs and drug targets to treat the flatworm disease schistosomiasis, protein kinases (PKs) have come under particular scrutiny because of their essential roles in developmental and physiological processes in schistosome parasites. In this context the application of the anti-cancer Abl tyrosine kinase (TK) inhibitor Imatinib (Gleevec/Glivec; STI-571) to adult Schistosoma mansoni in vitro has indicated negative effects on diverse physiological processes including survival. Motivated by these in vitro findings, we performed in vivo experiments in rodent models of S. mansoni infection. Unexpectedly, Imatinib had no effect on worm burden or egg-production. We found that the blood components serum albumin (SA) and alpha-1 acid glycoprotein (AGP or orosomucoid) negated Imatinib’s deleterious effects on adult S. mansoni and schistosomula (post-infective larvae) in vitro. This negative effect was partially reversed by erythromycin. AGP synthesis can increase as a consequence of inflammatory processes or infection; in addition upon infection AGP levels are 6–8 times higher in mice compared to humans. Therefore, mice and probably other rodents are poor infection models for measuring the effects of Imatinib in vivo. Accordingly, we suggest the routine evaluation of the ability of AGP and SA to block in vitro anti-schistosomal effects of small molecules like Imatinib prior to laborious and expensive animal experiments

Synchrotron X-Ray microtomography reveals interior microstructure of multicomponent food materials such as chocolate

The current contribution discusses the structure analysis of a solid multicomponent food product (which is in this case dark chocolate) using microtomography. The material consists of a continuous solid lipid phase, in which particles are suspended. A detailed analysis of the microstructure is needed to understand migration processes, which are e.g. responsible for major problems in the confectionery industry such as chocolate blooming. In this study it was possible to clearly distinguish the particles from the continuous phase. Particle arrangement and structural imperfections within the sample were made visible by using synchrotron radiation. The observed imperfections, which arise during the manufacturing process, might act as migration pathways, since they propagate throughout the entire sample. The captured microtomographic images proof the presence of cracks and voids within a common industrial made chocolate. Future research has to show if migration is happen along the identified microstructural defects

Discovery of Platyhelminth-Specific α/β-Integrin Families and Evidence for Their Role in Reproduction in <em>Schistosoma mansoni</em>

<div><p>In all metazoa, the response of cells to molecular stimuli from their environment represents a fundamental principle of regulatory processes controlling cell growth and differentiation. Among the membrane-linked receptors mediating extracellular communication processes are integrin receptors. Besides managing adhesion to the extracellular matrix or to other cells, they arrange information flow into the cells by activating intracellular signaling pathways often acting synergistically through cooperation with growth factor receptors. Although a wealth of information exists on integrins in different model organisms, there is a big gap of knowledge for platyhelminths. Here we report on the <em>in silico</em> detection and reconstruction of α and β integrins from free-living and parasitic platyhelminths, which according to structural and phylogenetic analyses form specific clades separate from each other and from further metazoan integrins. As representative orthologs of parasitic platyhelminths we have cloned one beta-integrin (Smβ-Int1) and four alpha-integrins (Smα-Int1 - Smα-Int4) from <em>Schistosoma mansoni</em>; they were characterized by molecular and biochemical analyses. Evidence is provided that Smβ-Int1 interacts and co-localizes in the reproductive organs with known schistosome cellular tyrosine kinases (CTKs), of which the Syk kinase SmTK4 appeared to be the strongest interaction partner as shown by yeast two-hybrid analyses and coimmunoprecipitation experiments. By a novel RNAi approach with adult schistosomes <em>in vitro</em> we demonstrate for the first time multinucleated oocytes in treated females, indicating a decisive role Smβ-Int1 during oogenesis as phenotypically analyzed by confocal laser scanning microscopy (CLSM). Our findings provide a first comprehensive overview about platyhelminth integrins, of which the parasite group exhibits unique features allowing a clear distinction from the free-living groups. Furthermore, we shed first lights on the functions of integrins in a trematode model parasite, revealing the complexity of molecular processes involved in its reproductive biology, which may be representative for other platyhelminths.</p> </div

Venus Kinase Receptors Control Reproduction in the Platyhelminth Parasite <i>Schistosoma mansoni</i>

<div><p>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 <i>Schistosoma mansoni</i>, then in a large variety of invertebrates. A single <i>vkr</i> gene is found in most genomes, except in <i>S. mansoni</i> in which two genes <i>Smvkr1</i> and <i>Smvkr2</i> 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 <i>S. mansoni</i>, particularly in the ovaries of female worms. By transcriptional analyses evidence was obtained that both SmVKRs fulfill different roles during oocyte maturation. Suppression of <i>Smvkr</i> 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 <i>Xenopus</i> oocytes, SmVKR1 and SmVKR2 receptors were shown to be activated by distinct ligands which are L-Arginine and calcium ions, respectively. Signalling analysis in <i>Xenopus</i> 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.</p></div

Phylogenetic analyses showing the unique status of plathyhelminth α-integrins.

<p>Phylogram of the analysis of the full-length sequences of the <i>S. mansoni</i> α-integrin receptors Smα-Int1, Smα-Int2, Smα-Int3, and other α-integrin receptors using CLUSTAL X (<a href="http://www.clustal.org" target="_blank">www.clustal.org</a>) and TreeViewX. The phylogenetic relationship was deduced using the Bootstrap Neighbour-Joining (N–J) method and the bootstrap values were generated based on 1000 bootstrap trails with a random number generator seed of 100. Sequences were obtained from the National Centre for Biotechnology Information using the WWW Entrez Browser (<a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a>), Swiss-Prot (<a href="http://www.uniprot.org" target="_blank">www.uniprot.org</a>), GeneDB (<a href="http://www.genedb.org" target="_blank">www.genedb.org</a>), and the <i>Schmidtea mediterranea</i> Genome Database (<a href="http://smedgd.neuro.utah.edu/" target="_blank">http://smedgd.neuro.utah.edu/</a>). The corresponding protein numbers are: Sha a1 (α-integrin 1 receptor, <i>S. haematobium</i>; Sha_102401), Sm a1 (α-integrin 1 receptor, <i>S. mansoni</i>; FR749887), Sjp a1 (α-integrin 1 receptor, <i>S. japonicum</i>; Sjp_0037690), Cs a5 (α-integrin 5 receptor, <i>Clonorchis sinesis</i>; GAA56616.1), Em a1 (α-integrin 1 receptor, <i>Echinococcus multilocularis</i>; EmuJ_000215000 ), Sm a4 (α-integrin 4 receptor, <i>S. mansoni</i>; Smp_1735401, Smp_181010), Sha a4 (α-integrin 4 receptor, <i>S. haematobium</i>; Sha_104436, Sha_106831), Sjp a4 (α-integrin 4 receptor, <i>S. japonicum</i>; Sjp_0046780, Sjp_0046790), Cs a4 (α-integrin 4 receptor, <i>Clonorchis sinesis</i>; GAA28731), Em a4 (α-integrin 4 receptor, <i>Echinococcus multilocularis</i>; EmuJ_000573500), Sha a2 (α-integrin 2 receptor, <i>S. haematobium</i>; Sha_106921), Sm a2 (α-integrin 2 receptor, <i>S. mansoni</i>; FR749888), Sjp a2 (α-integrin 2 receptor, <i>S. japonicum</i>; Sjp_0069490), Cs a-ps (α-integrin-ps receptor, <i>Clonorchis sinesis</i>; GAA54095, GAA49531, GAA49530), Em a2 (α-integrin 2 receptor, <i>Echinococcus multilocularis</i>; EmuJ_000192500 ), Smed a3 (α-integrin 3 receptor, <i>Schmidtea mediterranea</i>; lcl|mk4.000046.14.01), Smed a1 (α-integrin 1 receptor, <i>Schmidtea mediterranea</i>; lcl|mk4.001411.00.01), Smed a2 (α-integrin 2 receptor, <i>Schmidtea mediterranea</i>; lcl|mk4.003797. 00.01), Sha a3 (α-integrin 3 receptor, <i>S. haematobium</i>; Sha_102914), Sm a3 (α-integrin 3 receptor, <i>S. mansoni</i>; FR749889, Smp_156610, Smp_156620), Sjp a3 (α-integrin 3 receptor, <i>S. japonicum</i>; Sjp_0063430, Sjp_0063420), Cs a7 (α-integrin 7 receptor, <i>Clonorchis sinesis</i>; GAA52225.1), Em a3 (α-integrin 3 receptor, <i>Echinococcus multilocularis</i>; EmuJ_000782500), Sp aP (α-integrin P receptor, <i>Strongylocentrotus purpuratus</i>, AF177914), Dm aPS2 (α-integrin PS2 receptor, <i>Drosophila melanogaster</i>, Q24247), Mm a2b (α-integrin 2b receptor, <i>Mus musculus</i>; EDL34136.1), Hs a2b (α-integrin 2b receptor, <i>Homo sapiens</i>; EAW51595.1), Xl a2b (α-integrin 2b receptor, <i>Xenopus laevis</i>; NP_001088223.1), Mm a5 (α-integrin 5 receptor, <i>Mus musculus</i>; CAA55638.1), Rn a5 (α-integrin 5 receptor, <i>Rattus norvegicus</i>; NP_001101588.1), Hs a5 (α-integrin 5 receptor, <i>Homo sapiens</i>; NP_002196.2), Xl a5 (α-integrin 5 receptor, <i>Xenopus laevis</i>; NP_001081072.1), Hs aV (α-integrin V receptor, <i>Homo sapiens</i>; P06756), Hs a8 (α-integrin 8 receptor, <i>Homo sapiens</i>; P53708), Ce a-pat2 (α-integrin pat-2, <i>Ceanorhabditis elegans</i>; P34446), Gc a (α-integrin receptor, <i>Geodia cydonium</i>; X97283), Hs a1 (α-integrin 1 receptor, <i>Homo sapiens</i>; P56199), Hs a2 (α-integrin 2 receptor, <i>Homo sapiens</i>; P17301), Hs a10 (α-integrin 10 receptor, <i>Homo sapiens</i>; O75578), Hs a11 (α-integrin 11 receptor, <i>Homo sapiens</i>; Q9UKX5), Hs aD (α-integrin D receptor, <i>Homo sapiens</i>; Q13349), Hs aX (α-integrin X receptor, <i>Homo sapiens</i>; P20702), Hs aM (α-integrin M receptor, <i>Homo sapiens</i>; P11215), Hs aL (α-integrin L receptor, <i>Homo sapiens</i>; P20701), Hs aE (α-integrin E receptor, <i>Homo sapiens</i>; P38579), Hs a4 (α-integrin 4 receptor, <i>Homo sapiens</i>; P13612), Hs a9 (α-integrin 9 receptor, <i>Homo sapiens</i>; Q13797), Mm a7 (α-integrin 7 receptor, <i>Mus musculus</i>; AAA16600.1), Rn a7 (α-integrin 7 receptor, <i>Rattus norvegicus</i>; NP_110469.1), Hs a7 (α-integrin 7 receptor, <i>Homo sapiens</i>; EAW96822.1), Hs a6 (α-integrin 6 receptor, <i>Homo sapiens</i>; P23229), Hs a3 (α-integrin 3 receptor, <i>Homo sapiens</i>; P26006), Dm aPSI (α-integrin PSI receptor, <i>Drosophila melanogaster</i>, Q24247), and Ce a-ina1 (α-integrin ina1, <i>Ceanorhabditis elegans</i>; Q03600).</p