Buro C et al. (2013), Transcriptome analyses of inhibitor-treated sch...

XB-ART-47235

PLoS Pathog 2013 Jan 01;96:e1003448. doi: 10.1371/journal.ppat.1003448.

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Transcriptome analyses of inhibitor-treated schistosome females provide evidence for cooperating Src-kinase and TGFβ receptor pathways controlling mitosis and eggshell formation.

Buro C , Oliveira KC , Lu Z , Leutner S , Beckmann S , Dissous C , Cailliau K , Verjovski-Almeida S , Grevelding CG .

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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.

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Species referenced: Xenopus laevis
Genes referenced: ctss hsp70 hspa1l tspan1

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	Figure 2. Hierarchical clustering of differentially transcribed genes (q≤0.03) following TRIKI-treatment, Herbimycin A-treatment, and combined inhibitor treatment.Hierarchical clustering of (A) 2330 (TRIKI), (B) 1021 (Herbimycin A), and (C) 411 (combined inhibitors) differentially transcribed genes (q≤0.03) of treated and control female schistosomes. Columns represent three biological replicas as well as two technical replicas (A, TRIKI: columns TRIKI/DMSO 6 and 5, as well as TRIKI/DMSO 1 and 2 represent technical replicas of the first two biological replicas, respectively; TRIKI/DMSO 3 represents the 3rd biological replicate. B, Herbimycin A: columns Herb/DMSO 2 and 1, as well as Herb/DMSO 5 and 6 represent technical replicas of the first two biological replicas, respectively; Herb/DMSO 3 the 3rd biological replicate. C, combined inhibitors: columns H+T/DMSO 6 and 5, as well as H+T/DMSO 4 and 3 represent technical replicas of the first two biological replicas, respectively; H+T/DMSO 2 represents the 3rd biological replicate). Each line shows the calculated log2ratio (treated/control) of the transcription of a gene; genes with a repressed transcription were colored in green (A, TRIKI: 565 genes; B, Herbimycin A: 302 genes; C, combined inhibitors: 157 genes) and genes with an enhanced transcription in red (A, TRIKI: 1765 genes; B, Herbimycin A: 719 genes; C, combined inhibitors: 254 genes).
	Figure 3. Venn diagram of differentially transcribed genes identified by different inhibitor treatments.Numbers of differentially transcribed protein-coding genes of all three microarray experiments (“TRIKI”, “Herbimycin A”, “Combined treatment”). Each circle represents the microarray result of one of the inhibitor treatments. The intersections show the numbers of corresponding genes differentially transcribed following treatment with different inhibitors.
	Figure 4. Comparison of qPCR and microarray results following TRIKI-treatment.Transcriptional changes from the TRIKI-treatment of adult schistosome females detected in qPCR and microarray data were calculated as log2ratios (treated/control). The investigated genes were SmTβRI, SmActRIIb, a protein similar to fs800, eggshell precursor, and Na/K-pump as representative for enhanced transcription within the microarray data set (A). The selected genes representing the set of genes with repressed transcription within the microarray data set (B) were calmodulin-4, tetraspanin 18, SmSmad 4, and snurp. For each method three biological replicas were used, each with two technical replicas for the microarray analysis (except microarray 2) and three technical replicas for the qPCR. The mean of the technical replicas was calculated and is presented with standard deviations.
	Figure 5. Comparison of qPCR and microarray results following Herbimycin A-treatment.Transcriptional changes from the Herb A-treatment of adult schistosome females detected in qPCR and microarray data were calculated as log2ratios (treated/control). According to the microarray analysis SmActRIIb, calmodulin-4, and hsp70 showed enhanced transcription (A), whereas tetraspanin-1, SmSmad 4, and Smp48 were detected as transcriptionally repressed (B). For each method three biological replicas were used, each with two technical replicas for the microarray analysis (except microarray 2) and three technical replicas for the qPCR. The mean of the technical replicas was calculated and is presented with standard deviations.
	Figure 6. Comparison of qPCR and microarray results following the combined inhibitor treatment.Transcriptional changes from the simultaneous TRIKI- and Herb A-treatment of adult schistosome females detected in qPCR and microarray data were calculated as log2ratios (treated/control). The investigated genes with enhanced transcription, as determined by microarray analysis, were calmodulin-4 and hsp70 (A), the genes with repressed transcription comprised a gene similar to fs800, an eggshell precursor protein, tetraspanin 1, SmTYR1, and cathepsin S (B). For each method three biological replicas were used, each with two technical replicas for the microarray analysis (except microarray 2) and three technical replicas for the qPCR. The mean of the technical replicas was calculated and is presented with standard deviations.
	Figure 7. Comparison of qPCR and microarray results of genes involved in eggshell-formation processes.Summary of the log2ratio (treated/control) obtained by qPCR and microarray analyses of genes coding for proteins proven or hypothesised to be involved eggshell-formation processes following treatment of paired female schistosomes with either 300 nM TRIKI, 4.5 µM Herb A, or the combination of both inhibitors. The investigated genes were the eggshell precursor proteins Smp14 and Smp48 as well as a predicted eggshell precursor protein (precursor), the tyrosinase 1 (SmTYR1), and a gene similar to fs800. For each method three biological replicas were used, each with two technical replicas for the microarray analysis (except microarray 2) and three technical replicas for the qPCR. The mean of the technical replicas was calculated and is presented with standard deviations.
	Figure 8. In situ hybridization localizing the schistosome homologs of hippocalcin, ORAI-1, a predicted egg-shell precursor protein gene, and calmodulin-4.Sections (5 µm) of adult schistosome couples (males and females are indicated), were hybridized with DIG-labeled antisense-RNA probes of hippocalcin (A, B), ORAI-1 (D, E), eggshell precursor protein gene (G, H), and calmodulin-4 (J, K). For control, DIG-labeled sense-RNAs probes of hippocalcin (C), ORAI-1 (F), eggshell precursor protein gene (I), and calmodulin-4 (L) were used. Signals were observed in the ovary (o), the vitellarium (v), around the ootype (ot) where Mehli's gland is located, and the testes (t). Scale bar: 20 µm.
	Figure 9. Visual model of the inhibitor effects on transcription of genes involved in eggshell-formation.The inhibition of Src kinases targeted by Herb A and TβRI by TRIKI revealed an influence of both inhibitors on the transcription of genes known to play roles in eggshell formation such as Smp14, Smp48, eggshell precursor protein, SmTYR1 and fs800. Compared to TRIKI treatment, however, a stronger effect on transcription was observed when adult schistosome couples were treated with Herb A. From this we conclude cooperative signal- transduction activities during eggshell-formation with a more dominant role of Src kinases in promoting the expression of involved genes.
	Figure 1. Inhibition of SmTβRI activity by TRIKI.Inhibitory effect of TRIKI on SmTβRI kinase activity was monitored in Xenopus laevis oocytes expressing the intracellular domain of SmTβRI by measuring the level of GVBD induced by the kinase in the oocytes. Results of GVBD assays performed in TRIKI-treated oocytes (used concentrations: 0 nM, 3 nM, 30 nM, 300 nM), which expressed either the constitutively active intracellular part of SmTβRI (SmTβRI7D; the experiments were performed twice: 1°, dark grey squares; 2°, light grey rhombs), or an inactive kinase variant (SmTβRIVVAAAVV, black circles) as control.

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