Transcriptome analyses of inhibitor-treated schistosome females provide evidence for cooperating Src-kinase and TGFβ receptor pathways controlling mitosis and eggshell formation.
Transcriptome analyses of inhibitor-treated schistosome females provide evidence for cooperating Src-kinase and TGFβ receptor pathways controlling mitosis and eggshell formation.
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DOI：
10.1371/journal.ppat.1003448
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发表时间：
2013
期刊：
影响因子：
6.7
通讯作者：
Grevelding CG
中科院分区：
文献类型：
--
作者：
Buro C;Oliveira KC;Lu Z;Leutner S;Beckmann S;Dissous C;Cailliau K;Verjovski-Almeida S;Grevelding CG
血吸虫病是世界上影响人类和动物的最普遍的寄生虫之一。血吸虫的持续配对对雌性性成熟和产卵至关重要，这导致了发病。雌性成熟涉及控制生殖腺内有丝分裂和分化的信号通路。体外研究表明，src特异性抑制剂Herbimycin a （Herb a）和TGFβ受体（TβR）抑制剂TRIKI在配对雌性中具有抑制有丝分裂和产卵等生理作用。作为Herb A的一个靶点，我们确定了性腺特异性表达的Src激酶SmTK3。在这里，我们比较分析了草本A和triki处理的雌性的转录组谱，确定了src -激酶和t - β ri通路的转录靶点。通过在非洲爪蟾卵母细胞中进行激酶试验证明TRIKI抑制血吸虫tgf - β受体smt - β ri后，在体外同时使用Herb A、TRIKI或两种抑制剂对配对进行处理。从雌性中分离RNA进行微阵列杂交和转录分析。通过基因本体（GO）和匠心路径分析（IPA），以及人工分类和交叉分析对获得的数据进行评估。最后，进行了广泛的qPCR实验，以验证候选基因在抑制剂影响下的差异转录，并在功能上加强特定的生理效应。许多被发现受差异调控的基因与有丝分裂和分化有关。其中包括钙相关基因和蛋壳形成基因。原位杂交证实了钙传感器hippocalcin、钙转运蛋白ORAI-1和钙结合蛋白calmodulin-4在生殖系统中的编码基因转录，这表明钙在寄生虫繁殖中的作用。功能性qPCR结果证实了Smp14、Smp48、fs800（一种预测的蛋壳前体蛋白）和SmTYR1的转录活性受抑制的不同依赖性。结果表明，蛋壳形成受至少两种途径的调控，它们以一种平衡的方式共同作用，从而控制产蛋。血吸虫病是世界上最流行的寄生虫感染之一，由血吸虫属的血吸虫引起。病理与产蛋一致，产蛋开始于雌雄异株生活的成虫配对。需要持续的配对接触来诱导有丝分裂和分化过程，从而导致性腺的发育。虽然人们早就知道，控制血吸虫或其他扁形蠕虫性腺发育或产卵的分子过程在很大程度上是未知的。利用体外培养系统和特异性的化学抑制剂，我们在之前的研究中首次获得了信号转导过程在控制有丝分裂、分化和卵子产生中发挥重要作用的证据。在本研究中，我们将组合抑制剂处理与随后的微阵列和qPCR分析相结合，并首次证明了Src-Kinase-和tgf - β-信号通路的合作控制有丝分裂和卵子形成过程。除了管理蛋壳形成基因转录的直接证据外，还发现了这些途径的新靶分子。在这些基因中，钙相关基因为这种离子在生殖中的作用提供了第一个线索。我们的发现首次揭示了控制卵子形成的信号机制，这对生命周期和病理都很重要。
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. As one of the most prevalent parasitic infections worldwide, schistosomiasis is caused by blood-flukes of the genus Schistosoma. Pathology coincides with egg production, which is started upon pairing of the dioeciously living adults. A constant pairing contact is required to induce mitoses and differentiation processes in the female leading to the development of the gonads. Although long known, the molecular processes controlling gonad development or egg-production in schistosomes or other platyhelminths are largely unknown. Using an established in vitro-culture system and specific, chemical inhibitors we have obtained first evidence in previous studies for the participation of signal transduction processes playing essential roles in controlling mitoses, differentiation and egg production. In the present study we applied combinatory inhibitor treatments combined with subsequent microarray and qPCR analyses and demonstrate for the first time that cooperating Src-Kinase- und TGFβ-signaling pathways control mitoses and egg formation processes. Besides direct evidence for managing transcription of eggshell-forming genes, new target molecules of these pathways were identified. Among these are calcium-associated genes providing a first hint towards a role of this ion for reproduction. Our finding shed first light on the signaling mechanisms controlling egg formation, which is important for life-cycling and pathology.
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