Functional analyses of male-competence factors to unravel their role during the exceptional sex interplay in Schistosoma mansoni

男性能力因素的功能分析，以揭示其在曼氏血吸虫异常性别相互作用中的作用

• 批准号: 62522894
• 负责人: Professor Dr. Christoph G. Grevelding
• 金额: --
• 依托单位: Biomedizinisches Forschungszentrum Seltersberg (BFS)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/62522894?language=en
• 关键词: Functional; analyses; male; competence; factors

Schistosomes cause schistosomiasis, an infectious disease with worldwide meaning. A phenomenon of schistosomes, which are worm parasites, is the dependence of the development of the reproductive organs of the female on a continuous pairing contact with the male. Basis for this gender-interaction are molecular communication processes. Their consequences have been well studied in females, for which some knowledge exists. In males, however, knowledge about these processes is scarce, despite its relevance for basic research. Pairing is the prerequisite for egg production and thus essential for life-cycle maintenance. Furthermore, eggs are responsible for the clinical-pathological consequences of schistosomiasis, culminating in liver fibrosis. Based on transcriptomics with pairing-experienced males (bM) and pairing-unexperienced ones (sM), which were performed in previous funding periods, we have obtained a first comprehensive overview on the complexity of processes that are influence by pairing in males. Besides muscle and metabolism functions, stem cell-associated and particularly neural functions appeared to be important. Therefore, we focused on G protein-coupled receptors (GPCRs) and neuropeptides (Npps). Following a new description of all GPCRs (GPCRome), we analyzed their expression patterns on the basis of our transcriptomics data. We found many GPCRs to be more abundantly transcribed in bM, sM, and/or unpaired females compared to paired ones. This applied also to Npp-coding genes, which we identified by bioinformatics, and which we cloned. Next, we successfully established the MALAR yeast-2-hybrid system to identify Npp-GPCR interaction, and thus to “de-orphanize” GPCRs. We plan to confirm these interactions by a Fluorescence Resonance Energy Transfer (FRET) assays. By whole mount in situ hybridisation, we localised gpcr and npp transcripts, and Npps also by mass spectrometry imaging. First functional studies of Npp-GPCR partners showed interesting phenotypes such as deficits in egg production. For GPCR 9, which is testis-specific and pairing-dependently expressed, we discovered a role in spermatogenesis. We plan to extend these characterizations to all GPCRs, which exhibit this male-dominated expression profile. This allows a deeper insight into the biological relevance of these GPCRs and their ligands as well as their potential role(s) as male competence (MC) factors. Functional analyses of genes involved in dopamine synthesis, which are male-specific and pairing-dependently transcribed, confirmed their meaning for gonad differentiation in paired females. This is the first proof for the existence of MC factors. According to a meta-analysis, these genes start expression at the schistosomula stage. This motivates us to start KD of these genes also in this life stage using a new in vitro culture system, which allows rearing fully differentiated adult worms starting with schistosomula.

血吸虫引起血吸虫病，这是一种具有全球意义的传染病。血吸虫是一种寄生虫，其现象是雌性生殖器官的发育依赖于与雄性的持续配对接触。这种性别互动的基础是分子交流过程。它们的后果在女性身上已经得到了很好的研究，对此也有一些了解。然而，在男性中，关于这些过程的知识很少，尽管它与基础研究有关。配对是产卵的先决条件，因此对生命周期的维持至关重要。此外，虫卵对血吸虫病的临床病理后果负责，最终导致肝纤维化。基于之前资助时期对配对有经验的男性(BM)和配对-无经验的男性(SM)进行的转录本研究，我们首次全面概述了男性配对影响的过程的复杂性。除了肌肉和新陈代谢功能，干细胞相关的功能，特别是神经功能似乎也很重要。因此，我们重点研究了G蛋白偶联受体(GPCRs)和神经肽(NPPs)。在对所有GPCRs(GPC罗马)进行新的描述之后，我们基于我们的转录数据分析了它们的表达模式。我们发现许多GPCR在BM、SM和/或未配对的雌性中比配对的女性转录得更丰富。这也适用于NPP编码基因，我们通过生物信息学鉴定了这些基因，并进行了克隆。接下来，我们成功地建立了苹果酵母-2-杂交系统来鉴定NPP-GPCR的相互作用，从而实现了GPCRs的去孤儿。我们计划通过荧光共振能量转移(FRET)分析来确认这些相互作用。通过整装原位杂交，我们定位了GPCR和NPP转录本，也通过质谱学成像定位了NPP。首先，对NPP-GPCR合作伙伴的功能研究显示了有趣的表型，例如鸡蛋生产中的缺陷。对于GPCR9，它是睾丸特异的和配对依赖的表达，我们发现了在精子发生中的作用。我们计划将这些特征扩展到所有GPCR，这些GPCR表现出男性主导的表达模式。这使得对这些GPCR及其配体的生物学相关性以及它们作为男性能力(MC)因素的潜在作用(S)有了更深入的了解。对参与多巴胺合成的基因的功能分析证实了它们对配对女性性腺分化的意义。多巴胺合成是男性特有的，依赖于配对转录。这是MC因子存在的第一个证明。根据荟萃分析，这些基因在血吸虫阶段开始表达。这促使我们在这个生命阶段使用一种新的体外培养系统开始这些基因的KD，该系统允许从血吸虫开始饲养完全分化的成虫。