Methyl-CpG-binding (SmMBD2/3) and chromobox (SmCBX) proteins are required for neoblast proliferation and oviposition in the parasitic blood fluke Schistosoma mansoni

While schistosomiasis remains a significant health problem in low to middle income countries, it also represents a recently recognised threat to more economically-developed regions. Until a vaccine is developed, this neglected infectious disease is primarily controlled by praziquantel, a drug with a currently unknown mechanism of action. By further elucidating how Schistosoma molecular components cooperate to regulate parasite developmental processes, next generation targets will be identified. Here, we continue our studies on schistosome epigenetic participants and characterise the function of a DNA methylation reader, the Schistosoma mansoni methyl-CpG-binding domain protein (SmMBD2/3). Firstly, we demonstrate that SmMBD2/3 contains amino acid features essential for 5-methyl cytosine (5mC) binding and illustrate that adult schistosome nuclear extracts (females > males) contain this activity. We subsequently show that SmMBD2/3 translocates into nuclear compartments of transfected murine NIH-3T3 fibroblasts and recombinant SmMBD2/3 exhibits 5mC binding activity. Secondly, using a yeast-two hybrid (Y2H) screen, we show that SmMBD2/3 interacts with the chromo shadow domain (CSD) of an epigenetic adaptor, S. mansoni chromobox protein (SmCBX). Moreover, fluorescent in situ hybridisation (FISH) mediated co-localisation of Smmbd2/3 and Smcbx to mesenchymal cells as well as somatic- and reproductive- stem cells confirms the Y2H results and demonstrates that these interacting partners are ubiquitously expressed and found within both differentiated as well as proliferating cells. Finally, using RNA interference, we reveal that depletion of Smmbd2/3 or Smcbx in adult females leads to significant reductions (46–58%) in the number of proliferating somatic stem cells (PSCs or neoblasts) as well as in the quantity of in vitro laid eggs. Collectively, these results further expand upon the schistosome components involved in epigenetic processes and suggest that pharmacological inhibition of SmMBD2/3 and/or SmCBX biology could prove useful in the development of future schistosomiasis control strategies.

血吸虫病（schistosomiasis）在中低收入国家仍是重大公共卫生问题，同时近年来也被证实对经济更为发达的地区构成威胁。在疫苗研发成功之前，这种被忽视的传染病主要依靠吡喹酮（praziquantel）进行防控，而该药物的作用机制目前仍未阐明。通过进一步解析血吸虫（Schistosoma）的分子组分如何协同调控寄生虫的发育过程，有望鉴定出新一代药物靶点。本研究延续了我们在血吸虫表观遗传调控因子方面的研究，对曼氏血吸虫（Schistosoma mansoni）的DNA甲基化阅读器（DNA methylation reader）——甲基-CpG结合域蛋白（SmMBD2/3）的功能进行了表征。首先，本研究证实SmMBD2/3具备与5-甲基胞嘧啶（5mC）结合所必需的氨基酸特征，并发现成年血吸虫的核提取物（雌性提取物活性高于雄性）具有该结合活性。后续实验表明，SmMBD2/3可易位进入转染后的小鼠NIH-3T3成纤维细胞的核区室，且重组表达的SmMBD2/3具备5mC结合活性。其次，本研究通过酵母双杂交（yeast-two hybrid, Y2H）筛选实验，证实SmMBD2/3可与表观遗传衔接蛋白——曼氏血吸虫chromobox蛋白（SmCBX）的染色质阴影结构域（chromo shadow domain, CSD）相互作用。此外，通过荧光原位杂交（fluorescent in situ hybridisation, FISH）实验，我们发现Smmbd2/3与Smcbx共定位于间质细胞、体细胞干细胞及生殖干细胞中，这一结果验证了酵母双杂交实验的结论，同时证实这两种互作蛋白广泛表达于分化细胞与增殖细胞中。最后，本研究通过RNA干扰（RNA interference, RNAi）实验发现，在成年雌性血吸虫中敲低Smmbd2/3或Smcbx的表达，可使增殖性体细胞干细胞（PSCs或成体干细胞neoblasts）的数量以及体外产卵量显著降低46%至58%。综上，本研究结果进一步拓展了血吸虫表观遗传调控相关组分的研究范畴，同时提示靶向抑制SmMBD2/3与/或SmCBX的生物学功能，有望为未来血吸虫病防控策略的开发提供新的思路。