东方蜜蜂微孢子虫孢壁蛋白及其与侵染功能相关性研究

Insect microsporidia were well known as a class of fungi and their spore wall proteins were reported to play important roles in recognition and infection to the host, but their mechanism of function and other related scientific issues have not yet been resolved. So far, to systematically uncover the mode of action of spore wall proteins had became a bottleneck to clearly understanding the mechanism of microsporidia to infect their hosts. Hence, it was urgent to perform works associated with spore wall proteins in microsporidia. Microsporidiosis of honeybee is caused by Nosema ceranae，its rapidly-epidemic spreading in the colonies of bees brings great losses to the industry and bee products and it was regarded as an important quarantine disease of bees. The most noteworthy is that the works on spore wall proteins of Nosema ceranae was relatively weaker. Thus, it is necessary to systematically performing the studies on identification of Nosema ceranae spore wall proteins, the interaction with the host and furtherly the infection mechanism of the spores. In our previous study, we have obtained 14 hypothetical spore wall protein genes based on the reported genome data of Nosema ceranae and partial genes were expressed in prokaryotic cells and performed subcellular localization. Based on our established platforms and relative researches, we intend to systematically explore the functions of Nosema ceranae spore wall proteins in the followings: identification of spore wall proteins through genome scanning and protein technology, screening of infection related spore wall proteins, selection of interacted proteins in host with infection related spore wall protein and explore the mode of their interactions. This project's development has important reference value for illuminating the infection mechanism of Nosema ceranae and prevention and control of microsporidiosis of honeybee.

昆虫微孢子虫作为真菌类，在入侵宿主时，其孢壁蛋白在宿主识别与侵染过程中发挥重要作用，其机制等相关科学问题尚未解决，这已成为从整体上揭示微孢子虫侵染宿主机理的瓶颈问题，因而，开展微孢子虫孢壁蛋白的研究十分紧迫。蜜蜂微孢子虫病是由蜜蜂微孢子虫引起的蜜蜂重要检疫性病害，给蜜蜂产业造成严重危害。但目前对蜜蜂微孢子虫的研究，尤其对其孢壁蛋白的研究十分薄弱，系统开展蜜蜂微孢子虫孢壁蛋白鉴定及与宿主互作研究，进而分析其在入侵蜜蜂过程中的作用机制尤为必要。我们在对东方蜜蜂微孢子虫基因组数据分析中，获得14条孢壁蛋白候选序列，并进行了初步探讨。本项目聚焦蜜蜂微孢子虫孢壁蛋白的鉴定及其与侵染相关功能，从基因组与蛋白质水平上鉴定孢壁蛋白、筛选与侵染相关的组分，分析其与宿主特别是宿主中肠蛋白是否互作，并对其互作方式进行探讨。本项目的开展将为阐明蜜蜂微孢子虫的侵染机制及蜜蜂微孢子虫病的防治奠定基础。

昆虫微孢子虫作为真菌类，在入侵宿主时，其孢壁蛋白在宿主识别与侵染过程中发挥重要作用，开展微孢子虫孢壁蛋白的研究十分必要。蜜蜂微孢子虫病是由蜜蜂微孢子虫引起的蜜蜂重要检疫性病害，给蜜蜂产业造成严重危害。但目前对蜜蜂微孢子虫的研究，尤其对其孢壁蛋白的研究十分薄弱。本项目在基因组和蛋白质水平上共筛选获得20个蜜蜂微孢子虫候选孢壁蛋白，对其中的14个蛋白进行了外源重组表达，制备了14个候选孢壁蛋白的多克隆抗体；利用间接免疫荧光和免疫胶体金法完成了7个孢壁蛋白的亚细胞定位，其中6个主要定位于成熟孢子的内壁，1个主要定位于成熟孢子的外壁。生物学功能研究发现Ncswp7、NcSWP8和NcSWP12蛋白被抗体封闭后，能够导致孢子致病能力和侵染能力显著下降，表明Ncswp7、NcSWP8和NcSWP12蛋白在蜜蜂微孢子虫侵染宿主过程中扮演了重要的角色。孢壁蛋白与宿主中肠蛋白的Far-western blotting结果表明，Ncswp7、NcSWP8和rNcER_101151能够与宿主中肠蛋白发生相互作用，质谱法筛选获得了候选的中肠互作蛋白，将有助于深入了解Nc与宿主互作及其入侵宿主的机制。另外，NcSWP12和NcER_101151中存在肝素结合基序，肝素结合实验表明其能够与肝素结合，突变肝素结合关键位点能引起蛋白与肝素的结合力减弱，这表明二者具有与宿主细胞表面的糖胺聚糖结合的能力，进而介导孢子侵染宿主细胞。对孢壁蛋白参与孢子构成的研究表明，rAAJ76-140036761、rNcER_101151、rNcSWP7、rNcSWP8和rNcSWP12具备与孢子几丁质壳结合的能力，表明这些蛋白在微孢子虫孢壁骨架的构成，维持微孢子虫刚性和功能上至关重要。最后，通过Far-western blotting方法和Co-IP法构建了微孢子虫孢壁蛋白间的互作网络图，绘制了孢壁蛋白rNcSWP7、rNcSWP8 和rNcER_101151的孢壁蛋白互作网络与作用机制模式图。总的来讲，通过本项目的研究鉴定得到7个蜜蜂微孢子虫孢壁蛋白，筛选到5个与微孢子虫侵染宿主相关的孢壁蛋白，绘制了3个孢壁蛋白的互作网络与作用机制图，这能够为系统地揭示微孢子虫孢壁蛋白在介导侵染和孢子构建中的功能提供丰富的数据，进而为蜜蜂微孢子虫侵染机制的揭示和蜜蜂微孢子虫病的防治提供基础。