家蚕微粒子虫分泌蛋白己糖激酶NbHK对家蚕基因表达及相关通路的调控作用

Hexokinase of Nosema bombycis modulates gene expression and related pathways in silkworm

摘要:

【目的】微孢子虫是一种营专性细胞内寄生的微生物，它可以感染几乎所有动物种类，包括人类和重要的经济动物。本研究对家蚕微粒子虫分泌蛋白己糖激酶(Nosema bombycis hexokinase, NbHK)在家蚕胚胎细胞中表达特征、亚细胞定位、调控作用和宿主互作蛋白质进行了系统分析，为阐明该蛋白在侵染中的作用与机理提供参考。【方法】利用原核表达蛋白免疫小鼠，制备NbHK的多克隆抗体，并利用Western blotting和间接免疫荧光法分析家蚕微粒子虫在感染的家蚕胚胎细胞(Bombyx mori embryo, BmE)中的表达和定位；通过过表达和RNA干扰实验，分析NbHK对病原增殖的作用；利用RNA-seq分析NbHK调控的家蚕基因表达和通路；利用生物素-链霉亲和素系统和质谱技术，从NbHK::APEX2转基因细胞中分离鉴定NbHK的互作蛋白。【结果】在感染家蚕微粒子虫的BmE中，NbHK持续上调表达，主要被定位于宿主细胞核内。过表达NbHK显著促进了病原增殖，而敲低NbHK则明显抑制了病原增殖，说明在NbHK感染过程中发挥关键作用。利用RNA-seq分析鉴定了94个差异表达基因(differentially expressed genes, DEGs)，其中58个基因上调，36个基因下调。DEGs的富集分析显示，细胞寿命和内质网蛋白加工通路受到显著激活，而线粒体自噬途径受到明显抑制。互作蛋白鉴定分析发现，NbHK可能与宿主细胞核内的核蛋白易位启动子区(nucleoprotein translocated promoter region, NTPR)等蛋白间存在相互作用。【结论】NbHK主要被定位至家蚕细胞核中，调控家蚕细胞寿命等多个重要通路的基因表达，以利于病原增殖。本研究为深入解析NbHK在感染过程中的功能及其调控机理提供了新的参考。

Abstract:

[Objective] Microsporidia are obligate intracellular parasites capable of infecting a wide range of animal species, including both humans and animals of economic interests. We explored Nosema bombycis hexokinase (NbHK) in terms of the expression, subcellular localization, regulatory functions, and interacting proteins in Bombyx mori embryo cells, aiming to provide insights into the function and mechanism of this protein during infection. [Methods] We prepared a polyclonal antibody against NbHK to analyze the expression and localization of NbHK in N. bombycis-infected BmE cells by using Western blotting and the indirect immunofluorescent assay. Overexpression and RNA interference experiments were performed to assess the impact of NbHK on pathogen proliferation. RNA-seq was employed to analyze the transcriptional responses of the NbHK-transgenic BmE cells. A biotin-streptavidin system and mass spectrometry were employed to identify the interacting proteins of NbHK from NbHK::APEX2-transgenic BmE cells. [Results] NbHK was predominantly localized in the nucleus of infected cells, with consistently upregulated expression during infection. The overexpression of NbHK significantly increased the pathogen load, while the knock-down of NbHK suppressed pathogen proliferation, which indicated the crucial roles of NbHK during infection. RNA-seq analysis identified 94 differentially expressed genes (DEGs) responsive to infection, comprising 58 up-regulated genes and 36 down-regulated genes. The enrichment analysis of DEGs revealed significant activation of pathways related to cell lifespan regulation and protein processing in the endoplasmic reticulum while significant inhibition of the mitophagy pathway. Additionally, we identified host proteins including nucleoprotein translocated promoter region (NTPR) in the nucleus that potentially interacted with NbHK. [Conclusion] NbHK is secreted into silkworm nucleus to modulate the expression of genes involved in multiple pathways for promoting pathogen proliferation. Our study offers novel insights into the roles of NbHK in the infection of N. bombycis.