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

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.