Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, Jinzhong 030801, China.
State Key Laboratory for Animal Disease Control and Prevention, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
Int J Mol Sci. 2024 Jul 17;25(14):7834. doi: 10.3390/ijms25147834.
Eukaryotic translation initiation factors (eIFs) are crucial for initiating protein translation and ensuring the correct assembly of mRNA-ribosomal subunit complexes. In this study, we investigated the effects of deleting six eIFs in the apicomplexan parasite using the CRISPR-Cas9 system. We determined the subcellular localization of these eIFs using C-terminal endogenous tagging and immunofluorescence analysis. Four eIFs (RH::315150-6HA, RH::286090-6HA, RH::249370-6HA, and RH::211410-6HA) were localized in the cytoplasm, while RH::224235-6HA was localized in the apicoplast. Additionally, RH::272640-6HA was found in both the basal complex and the cytoplasm of . Functional characterization of the six RHΔ strains was conducted using plaque assay, cell invasion assay, intracellular growth assay and egress assay in vitro, and virulence assay in mice. Disruption of five eIF genes (RHΔ, RHΔ, RHΔ, RHΔ, and RHΔ) did not affect the ability of the RH strain to invade, replicate, form plaques and egress in vitro, or virulence in Kunming mice ( > 0.05). However, the RHΔ strain showed slightly reduced invasion efficiency and virulence ( < 0.01) compared to the other five RHΔ strains and the wild-type strain. The disruption of the TGGT1_286090 gene significantly impaired the ability of tachyzoites to differentiate into bradyzoites in both type I RH and type II Pru strains. These findings reveal that the eukaryotic translation initiation factor TGGT1_286090 is crucial for bradyzoite differentiation and may serve as a potential target for drug development and an attenuated vaccine against .
真核翻译起始因子 (eIFs) 对于启动蛋白质翻译和确保 mRNA-核糖体亚基复合物的正确组装至关重要。在这项研究中，我们使用 CRISPR-Cas9 系统研究了删除 顶复门寄生虫中的六个 eIF 的影响。我们通过 C 末端内源性标记和免疫荧光分析确定了这些 eIF 的亚细胞定位。四个 eIF（RH::315150-6HA、RH::286090-6HA、RH::249370-6HA 和 RH::211410-6HA）定位于细胞质中，而 RH::224235-6HA 定位于顶质体中。此外，RH::272640-6HA 存在于 的基底复合物和细胞质中。我们使用噬菌斑测定、细胞入侵测定、细胞内生长测定和体外出芽测定以及在小鼠中的毒力测定对六个 RHΔ 株系进行了功能表征。五个 eIF 基因（RHΔ、RHΔ、RHΔ、RHΔ 和 RHΔ）的破坏不影响 RH 株系在体外入侵、复制、形成噬菌斑和出芽以及昆明小鼠中的毒力（ > 0.05）。然而，与其他五个 RHΔ 株系和野生型株系相比，RHΔ 株系的入侵效率和毒力略低（ < 0.01）。TGGT1_286090 基因的破坏显著降低了 RH 和 Pru 型 I 和 II 两种类型中的速殖子分化为缓殖子的能力。这些发现表明，真核翻译起始因子 TGGT1_286090 对缓殖子分化至关重要，可能成为药物开发和针对 的减毒疫苗的潜在靶标。
