Functional Characterization of Six Eukaryotic Translation Initiation Factors of Toxoplasma gondii Using the CRISPR-Cas9 System

文献类型： 外文期刊

第一作者： Kou, Yong-Jie

作者： Kou, Yong-Jie;Gao, Jin;Ma, Zhi-Ya;Wu, Xiao-Jing;Zheng, Xiao-Nan;Zhu, Xing-Quan;Kou, Yong-Jie;Gao, Jin;Li, Rui;Wu, Xiao-Jing;Wang, Meng;Elsheikha, Hany M.;Wang, Meng

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关键词： Toxoplasma gondii; eukaryotic protein translation initiation factors; CRISPR-Cas9; differentiation

期刊名称：INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES （ 影响因子：4.9； 五年影响因子：5.6 ）

ISSN： 1661-6596

年卷期： 2024 年 25 卷 14 期

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收录情况： SCI

摘要： 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 Toxoplasma gondii 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 T. gondii. Functional characterization of the six RH Delta eIFs 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 (RH Delta 315150, RH Delta 272640, RH Delta 249370, RH Delta 211410, and RH Delta 224235) did not affect the ability of the T. gondii RH strain to invade, replicate, form plaques and egress in vitro, or virulence in Kunming mice (p > 0.05). However, the RH Delta 286090 strain showed slightly reduced invasion efficiency and virulence (p < 0.01) compared to the other five RH Delta eIFs 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 T. gondii bradyzoite differentiation and may serve as a potential target for drug development and an attenuated vaccine against T. gondii.

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