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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\u0394eIFs 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\u0394315150, RH\u0394272640, RH\u0394249370, RH\u0394211410, and RH\u0394224235) 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 &gt; 0.05). However, the RH\u0394286090 strain showed slightly reduced invasion efficiency and virulence (p &lt; 0.01) compared to the other five RH\u0394eIFs 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.<\/jats:p>","DOI":"10.3390\/ijms25147834","type":"journal-article","created":{"date-parts":[[2024,7,17]],"date-time":"2024-07-17T16:35:58Z","timestamp":1721234158000},"page":"7834","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Functional Characterization of Six Eukaryotic Translation Initiation Factors of Toxoplasma gondii Using the CRISPR-Cas9 System"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-1569-3890","authenticated-orcid":false,"given":"Yong-Jie","family":"Kou","sequence":"first","affiliation":[{"name":"Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, Jinzhong 030801, China"},{"name":"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"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jin","family":"Gao","sequence":"additional","affiliation":[{"name":"Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, Jinzhong 030801, China"},{"name":"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"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rui","family":"Li","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhi-Ya","family":"Ma","sequence":"additional","affiliation":[{"name":"Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, Jinzhong 030801, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3303-930X","authenticated-orcid":false,"given":"Hany M.","family":"Elsheikha","sequence":"additional","affiliation":[{"name":"Faculty of Medicine and Health Sciences, School of Veterinary Medicine and Science, Sutton Bonington Campus, University of Nottingham, Loughborough LE12 5RD, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiao-Jing","family":"Wu","sequence":"additional","affiliation":[{"name":"Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, Jinzhong 030801, China"},{"name":"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"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiao-Nan","family":"Zheng","sequence":"additional","affiliation":[{"name":"Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, Jinzhong 030801, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Meng","family":"Wang","sequence":"additional","affiliation":[{"name":"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"},{"name":"Institute of Urban Agriculture, Chinese Academy of Agricultural Sciences, Chengdu 610213, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2530-4628","authenticated-orcid":false,"given":"Xing-Quan","family":"Zhu","sequence":"additional","affiliation":[{"name":"Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, Jinzhong 030801, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,7,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1038\/s41579-021-00518-7","article-title":"Toxoplasma gondii infection and its implications within the central nervous system","volume":"19","author":"Matta","year":"2021","journal-title":"Nat. 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