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

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 RHΔ 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 (RHΔ 315150 , RHΔ 272640 , RHΔ 249370 , RHΔ 211410 , and RHΔ 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 RHΔ 286090 strain showed slightly reduced invasion efficiency and virulence ( p < 0.01) compared to the other five RHΔ eIFs strains and the wild-type strain. The disruption of the TGGT1&#95;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&#95;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 .