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 RHΔeIFs 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 (RHΔ315150, RHΔ272640, RHΔ249370, RHΔ211410, and RHΔ224235) 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 RHΔ286090 strain showed slightly reduced invasion efficiency and virulence (p < 0.01) compared to the other five RHΔeIFs 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.

The splicing factor SR2 is an important virulence factor of Toxoplasma gondii.

Serine/arginine-rich (SR) proteins are key factors with important roles in constitutive and alternative splicing (AS) of pre-mRNAs. However, the role of SR splicing factors in the pathogenicity of T. gondii remains largely unexplored. Here, we investigated the role of splicing factor SR2, a homolog of Plasmodium falciparum SR1, in the pathogenicity of T. gondii. We functionally characterized the predicted SR2 in T. gondii by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The results showed that SR2 was localized in the nucleus and expressed in the tachyzoite and bradyzoite stages. In vitro studies including plaque formation, invasion, intracellular replication, egress and bradyzoite differentiation assays showed that deletion of SR2 in type I RH strain and type II Pru strains had no significant effect on the parasite growth and bradyzoite differentiation (p > 0.05). Interestingly, the disruption of SR2 in RH type I (p < 0.0001) and Pru type II (p < 0.05) strains resulted in varying degrees of attenuated virulence. In addition, disruption of SR2 in type II Pru strain significantly reduced brain cyst burden by ~80% (p < 0.0001). Collectively, these results suggest that splicing factor SR2 is important for the pathogenicity of T. gondii, providing a new target for the control and treatment of toxoplasmosis.

Functional Characterization of Eight Zinc Finger Motif-Containing Proteins in Toxoplasma gondii Type I RH Strain Using the CRISPR-Cas9 System.

The Zinc finger protein (ZFP) family is widely distributed in eukaryotes and interacts with DNA, RNA, and various proteins to participate in many molecular processes. In the present study, the biological functions of eight ZFP genes in the lytic cycle and the pathogenicity of Toxoplasma gondii were examined using the CRISPR-Cas9 system. Immunofluorescence showed that four ZFPs (RH248270-HA, RH255310-HA, RH309200-HA, and RH236640-HA) were localized in the cytoplasm, and one ZFP (RH273150-HA) was located in the nucleus, while the expression level of RH285190-HA, RH260870-HA, and RH248450-HA was undetectable. No significant differences were detected between seven RHΔzfp strains (RHΔ285190, RHΔ248270, RHΔ260870, RHΔ255310, RHΔ309200, RHΔ248450, and RHΔ236640) and the wild-type (WT) strain in the T. gondii lytic cycle, including plaque formation, invasion, intracellular replication, and egress, as well as in vitro virulence (p > 0.05). However, the RHΔ273150 strain exhibited significantly lower replication efficiency compared to the other seven RHΔzfp strains and the WT strain, while in vivo virulence in mice was not significantly affected. Comparative expression analysis of the eight zfp genes indicates that certain genes may have essential functions in the sexual reproductive stage of T. gondii. Taken together, these findings expand our current understanding of the roles of ZFPs in T. gondii.