*Toxoplasma gondii* is an obligate intracellular parasite infecting a significant portion of the human population. Infection begins with rapidly replicating tachyzoites, but some differentiate into latent bradyzoites forming tissue cysts, leading to chronic infection. These bradyzoites can reactivate, causing severe disease in immunocompromised individuals. Currently, there are no effective treatments to eliminate chronic *T. gondii* infection. While stressors like alkaline pH can trigger in vitro differentiation, the underlying molecular mechanisms remain largely unknown. The tachyzoite-to-bradyzoite transition involves substantial changes, including parasitophorous vacuole membrane remodeling, metabolic shifts, starch granule accumulation, and altered gene and protein expression. mRNA translation plays a vital role, with translational control being critical for differentiation in many protozoan parasites. Key examples include the regulation of eIF2α phosphorylation and the role of eIF4E1, highlighting the complex translation-directed regulatory landscape. This study aimed to identify additional factors involved in *T. gondii* differentiation, focusing on the translation initiation factor eIF1.2.

Previous research highlighted the importance of autophagy in bradyzoite viability and identified cathepsin protease L (CPL) and ATG9 as key genes involved in bradyzoite autophagy. The master regulator of bradyzoite differentiation, BFD1, is known to be regulated by the RNA-binding protein BFD2/ROCY. Studies on other parasites have shown that the phosphorylation of eIF2α is crucial for stage conversion in *Leishmania*, *Plasmodium*, *Trypanosoma*, and *T. gondii*. The absence of eIF4E1 has also been shown to trigger *T. gondii* differentiation, suggesting a complex interplay of translational control mechanisms.

The study employed several techniques to investigate the role of eIF1.2 in *T. gondii* differentiation. A chemical mutagenesis screen using ENU, coupled with FACS sorting, identified mutants with altered bradyzoite formation. Whole genome sequencing identified mutations in the candidate genes. CRISPR-Cas9 gene editing was used to introduce individual mutations into WT parasites for validation. Single-molecule scanning assays were used to analyze the effects of the eIF1.2 F97L mutation on ribosome preinitiation complex scanning dynamics. Gel shift assays assessed eIF1.2 binding to the 40S ribosomal subunit. RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) were conducted to identify differentially expressed genes and changes in translational efficiency. In vivo experiments using mice were performed to assess the impact of eIF1.2 mutations on acute virulence and chronic infection. Conditional expression of BFD1 and BFD2 was used to determine if these factors could rescue the differentiation defects in eIF1.2-deficient parasites. Immunofluorescence assays and western blotting quantified protein levels and cyst formation. qPCR was used to measure parasite burden in mice brains. Polysome profiling was used to assess the overall impact of eIF1.2 loss on translation. Finally, phylogenetic analysis examined the evolution of eIF1 in apicomplexan parasites. Statistical analyses included Student’s two-tailed t-test, Poisson model, linear regression model, linear mixed model, Wilcoxon rank-sum test, and Welch’s two-tailed t-test, among others.

A mutagenesis screen unexpectedly revealed a mutant with decreased GFP expression (driven by the bradyzoite-specific *LDH2* promoter) and increased tdTomato-ATG8 signal (autophagy marker). The mutant clone (5E4) contained a F97L mutation in eIF1.2. The F97L mutation in eIF1.2 did not affect tachyzoite growth but significantly impaired bradyzoite cyst formation in vitro and in vivo, resulting in substantially lower parasite burden in mice brains. Single-molecule scanning assays showed the F97L mutation altered preinitiation complex scanning, potentially affecting start codon selection. Deletion of *eIF1.2* also resulted in impaired bradyzoite formation, confirming the loss-of-function nature of the F97L mutation. RNA-seq and Ribo-seq revealed differential expression of many stage-specific genes in both unstressed and stressed ΔeIF1.2 parasites, including downregulation of bradyzoite genes and upregulation of tachyzoite genes. Specifically, eIF1.2 deficiency reduced BFD1 and BFD2 induction. Forced expression of BFD1 or BFD2 rescued differentiation in ΔeIF1.2 parasites. The study also revealed that the absence of eIF1.2 affected the translational efficiency of many mRNAs, including BFD1 and CST4.

The findings demonstrate that eIF1.2 is essential for *T. gondii* differentiation by regulating the expression of key differentiation factors like BFD1 and BFD2. The F97L mutation disrupts preinitiation complex scanning, impacting the translation of these factors. The observation that eIF1.2 deficiency affects both transcription and translation suggests a multifaceted regulatory role. This study also highlights the importance of translational control in *T. gondii* differentiation. The differences in dwell times observed in single-molecule assays show how the F97L mutation alters scanning dynamics. The rescue of differentiation by forced expression of BFD1 or BFD2 supports the role of eIF1.2 in regulating these specific factors. The observation that only one eIF1 paralog is found in non-cyst-forming apicomplexans suggests that this paralog may have evolved to facilitate cyst formation.

This study identified eIF1.2 as a key regulator of *T. gondii* differentiation, acting through regulation of BFD1 and BFD2 translation. The findings provide valuable insight into the complex molecular mechanisms governing stage conversion and could inform the development of novel strategies to target chronic toxoplasmosis. Future research should focus on elucidating how eIF1.2 is itself regulated and on identifying additional factors involved in this regulatory pathway. Further investigations into the precise mechanistic interactions of eIF1.2 with the translational machinery and mRNA regulatory elements will further advance our understanding of *T. gondii* pathogenesis.

The study primarily focused on two key differentiation factors, BFD1 and BFD2, while other factors are undoubtedly involved. While the in vivo studies provided valuable insights into chronic infection, it is not fully elucidated what impact the mutation in eIF1.2 could have on the acute phase of infection. The single-molecule scanning assays were performed using a model mRNA. While this approach provided valuable insights into the mechanistic effects of the mutations, the results may not fully capture the complexity of in vivo translation initiation. The effect of the HA tag in complementation experiments could have some influence on the results.