Translation initiation factor eIF1.2 promotes *Toxoplasma* stage conversion by regulating levels of key differentiation factors

Toxoplasma gondii infects felids and a wide range of warm-blooded vertebrates, including humans, with approximately one-third of the global human population seropositive. Following infection, rapidly replicating tachyzoites disseminate and a subset differentiates into latent bradyzoites within tissue cysts, enabling lifelong persistence and posing risks of recrudescence in immunocompromised individuals. Despite external stresses (e.g., alkaline pH) triggering in vitro differentiation, the molecular mechanisms orchestrating the tachyzoite-to-bradyzoite transition remain incompletely understood. The transition entails extensive remodeling of parasite biology and gene expression, implying a critical role for translational control. This study tests the hypothesis that translation initiation factor eIF1.2 is essential for stage conversion by regulating expression of master differentiation regulators and other bradyzoite determinants.

Translational control is pivotal in protozoan parasite differentiation. The eukaryotic initiation factor 2α (eIF2α) is a well-studied regulator; stress-induced phosphorylation reduces global protein synthesis while favoring translation of adaptive mRNAs and is crucial for stage conversion in Leishmania, Plasmodium, Trypanosoma, and T. gondii. More recently, loss of the cap-binding protein eIF4E1 was shown to trigger T. gondii differentiation, acting in parallel with eIF2α-dependent control, indicating a complex translational regulatory landscape. The master regulator BFD1 is translationally regulated in part by the RNA-binding protein BFD2/ROCY, forming a positive feedback loop controlling chronic differentiation. Apicomplexans that form tissue cysts often harbor two eIF1 paralogs, suggesting subfunctionalization for differentiation-specific roles. These prior findings support investigating additional initiation factors, such as eIF1.2, in controlling start-site selection and translation of differentiation factors.

• ENU mutagenesis and FACS screen: Performed chemical mutagenesis (3.0 mM ENU) on PruΔKu80 LUC tdTomato-ATG8 parasites expressing GFP under the bradyzoite-specific LDH2 promoter. After 7 days of alkaline stress (RPMI, pH 8.2–8.3), parasites were sorted by FACS for GFP(+) with top 5% tdTomato-ATG8 signal across multiple rounds. Mutant populations were subcloned and subjected to whole-genome sequencing and SNP calling.
• CRISPR-Cas9 editing: Introduced candidate SNVs from mutant clone 5E4 into WT; generated targeted eif1.2 F97L mutation; created Δeif1.2 knockout by replacing the gene with a GFP cassette in ME49ΔKu80; generated an HA-tagged eIF1.2 complementation at the endogenous locus; additional genetic constructs for conditional expression and tagging of BFD1 (DD-BFD1-Ty) and BFD2 (DD-HA-BFD2) and combined with Δeif1.2 background.
• In vitro differentiation assays: Induced bradyzoite formation by alkaline stress for 7 days; assessed cyst-like structures by phase-contrast microscopy and cyst wall formation by Dolichos biflorus agglutinin (DBA) staining; quantified BAG1 (bradyzoite) and SAG1 (tachyzoite) proteins by immunoblot; quantified GFP (LDH2 promoter) and tdTomato-ATG8 by flow cytometry and IF.
• Plaque assays: Measured parasite growth (plaque number and size) for WT, eIF1.2 F97L, Δeif1.2, and complemented lines.
• In vivo mouse studies: Assessed acute-stage replication by luciferase bioluminescence imaging (C57BL/6) after infection with Pru WT or eIF1.2 F97L; measured survival and body weight. Quantified brain parasite burden at 5 weeks by qPCR (B1 gene). In CBA/J mice, quantified brain cyst burden at 5 weeks after infection with ME49 WT, Δeif1.2, or complemented strain with blinded counting.
• Biochemistry and single-molecule assays: Native gel shift assays to evaluate binding of T. gondii eIF1.2 (WT and F97L) to yeast 40S ribosomal subunits with/without yeast eIF1A. Single-molecule zero-mode waveguide (ZMW) scanning assays using a model yeast RPL41A mRNA with a near-cognate CUG at +25 and a downstream AUG at +110; monitored dwell times of Cy5-labeled eIF1.2 in chimeric PICs (yeast 40S+factors) to infer scanning dynamics and start-site selection.
• Polysome profiling: Assessed global translation under unstressed and 1-day alkaline stress conditions across genotypes.
• Ribosome profiling (Ribo-seq) and RNA-seq: Performed in triplicate on WT and Δeif1.2 parasites under unstressed and 1-day stressed conditions; quantified differential RNA abundance, ribosome-protected fragments (RPFs), and translational efficiency; compared results to published in vivo acute vs chronic datasets.
• Conditional expression rescue: Stabilized DD-BFD1 or DD-HA-BFD2 with Shield-1 to test for rescue of differentiation (BAG1 and DBA induction) in WT and Δeif1.2 backgrounds.
• Statistics: Applied linear/mixed models, Poisson models (cyst counts), Wilcoxon rank-sum (single-molecule dwell times), DESeq2 for RNA/Ribo-seq with multiple-testing correction, and other appropriate analyses as reported.

• Identification of eIF1.2 as a differentiation factor: ENU mutagenesis and FACS uncovered mutants with increased tdTomato-ATG8 and decreased LDH2-driven GFP. Clone 5E4 exhibited impaired bradyzoite formation; an SNV (F97L) in eIF1.2 (TGME49_286090) reproduced the phenotype.
• eIF1.2 F97L phenotype in vitro: Normal tachyzoite growth in plaque assays (NS). Under 7-day alkaline stress, F97L showed reduced cyst-like structures and markedly decreased DBA staining, reduced LDH2-GFP and BAG1, with moderately increased SAG1 (multiple tests with P<2e-16 for several readouts). Flow cytometry indicated fewer GFP(+) cells and lower GFP intensity, with increased ATG8 signal.
• In vivo effects of F97L: Similar acute infection kinetics, weight loss, and survival compared to WT (NS), with transiently higher burden at day 5 (bioluminescence, P=0.0221). At 5 weeks, significantly lower parasite burden in brain by qPCR (P=3.13e-05), indicating impaired establishment of chronic infection.
• Mechanism via scanning fidelity: Gel shifts showed similar eIF1.2 WT and F97L 40S binding (no significant Kd change), with yeast eIF1A enhancing F97L binding. Single-molecule ZMW assays revealed longer dwell times for F97L, indicating increased bypass of the near-cognate +25 CUG and commitment to downstream start sites; simulations favored altered start-site selection rather than slower scanning.
• eIF1.2 is essential for differentiation: Δeif1.2 parasites formed fewer but larger plaques; displayed a near-complete inability to form cyst-like structures in vitro after 7-day stress and drastically reduced DBA and BAG1 with increased SAG1. HA-eIF1.2 complementation restored in vitro differentiation (DBA, BAG1) and partially rescued plaque number and in vivo cyst burden.
• Temporal regulation of eIF1.2: Protein levels unchanged after 1 day stress but decreased after 7 days in WT; bradyzoite-specific mRNAs (LDH2, ENO1) were significantly lower at day 1 in Δeif1.2 vs WT.
• Transcriptome and translatome reprogramming: Ribo-seq/RNA-seq showed that Δeif1.2 parasites under stress exhibit gene expression patterns resembling tachyzoites. In WT upon stress, BFD1 translation increased 9.3-fold (Padj=2.7×10^-24) and BFD2 increased at RNA and translation levels. In Δeif1.2 upon stress, BFD1 translation increased 4.8-fold (Padj=7.3×10^-7), BFD2 showed no significant change, and SAG1 and eIF1.1 remained unchanged. Translational efficiency of BFD1 increased 8.4-fold in WT (Padj=4.2×10^-12) and 6.1-fold in Δeif1.2 (Padj=1.1×10^-12). CST4 TE increased 3.15-fold in stressed WT (Padj=0.0031) but was unchanged in stressed Δeif1.2.
• Direct validation of BFD1/BFD2 regulation: In a tagged strain, Δeif1.2 parasites showed markedly reduced induction of BFD2 protein and modestly reduced BFD1 induction during days 1–3 of stress; BAG1 failed to accumulate by day 3 in Δeif1.2.
• Functional rescue by forced expression: Stabilization of DD-BFD1 or DD-HA-BFD2 with Shield-1 induced BAG1 and DBA signals in WT and Δeif1.2 backgrounds. BFD2 stabilization elicited stronger BAG1/DBA induction than BFD1. Loss of eIF1.2 dampened the magnitude of induction but differentiation markers were still significantly elevated, indicating that elevating BFD1 or BFD2 can overcome the differentiation defect.
• In vivo cyst burden with gene deletion: In CBA/J mice at 5 weeks, Δeif1.2 showed markedly reduced brain cysts compared to WT, with partial rescue by HA-eIF1.2 complementation; survival did not differ significantly among groups.

The study demonstrates that eIF1.2 is a key translation initiation factor required for efficient tachyzoite-to-bradyzoite conversion. Loss-of-function via the F97L mutation or gene deletion disrupts differentiation without severely impacting acute-stage replication, explaining reduced chronic infection. Single-molecule assays indicate that F97L alters start-site selection during scanning, likely affecting fidelity at near-cognate codons and thereby modulating translation of critical differentiation regulators. Multi-omic data show that eIF1.2 promotes the stress-induced upregulation of the master regulator BFD1 at the translational level and robustly enables induction of BFD2 at both RNA and translation levels. Consequently, Δeif1.2 parasites maintain a tachyzoite-like expression state under stress. Forced expression of BFD1 or BFD2 restores differentiation markers in Δeif1.2 parasites, functionally linking eIF1.2 to the BFD1/BFD2 axis. The presence of two eIF1 paralogs in tissue cyst-forming apicomplexans suggests subfunctionalization, with eIF1.2 assuming a specialized role during stress and differentiation while eIF1.1 likely supports homeostatic translation in unstressed conditions. These findings position eIF1.2 as a central node integrating translational control with developmental fate decisions in T. gondii.

This work identifies eIF1.2 as an essential regulator of Toxoplasma gondii stage conversion, acting through modulation of translation initiation and start-site selection to elevate key differentiation factors, notably BFD1 and BFD2. Genetic disruption (F97L or deletion) impairs cyst formation in vitro and in vivo, while conditional expression of BFD1 or BFD2 can bypass the defect and trigger differentiation. The results underscore a broader role for translational control in apicomplexan development and highlight eIF1.2 as a potential target to manipulate parasite latency. Future directions include global mapping of start-site selection (e.g., QTI-seq) in eIF1.2 mutants, delineating direct versus indirect effects on BFD2, mechanistic dissection of interactions with eIF1A and other initiation factors, and defining the roles and regulation of the eIF1 paralogs during stress and differentiation.

• Single-molecule scanning used a chimeric system (T. gondii eIF1.2 with yeast 40S and factors) and a yeast model mRNA; findings on start-site selection may not fully capture parasite-native contexts.
• The study did not perform genome-wide start-site mapping (e.g., QTI-seq) to directly quantify global effects of eIF1.2 loss or F97L on initiation codon usage.
• HA tagging and prolonged passaging may influence complementation phenotypes (partial rescue of plaque number and elevated BAG1 upon stress), complicating interpretation.
• Discrepancies between western blot and IF quantification (e.g., BAG1 induction under DD-BFD1 stabilization) reflect methodological sensitivity and sampling limitations.
• The role of the eIF1.1 paralog remains unresolved; its downregulation under stress and functional division from eIF1.2 require further investigation.
• eIF1.2 protein decreased after 7 days of stress; mechanisms of eIF1.2 regulation over differentiation timecourses were not defined.