Cross-lineage 5-methylcytosine methylome profiling reveals methylated divergence among Toxoplasma gondii tachyzoites of the three major clonal lineages.

Background: Toxoplasma gondii is a globally widespread zoonotic parasite, infecting nearly one-third of the human population, often leading to chronic, latent infections. Among the emerging layers of gene regulation, 5-methylcytosine (m⁵C) has emerged as a pivotal post-transcriptional modification in eukaryotes. Despite its growing recognition in various species, the epitranscriptomic landscape of m⁵C in the tachyzoite stage of T. gondii remains largely unexplored. To address this gap, we performed the first comprehensive m⁵C methylation profiling across three major T. gondii genotypes-RH (type I), ME49 (type II), and VEG (type III).

Methods: The comparative m⁵C methylation analysis was carried out using methylated RNA immunoprecipitation sequencing (MeRIP-Seq) combined with RNA sequencing (RNA-Seq). Differentially m⁵C-methylated genes (DMMGs) were functionally annotated via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. By combining methylation and transcriptomic data, we uncovered strain-specific correlations between m⁵C modifications and gene expression. Additionally, expression and methylation patterns of potential regulators identified via BLASTP searches were examined. Statistical analyses were determined by one-way ANOVA.

Results: Our analysis revealed a total of 5129, 4968, and 4577 m⁵C-methylated genes in RH, ME49, and VEG tachyzoites, respectively, with methylation predominantly enriched in the coding sequences. Comparative analysis across different strains uncovered 1710, 1131, and 784 DMMGs in RH versus ME49, RH versus VEG, and ME49 versus VEG, respectively. Functional enrichment analysis highlighted key biological processes, including catalytic activity, transport, phospholipid metabolism and transcription regulation. Furthermore, KEGG pathway analysis identified critical m⁵C-regulated processes such as nucleocytoplasmic transport, DNA replication, and ATP-dependent chromatin remodeling. Virulence-associated secretory effectors exhibited hypermethylation in more virulent strains, such as GRA39 and ROP35. Additionally, several putative m⁵C regulators displayed genotype-specific or conserved expression and methylation patterns.

Conclusions: This study presents the first m⁵C epitranscriptomic atlas of T. gondii tachyzoites, revealing both conserved and genotype-specific mRNA modification networks. These insights significantly increased the understanding of the regulatory role of m⁵C in T. gondii pathogenesis and open promising avenues for the development of vaccines and therapeutics aimed at combating zoonotic toxoplasmosis.