Cross-lineage m5C methylome profiling reveals methylated divergence in Toxoplasma tachyzoites
Toxoplasma gondii is a globally prevalent zoonotic parasite that chronically infects nearly one-third of the human population. Among emerging layers of gene regulation, 5-methylcytosine (m5C) has been recognized as a key post-transcriptional modification that modulates mRNA stability and translation in eukaryotes. However, the epitranscriptomic landscape of m5C in the tachyzoite stage of T. gondii remains uncharacterized. This study presents the first comprehensive profiling of m5C methylation across three major T. gondii genotypes—RH (type I), ME49 (type II), and VEG (type III)—using methylated RNA immunoprecipitation sequencing (MeRIP-seq) combined with 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. Integration of methylation and transcriptomic data revealed strain-specific correlations between m5C modification and gene expression. Candidate m5C regulatory factors were identified through BLASTP searches in the ToxoDB database and further examined for expression and methylation patterns. We identified 5,129, 4,968, and 4,577 m5C-methylated genes in RH, ME49, and VEG tachyzoites, respectively, with methylation predominantly enriched within coding sequences (CDS). Comparative analysis revealed 1,710, 1,131, and 784 DMMGs across RH vs. ME49, RH vs. VEG, and ME49 vs. VEG comparisons. Functional enrichment highlighted DMMGs involved in catalytic activity, transport, phospholipid metabolism and transcription regulation. KEGG pathway analysis identified nucleocytoplasmic transport, DNA replication, and ATP-dependent chromatin remodeling as key m5C-regulated processes. Additionally, several putative m5C regulators were identified, displaying genotype-specific or conserved expression and methylation patterns, suggesting their potential roles in strain-specific phenotypic variation and as targets for therapeutic intervention. This study presents the first m5C epitranscriptomic atlas of T. gondii tachyzoites, revealing both conserved and genotype-specific mRNA modification networks. These findings offer critical insights into the regulatory role of m5C in T. gondii pathogenesis and open new avenues for the development of vaccines and therapeutics against zoonotic toxoplasmosis. Overall design: MeRIP-seq and RNA-seq of  tachyzoites from three major T. gondii genotypes—RH (type I), ME49 (type II), and VEG (type III).