Differences in N⁶-methyladenosine (m⁶A) methylation among the three major clonal lineages of Toxoplasma gondii tachyzoites

Toxoplasma gondii is an important zoonotic parasite which has over 200 genotypes worldwide. N⁶-methyladenosine (m⁶A) methylation is a common epigenetic modification in messenger RNAs (mRNAs), and has been implicated in many aspects of mRNA biology. However, little is known about the difference in m⁶A methylation among different genotypes of T. gondii. In the present study, we employed methylated RNA immunoprecipitation sequencing (MeRIP-seq) technology to identify key genes exhibiting m⁶A methylation in the three major clonal lineages (Types I, II and III) of T. gondii tachyzoites. A total of 7,650, 8,359 and 7,264 m⁶A peaks were identified in 5,211, 5,607 and 4,974 genes in tachyzoites of RH (Type I), ME49 (Type II) and VEG strain (Type III), respectively. By comparing RH vs. ME49, RH vs. VEG, and ME49 vs. VEG, 735, 192 and 615 differentially methylated peaks (DMPs) were identified in 676, 168 and 553 genes, respectively. A combined MeRIP-seq and RNA-seq analysis revealed 172, 41 and 153 differentially methylated genes (DMGs) at both the m⁶A methylation and transcriptional level. Gene Ontology term enrichment analysis of the DMPs identified differences related to Golgi apparatus, plasma membrane, signal transduction, RNA processing and catalytic step 2 spliceosome. KEGG pathway enrichment analysis showed that the DMGs are mainly involved in endocytosis, systemic lupus erythematosus and mTOR signaling pathway. These findings reveal genotype-specific differences in m⁶A methylation, which provide new resources for further investigations of the role of m⁶A in the pathobiology of T. gondii.