Wolbachia elevates host methyltransferase expression and alters the mA methylation landscape in Aedes aegypti mosquito cells.

Wolbachia pipientis is an intracellular endosymbiotic bacterium that blocks the replication of several arboviruses in transinfected Aedes aegypti mosquitoes, yet its antiviral mechanism remains unknown. For the first time, we employed Nanopore direct RNA sequencing technology to investigate the impact of wAlbB strain of Wolbachia on the host's N-methyladenosine (mA) machinery and post-transcriptional modification landscape. Our study revealed that Wolbachia infection elevates the expression of genes involved in the mosquito's mA methyltransferase complex. However, knocking down these mA-related genes did not affect Wolbachia density. Nanopore sequencing identified 1,392 differentially modified mA DRACH motifs on mosquito transcripts, with 776 showing increased and 616 showing decreased mA levels due to Wolbachia. These mA sites were predominantly enriched in coding sequences and 3'-untranslated regions. Gene Ontology analysis revealed that genes with reduced mA levels were over-represented in functional GO terms associated with purine nucleotide binding functions critical in the post-transcriptional modification process of mA. Differential gene expression analysis of the Nanopore data uncovered that a total of 643 protein-coding genes were significantly differentially expressed, 427 were downregulated, and 216 were upregulated. Several classical and non-classical immune-related genes were amongst the downregulated DEGs. Notably, it revealed a critical host factor, transmembrane protein 41B (TMEM41B), which is required for flavivirus infection, was upregulated and methylated in the presence of Wolbachia. Indeed, there is a strong correlation between gene expression being upregulated in genes with both increased and decreased levels of mA modification, respectively. Our findings underscore Wolbachia's ability to modulate many intracellular aspects of its mosquito host by influencing post-transcriptional mA modifications and gene expression, and it unveils a potential link behind its antiviral properties.