Wolbachia elevates host methyltransferase expression and alters the m6A 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 N⁶-methyladenosine (m⁶A) machinery and post-transcriptional modification landscape. Our study revealed that Wolbachia infection elevates the expression of genes involved in the mosquito's m⁶A methyltransferase complex. However, knocking down these m⁶A-related genes did not affect Wolbachia density. Nanopore sequencing identified 1,392 differentially modified m⁶A DRACH motifs on mosquito transcripts, with 776 showing increased and 616 showing decreased m⁶A levels due to Wolbachia. These m⁶A sites were predominantly enriched in coding sequences and 3'-untranslated regions. Gene Ontology analysis revealed that genes with reduced m⁶A levels were over-represented in functional GO terms associated with purine nucleotide binding functions critical in the post-transcriptional modification process of m⁶A. 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 m⁶A modification, respectively. Our findings underscore Wolbachia's ability to modulate many intracellular aspects of its mosquito host by influencing post-transcriptional m⁶A modifications and gene expression, and it unveils a potential link behind its antiviral properties.