Transcriptome and m6A epitranscriptome profiling reveal CCL20 as a key mediator of inflammation in Staphylococcus aureus-induced bovine mastitis.

Mastitis caused by Staphylococcus aureus (S. aureus) poses a significant challenge in dairy farming because of its impact on animal health and milk production. Understanding the regulatory role of N6-Methyladenosine (m6A) modification in this disease remains crucial. Here, we used direct RNA sequencing (DRS) to explore transcriptomic changes in bovine mammary epithelial cells (BMECs) exposed to S. aureus (SA) compared to untreated controls (MEC). We identified 178 differentially expressed isoforms (DEIs) enriched in pathways such as the TNF signaling pathway, Kaposi's sarcoma-associated herpesvirus infection, IL-17 signaling pathway, NF-kappa B signaling pathway, and rheumatoid arthritis. Most DEIs in these pathways exhibited m6A modifications, with 18 DEIs implicated in inflammation. Functional assays revealed that S. aureus increased the m6A levels of CCL20 mRNA, enhancing its stability via an m6A-YTHDF2-dependent mechanism, which led to elevated CCL20 expression. Silencing YTHDF2 increased IL-6 and TNF-α levels, whereas IL-10 expression decreased. Western blotting confirmed the role of YTHDF2 by showing elevated PI3K, Akt, and NF-κB levels in SA-treated cells following YTHDF2 silencing. These findings suggest that m6A-mediated regulation of CCL20 in S. aureus-induced BMECs operates via the PI3K/Akt/NF-κB pathway. These findings underscore the significance of m6A-mediated CCL20 regulation in mastitis pathogenesis and suggest potential therapeutic strategies for managing this economically significant disease in dairy farming.