m6A writer METTL14 restricts Mycobacterium tuberculosis infection by promoting TAX1BP1-mediated autophagy in macrophages

N6-methyladenosine (m6A) modification plays a crucial role in regulating immune responses against Mycobacterium tuberculosis (Mtb) infection, but the underlying mechanisms remain unclear. Here, we analyzed m6A regulatory gene expression in GEO dataset GSE139825 and validated the finding in THP-1-derived macrophages. Bioinformatics analysis showed that METTL14, ALKBH5, and YTHDC1 were significantly downregulated, while METTL3 was upregulated in Mtb-infected alveolar macrophages compared to controls. In vitro experiments confirmed METTL14 expression was dose-dependently reduced with Mtb infection in macrophages, and METTL14 overexpression reversed Mtb-induced decreases in cell viability, reduced LDH release, and diminished intracellular Mtb burden. Mechanistically, METTL14 overexpression restored Mtb-suppressed autophagy, evidenced by increased LC3II/I ratio and Beclin1 expression as well as decreased p62 levels, which was abrogated by the autophagy inhibitor 3-MA. Further, as validated by bioinformatic prediction, RIP assay and actinomycin D experiment, METTL14 directly bound to TAX1BP1 mRNA and enhanced its m6A modification, thereby stabilizing TAX1BP1 mRNA and upregulating its expression. TAX1BP1 knockdown abolished the protective effects of METTL14 overexpression on macrophage viability, autophagy, and anti-Mtb capacity. Collectively, our findings demonstrate that METTL14, downregulated by Mtb infection, promotes macrophage autophagy and antibacterial activity via m6A-mediated stabilization of TAX1BP1. This METTL14-TAX1BP1-m6A pathway provides a novel therapeutic target for tuberculosis treatment. • Mycobacterium tuberculosis infection alters the expression of multiple m6A regulatory proteins in macrophages. • Mycobacterium tuberculosis suppresses METTL14 expression in a dose-dependent manner in macrophages. • METTL14 overexpression enhances macrophage viability and reduces intracellular mycobacterial load. • METTL14 promotes macrophage autophagy and antibacterial activity by stabilizing TAX1BP1.