Direct RNA sequencing reveals selective remodeling of the host m6A epitranscriptome during Leishmania infection
N6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNA, yet its role in host responses to protozoan infection remains poorly explored. Here, we provide the first characterization of the host m6A epitranscriptome during Leishmania amazonensis infection, integrating clinical data, in vitro macrophage models, and direct RNA sequencing. Analysis of publicly available transcriptomic datasets from patients with cutaneous and visceral leishmaniasis, validated in an independent cohort of patients with cutaneous leishmaniasis, revealed coordinated remodeling of the m6A regulatory machinery, characterized by significant upregulation of the writer METTL3 and downregulation of the eraser ALKBH5 and the reader YTHDF3. These changes were recapitulated in RAW264.7 macrophages infected in vitro, where METTL3 protein levels increased progressively between 2 and 8 hours post-infection (hpi), while ALKBH5 abundance declined, resulting in elevated global m6A levels detectable as early as the first post-internalization time point. Transcriptome-wide mapping by direct RNA sequencing at 8 hpi identified 1,998 m6A-modified transcripts harboring 7,132 high-confidence sites in non-infected macrophages, compared with 2,956 modified transcripts and 13,495 sites in infected cells. Beyond this quantitative expansion, infection was associated with increased modification occupancy at shared sites, a greater prevalence of multi-site and multi-region methylation, and selective remodeling of immune-related and immunometabolic transcripts, including Tlr4, Ccl2, Hmox1, Socs3, Rela, Hk1, Hk2, and Dicer1. Altogether, these findings establish that Leishmania infection drives extensive yet selective remodeling of the host m6A landscape and position the host epitranscriptome as a previously unrecognized regulatory axis in Leishmania-macrophage interactions, with implications for the development of host-directed therapeutic strategies. Impact Statement N6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNA, and emerging evidence links epitranscriptomic remodeling to host responses against diverse pathogens. However, whether m6A regulates host-macrophage responses during Leishmania infection remains unknown. Using Oxford Nanopore Direct RNA sequencing, we provided the first transcriptome-wide map of host m6A modifications during Leishmania amazonensis infection. Infection selectively remodels the m6A landscape of macrophages, targeting immune-regulatory and immunometabolic transcripts, establishing the host epistranscriptome as a previously unrecognized regulatory layer in Leishmania-macrophage interactions.