TLR18 mediates antibacterial immunity via MyD88-dependent NF-κB and IRF pathways in turbot (Scophthalmus maximus)

Toll-like receptors (TLRs), as core pattern recognition receptors (PRRs), play pivotal roles in pathogen detection during innate immune defense and are critical for initiating immune responses. In this study, we investigated a novel member of the TLR1 subfamily, TLR18, in the economically significant species Scophthalmus maximus, aiming to elucidate its molecular mechanisms in antibacterial immunity and provide insights into innate immune regulation in fish. In our results, SmTLR18 (2652 bp) ‌encoded‌ an 883-amino-acid protein sharing high identity with Paralichthys olivaceus TLR14 (84.15%). SmTLR18 localizes to the cytoplasm and cell membrane and is ubiquitously expressed with the highest expression level in spleen and lowest expression level in ‌the‌ blood. In vitro, SmTLR18 was upregulated by PGN and LTA but downregulated by LPS. The extracellular domain of SmTLR18 bound all tested ligands and bacteria, exhibiting the highest affinity for Vibrio anguillarum, and immunohistochemical analysis revealed strong positive SmTLR18 signals within hyperplastic hemocytes. Molecular docking, co-localization, and co-immunoprecipitation assays revealed that SmTLR18 recruits MyD88 to activate downstream signaling. Transcriptomics following SmTLR18 overexpression suggested regulation of IRF, ZBTB, MYSM, STAT, and zf-C2H pathways, upregulating MyD88 and IL-1β while downregulating TNF-α. Conversely, knockdown increased TNF-α and downregulated MyD88 and IL-1β. Mechanistically, dual-luciferase assays demonstrated SmTLR18-mediated NF-κB activation. Results demonstrated SmTLR18 recognizes PAMPs, specifically Vibrio anguillarum, and recruits MyD88 to activate NF-κB and IRF pathways driving cytokine production. These findings underscore the critical role of SmTLR18 in turbot antibacterial immunity.