TLR5M cooperates with TLR5S to activate NF-κB in Nile tilapia (Oreochromis niloticus)

The binding of flagellin to receptor TLR5 (Toll-like receptor 5) on the cell surface is a prerequisite for the pathogen infection to the body, which has important implication for disease prevention. Unlike mammals, teleost TLR5 exists in two different forms: membrane-bound TLR5M (membrane TLR5, TLR5M) and secreted TLR5S (soluble TLR5, TLR5S). In this study, TLR5M and TLR5S from Nile tilapia (Oreochromis niloticus), named OnTLR5M and OnTLR5S, were cloned and identified. The open reading frame (ORF) of OnTLR5M was 2658 base pair (bp), encoding 885 amino acids (aa), while the ORF of OnTLR5S was 1926 bp, encoding 641 aa. Under normal physiological conditions, both OnTLR5M and OnTLR5S are expressed in the tested tissues, among which OnTLR5M has the highest expression in the liver and the lowest expression in the brain, while OnTLR5S has the highest expression in peripheral blood and the lowest expression in thymus. After Aeromonas hydrophila stimulation in vivo, OnTLR5M and OnTLR5S were significantly up-regulated in peripheral blood and head kidney, and relatively insignificant in spleen. After A. hydrophila stimulation in monocytes/macrophages, OnTLR5M and OnTLR5S were also significantly up-regulated. The results of subcellular localization analysis showed that OnTLR5M was distributed on the cell membrane, while OnTLR5S was distributed in the whole cell. Both OnTLR5M and OnTLR5S can significantly activate NF-kappa B signal, and the activation degree is further increased after the synergy of A. hydrophila or flagellin. In addition, co-transfection of OnTLR5M and OnTLR5S showed a stronger ability to activate NF-kappa B than expression of OnTLR5M alone. Further, the response to NF-kappa B after transferring the hotspot site of zebrafish TLR5 into tilapia TLR5M and TLR5S was not significantly different from that of wild type, suggesting that the hotspot site may be conserved in teleost fish. Moreover, OnTLR5M interacts with OnTLR5S and OnMyD88, respectively. It is speculated that OnTLR5S cooperates with OnTLR5M to detect pathogens, and then OnTLR5M recruits OnMyD88, which activates NF-kappa B through a signaling cascade, prompting cells to express cytokines, thereby resisting pathogens. This study may provide a theoretical support for the understanding of the flagellin recognition system and TLR5-mediated signaling pathway in teleost fish.