Transcriptional activation of membrane-related Toll-like receptor 5 through the NF-kappa B subunit p65 and Vibrio parahaemolyticus flagellin in orange-spotted grouper (Epinephelus coioides)

Toll-like receptor 5 (TLR5) is an important pattern recognition receptor (PRR) for bacterial flagellin. There are two kinds of subtypes in fish, the membrane-associated Toll-like receptor 5 (TLR5M) and the soluble form of TLR5 (TLR5S). Among them, TLR5M is a homolog of mammalian TLR5, and TLR5S is fish-specific. TLR5M in orange-spotted grouper (Epinephelus coioides) was involved in recognizing flagellin and activating the nuclear factor (NF)-?B signalling pathway while EcTLR5S negatively regulated the NF-?B pathway. In mammals, the NF-kappa B pathway is critical to the expression of inflammatory cytokines and effectors. To better understand the activity of the TLR5M gene, we characterized the TLR5M 5' flanking sequence region from E. coioides and assayed the TLR5M promoter luciferase reporter activity in HEK 293 T cells and grouper spleen cells (GS). The 5'-flanking region of TLR5M included three NF-kappa B binding sites. To elucidate the molecular basis of TLR5M gene expression, the activity of the TLR5M gene promoter was characterized and the deleted or site-directed mutants were generated to explore the functional significance of these kinds of binding sites. The luciferase activity of the TLR5M promoter in HEK 293 T cells was detected after flagellin was treated. The overexpression of NF-kappa B/p65 greatly increased the wild-type TLR5M promoter luciferase activity higher than the mutants. TLR5M promoter luciferase activity was increased more in the presence of p65 overexpression and Vibrio parahaemolyticus Flagellin C (vpFlaC) stimulation. These results suggest that NF-kappa B may be the important transcription factor and vpFlaC may act as a ligand, they could maximally increase the TLR5M promoter luciferase activity.

Automated summary

Toll-like receptor 5 (TLR5) has two subtypes in fish, the membrane-associated TLR5 (TLR5M) and soluble TLR5 (TLR5S). TLR5M in orange-spotted grouper recognizes flagellin and activates the NF-κB signaling pathway, while EcTLR5S negatively regulates this pathway. The NF-κB binding sites in the 5'-flanking region of TLR5M play a significant role in its gene expression and activation.