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

Abstract

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)-κB signalling pathway while EcTLR5S negatively regulated the NF-κB pathway. In mammals, the NF-κB 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-κB 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-κB/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-κB may be the important transcription factor and vpFlaC may act as a ligand, they could maximally increase the TLR5M promoter luciferase activity.