Vibrio parahaemolyticus flagellin F (FlaF) induces the inflammatory response of the Tetraodon nigroviridis through the TLR5M

Abstract

Vibrio parahaemolyticus, one of the major causes of vibriosis in aquatic animals, causes inflammation that may be related to tissue damage. Flagellin is the major structural component of the flagellum, and also an important virulence factor of bacteria. The filament of V. parahaemolyticus flagellum is composed of multiple flagellin subunits, including FlaA, FlaB, FlaC, FlaD, FlaE, and FlaF which are related to virulence. We produced six V. parahaemolyticus recombinant flagellin through prokaryotic expression including rV.pFlaA, rV.pFlaB, rV.pFlaC, rV.pFlaD, rV.pFlaE, and rV.pFlaF. The primary spleen and head kidney cells (including leukocytes) of Tetraodon nigroviridis were obtained, and the recombinant flagellins were used as stimulants. In this study, the recombinant V. parahaemolyticus flagellin on pro-inflammatory mediators including the release of nitric oxide (NO) and reactive oxygen species, as well as the expression of inflammatory cytokines IL-1β and IFNγ2 etc were detected. The results showed that the NO production and respiratory burst response were significantly induced after stimulation with rV.pFlaF in T. nigroviridis head kidney and spleen cells. Our results also showed that the mRNA levels of T. nigroviridis toll-like receptor 5 M (TnTLR5M), TnTLR5S, and downstream genes, such as myeloid differentiation factor 88 (MyD88), TNF receptor-associated factor 6 (TRAF6), interleukin-1β (IL-1β), and IFNγ2 were all significantly enhanced after stimulation with rV.pFlaF in T. nigroviridis primary cells. In addition, the NO production and alkaline phosphatase (ALP) activity were significantly increased in the liver of T. nigroviridis after the rV.pFlaF challenge in vivo. Meanwhile, in TnTLR5M-overexpressed HEK 293 T and Cos-7 cells, the luciferase activity of the nuclear factor-kappa B (NF-κB) reporter gene was significantly enhanced after rV.pFlaF stimulation, in a time- and dose-dependent manner. However, the other five flagellin components have no inflammation-inducing ability or the effect is relatively weak. Overall, our results suggested that rV.pFlaF activates the pro-inflammatory mediators including the release of NO and reactive oxygen species, as well as the expression of inflammatory cytokines IL-1β and IFNγ2, through the TnTLR5M-NF-κB signaling pathway in T. nigroviridis. In conclusion, this study indicated that the rV.pFlaF has a significant role in inducing the inflammation of T. nigroviridis.