Recombinant Vibrio harveyi flagellin A protein and partial deletions of middle variable region and D0 domain induce immune related genes in Epinephelus coioides and Cyprinus carpio

Abstract

Vibrio harveyi is a Gram-negative bacterium that causes vibriosis in various aquaculture species, including the orange-spotted grouper (Epinephelus coioides). Bacterial flagellin is a potent pathogen-associated molecule that stimulates the innate and adaptive immune systems through toll-like receptor 5 (TLR5) signaling. In this study, we isolated V. harveyi flagellin A (VhFliA) gene from V. harveyi (originated from orange-spotted grouper) and investigated the in vivo activities of recombinant VhFliA protein. Multiple sequence alignment showed that the amino acid sequence of VhFliA has conserved domains of N- and C-terminals (D0 and D1) and a middle variable (MV) region. We produced the VhFliA recombinant protein (wild type (WT)-VhFliA) by Escherichia coli and investigated its in vivo biological activity. Additionally, we prepared the VhFliA recombinant proteins with deletion of domains (ΔMV-VhFliA and ΔD0MV-VhFliA) to identify the domain for biological activity in the orange-spotted grouper. WT and ΔMV-VhFliA induced the expression of inflammatory cytokines (IFNγ, IL-1β, and IL-8) in groupers. However, ΔD0MV-VhFliA did not induce the expression of inflammatory cytokines. Additionally, to demonstrate the applicability of recombinant VhFliA to teleost species, we performed an in vivo assay of the recombinant proteins in koi carp (Cyprinus carpio). WT-VhFliA stimulates the expression of inflammatory cytokines (IL-1β, IL-6, and IL-8) in carp. ΔMV-VhFliA did not upregulate IL-1β and IL-6, whereas ΔD0MV-VhFliA induced expression in carp. These findings showed the potential of VhFliA as an effective immune stimulant adjuvant and comparative studies of flagellin - TLR5 signaling in teleosts.