Molecular cloning, phylogenetic analysis and functional characterization of soluble Toll-like receptor 5 in gilthead seabream, Sparus aurata

• A gene encoding a soluble TLR5 (sbTLR5S) was cloned in the gilthead seabream.
• sbTLR5S expression is highly induced in immune tissues after bacterial infection.
• Phylogenetic analyses reveals co-evolution of membrane and soluble TLR5 genes.
• Recombinant sbTLR5S is able to physically interact with flagellin.

Two forms of TLR5, one membrane-anchored and one soluble, have been described in some teleost fish species. However, the exact role of each form has been poorly studied. In the present study, we show that the mRNA levels of soluble gilthead seabream TLR5 (sbTLR5S) are highly induced in head kidney, spleen, liver and blood after Vibrio anguillarum infection, suggesting an important role for sbTLR5S in the innate immune response against bacteria. Comparative genomic and phylogenetic analyses revealed a co-evolution pattern of both genes across fish species and a proximal location in their genomes, further suggesting a functional link between them. To further investigate this issue, the coding sequence of the sbTLR5S was cloned and the corresponding recombinant protein was produced in HEK293 cells. The gene product was secreted to the culture medium as a soluble factor and a physical interaction between flagellin and sbTLR5S was demonstrated. Collectively, these results suggest that sbTLR5S plays an important role in modulating the flagellin-mediated immune response in seabream.