Identification and characterization of TLR7, TLR8a2, TLR8b1 and TLR8b2 genes in Atlantic salmon (Salmo salar)

Abstract

Mammalian Toll-like receptor (TLR) 7 and 8 are responsible for recognizing viral single-stranded RNA (ssRNA) and are activated by anti-viral imidazoquinoline compounds, leading to a series of defensive mechanisms being launched to protect the host against viruses. In this study, we identified two TLR7 (with one probably a pseudogene) and three TLR8 genes, namely TLR8a2, TLR8b1 and TLR8b2 from Atlantic salmon (Salmo salar) whole-genome shotgun (WGS) contigs. Bioinformatics analysis showed that salmon TLR7 and TLR8a2 are closely related to the corresponding trout orthologs, however, salmon TLR8b1 and TLR8b2 share the highest amino acid sequence similarity to zebrafish TLR8b and formed a subfamily of the piscine TLR8 molecules in phylogenetic tree analysis. A conserved gene synteny was found with the salmon TLR7/8a members as seen in other vertebrate loci. Deduced domain organisation of salmon TLR7 and TLR8 molecules showed similar structural features, with equal numbers of leucine-rich repeats (LRRs) and insertion motifs. Individual TLR molecules were expressed in a similar pattern between parr and post-smolts, with a high expression level in immune tissues. Promoter analysis predicted several transcription factor binding sites in the TLR8a1/2 and TLR8b1 5′ flanking regions, namely C/EBP, AP-1, STAT, NFκB, and IRF family, suggesting cytokine regulation of the genes. Hence, three recombinant cytokines, type I IFN, IFNγ and IL-1β were used to study the regulation of the salmon TLR gene expression levels in primary head kidney cells and the Salmon Head Kidney-1 (SHK-1) cell line. Salmon TLR7 and TLR8a1 gene expression was more sensitive to type I IFN and IFNγ treatment in primary head kidney cells and SHK-1 cells respectively, with no significant up-regulation of TLR8a2 and TLR8b2 by any of the treatments. On the other hand, salmon TLR8a1 and TLR8b1 were most sensitive to IL-1β treatment in SHK-1 cells and primary head kidney cells, respectively. TLR8b2 was undetectable in SHK-1 cells under these same conditions.