Identification and characterization of three TLR1 subfamily members from the orange-spotted grouper, Epinephelus coioides

Toll-like receptors (TLRs), which play important roles in host defense against pathogen infection, are the most intensively studied pattern recognition receptors (PRRs). In this study, we identified three novel TLR1 subfamily members, including TLR1 (EcTLR1b), TLR2 (EcTLR2b) and TLR14 (EcTLR14), from the orange-spotted grouper (Epinephelus coioides). EcTLR1b and EcTLR2b displayed low sequence identity with the previously reported grouper TLR1 (EcTLR1a) and TLR2 (EcTLR2a), respectively. The open reading frames (ORFs) of EcTLR1b, EcTLR2b and EcTLR14 contain 2484 bp, 2394 bp and 2640 bp, which encode the corresponding 827 amino acids (aa), 797 aa and 879 aa, respectively. All three TLRs have leucine-rich repeat (LRR) domains (including an LRR-NT (except for EcTLR1b), several LRR motifs and an LRR-CT), a trans-membrane region and a Toll/interleukin-1 receptor (TIR) domain. The TIR domains of the three TLRs exhibited conserved boxes, namely box1, box2 and box3, and their 3D models were similar to those of human TLR1 or TLR2. Sequence alignment demonstrated that the TIR domains of the three TLRs shared higher sequence identity with those of other species than the full-length receptors. Phylogenetic analysis indicated that EcTLR1s and EcTLR2s are characterized by their differing evolutionary status, whereas EcTLR14 was found to be in the same group as other piscine TLR14/18s. The three TLRs were ubiquitously expressed in seven tested tissues of healthy groupers, although their expression profiles were different. Post Cryptocaryon irritans infection, TLR1s expression was up-regulated in the gills. The expression of TLR2b was mainly increased in the spleen, but decreased in the gills, which was similar to the expression pattern of TLR2a post C. irritans infection. Unlike EcTLR1b and EcTLR2b, however, the grouper TLR14 transcript was substantially induced in both tissues post challenge. These findings may be helpful in understanding the innate immune mechanism of host anti-parasite infection.