A Crassostrea gigas Toll-like receptor and comparative analysis of TLR pathway in invertebrates

Toll-like receptor (TLR) signaling pathway was an important and evolutionarily conserved innate immune pathway. Evolutionary lineage of this pathway in the Lophotrochozoans is still less understood. In this study, we cloned a novel TLR, a key component of TLR pathway, from Crassostrea gigas, and named it CgToll-1. The 4343 base pairs full-length cDNA was assembled with the 3' and 5' RACE (rapid amplification of cDNA ends) PCR results, and containing a 3540 bp open reading frame, which encoding a putative TLR protein of 1179 amino acid residues. Real-time reverse transcription polymerase chain reaction analysis revealed that the highest CgToll-1 expression level was in hemolymph, and the expression pattern in hemolymph dramatically increased in the presence of bacteria Vibrio anguillarum. Furthermore, TLR pathway core genes of mollusks were searched and compared with model invertebrates. Phylogenetic trees of two downstream genes (IκB, Rel) showed that mollusks genes were closer to Drosophila melanogaster than Strongylocentrotus purpuratus, while three upstream genes (MyD88, IRAK, TRAF6) showed the opposite propensity. We have also detected that these two downstream genes were significantly more conservative than the three upstream genes based on amino acid sequence alignment. We found no significant difference between the codon usage biases of TLR pathway genes. This study suggests that CgToll-1 was a constitutive and inducible protein and thus could play an important role in the immune responses against bacterium infection. Besides, comparative analysis of TLR pathway showed that gene loss and divergence might exist during evolution in invertebrate.