Molecular cloning and expression study on Toll-like receptor 5 paralogs in Japanese flounder, Paralichthys olivaceus

Toll-like receptor (TLR) 5 is responsible for the bacterial flagellin recognition in vertebrates. Synergistic role of TLR 5 membrane form (TLR 5M) and TLR 5 soluble form (TLR 5S) have been reported from the study on rainbow trout. This system is regarded as the unique system in teleost fish. However, systemic response of TLR 5 genes in teleost fish has not been fully understood. Hence, we cloned Japanese flounder (Paralichthys olivaseus) TLR 5M and TLR 5S genes and their expressions were analyzed. The coding region of Japanese founder TLR 5M and TLR 5S cDNA were 2670 bp and 1923 bp, encoding 889 and 640 amino acid residues, respectively. The Japanese flounder TLR 5M was composed of an extracellular leucine rich repeats (LRRs), a transmembrane and an intracellular Toll/interleukin-1 receptor (TIR) domains, whereas TLR 5S possessed only the LRR domain. TLR 5M was highly expressed in the gill, head kidney, heart and liver. TLR 5S was highly expressed in the brain, head kidney and heart. Flagellin stimulation (1 and 5 microg/ml) led to strong gene expression of TLR 5S in peripheral blood leukocytes (PBLs) and liver cells. In contrast to TLR 5S, TLR 5M was down-regulated until 3 h after flagellin stimulation in PBLs and liver cells. The flagellin stimulation also resulted in the production of the flounder IL-1beta and IL-6 from the liver cells and PBLs. The gene expression of TLR 5M was highly induced in the liver, while TLR 5S gene expression was drastically increased in the intestine following challenge with Edwardsiella tarda. Increased number of TLR 5M- and 5S-expressing cell populations were detected by in situ hybridization in the lamina propria of the intestine and liver after E. tarda infection, respectively. These results imply that the expression of these TLR 5 paralogs in Japanese flounder are differently regulated in the whole body and play important roles in the immune response against bacterial pathogens.