Study On The Toll Like Receptor Signal Pathway And Its Immune Function In Zhikong Scallop Chlamys Farreri

Scallop culture is an important marine aquaculture industry in China. But the frequently outbreak of disease has been threatening the sustainable development of this culture industry. A better understanding of immune defense has been considered as a basic solution in the disease control because of the potential in the development of therapeutic agents, and genetic improvement to increase the resistance to disease.Toll-like receptors (TLR) are an ancient family of pattern recognition receptors (PRR), which show homologies with the Drosophila Toll protein and play key roles in detecting various non-self substances and then initiating and activating immune system. In addition, TLRs can induce DC maturation, participate in immune tolerance and apoptosis, mediate recognition of non-infection factors and etc. However, most of the research about TLRs and their signal pathways were focused on the model organisms, such as Drosophila melanogaster, Caenorhabditis elegans and mammals. Rare reports were available in other animals, especially in marine invertebrates.In the present study, several key components of TLR signal pathway identified from Zhikong scallop were analyzed by bioinformatics tools. These genes including TLR, Myd88 (Myeloid differentiation primary response gene 8), TRAF6 (TNF receptor associated factor 6), NF-κB (nuclear factor kappa-B factor) and IκB (nuclear factor kappa-B factor inhibitor) exhibited significant similarity with their orthologs in other species. The identities were: 23%-38% for TLR, 23%-34% for MyD88, 28%-45% for TRAF6, 23%-41% for IκB and 43%-52% for NF-κB. In addition, the typical motifs or domains were also identified in the predicted protein sequences of these genes. Besides, a strong interaction between the TIR domain of TLR and MyD88 was detected via ELISA assays. So the key components of TLR signal pathway identified from C. farreri constituted a rather canonical MyD88-denpendent TLR pathway. After the treatments of LPS (Lipopolysaccharide), the mRNA expression of all the key components in TLR signal pathway were all up-regulated: the mRNA expression of TLR, MyD88 and TRAF6 were reached to 2.95-fold, 8.07-fold and 13.98-fold of the normal expression level separately, while the mRNA expression of IκB and NFκB reached to 49.09-fold and 63.31-fold of the normal expression level separately, indicating that C. farreri TLR signal pathway could be activated by LPS and may play a role in the immune response.To confirm this hypothesis, the RNAi technology was applied to inhibit the expression of TLR gene, and the expression profiles and enzyme activity of related genes were detected via realtime PCR. During the course of RNAi, all the key components of TLR signal pathway were down-regulated, and the expression level and enzyme activity of SOD (Super Oxide Dismutase) and CAT (Catalase) were up-regulated, but expression level of G-type lysozyme was down-regulated. It can be inferred that the TLR signal pathway could control the anti-oxidant system and regulate the expression of anti-bacteria proteins in C. farreri. During the course of RNAi, the expression level of three LRR (leucine-rich repeat) genes were all upregualted, indicating these genes, which shared a high similarity with the extracellular part of C. farreri TLR (CfTLR), may stand a compensatory mechanism for the TLR signal pathway.After the challenge of Listonella anguillara, the apoptosis level in scallops with CfTLR knocked-down was significantly lower than that in other groups. It can be inferred that the apoptosis signal could not be transduce well. The cumulative mortality of scallops with CfTLR knocked-down was significantly increased after the challenge of Listonella anguillara. CfTLR was suspected to be involved in the activating downstream signaling pathways that lead to the expression of defense molecules and regulate the anti-oxidant system to defend bacteria challenge.In conclusion, a primitive and conserved TLR signal pathway was found in the Zhikong Scallop C. farreri and this pathway played an important role in the defense of scallop to L. anguillarum challenge via several mechanisms, such as activating the expression of defense molecules, decreasing the oxidative stress and so on.