Study On The Mechanism Of Immune Response Mediated By Key Pattern Recognition Receptors 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.The immune recognition mediated by pattern recognition receptors (PRRs) is the first and crucial step of innate immunity. However, knowledge on immune recognition and immune response mediated by PRRs in mollusk is still limit. In the present study, the immune recognition and immune responses mediated by Zhikong scallop Chlamys farreri PRRs, including C-type lectin (CfLec-1, CfLec-2 and CfLec-3), peptidoglycan recognition protein (CfPGRP-S1) and lipopolysaccharide andβ-1, 3-glucan binding protein (CfLGBP) were performed by molecular biological and immunological technology.The mRNA expression of CfLec-1 was significantly up-regulated towards the stimulation of LPS andβ-glucan, but significantly down-regulated towards stimulation of PGN, while expression of CfLec-2 was significantly up-regulated towards all the three PAMPs. Both the recombinant protein of CfLec-1 and CfLec-2 could bind LPS, PGN and mannan in vitro, besides rCfLec-2 could also bind zymosan, indicating that CfLec-1 and CfLec-2 could mediate the immune recognition toward different invaders. Immunofluorescence assay revealed that CfLec-1 and CfLec-2 exhibited different tissue distribution specificity, for CfLec-1 was located in mantle and gill, while CfLec-2 was located in the mantle, kidney and gonad. Though CfLec-1 and CfLec-2 were predicted to be secreted with the instruction of signal peptide, they were observed binding to hemocytes after secreting into hemolymph, and such interaction was subsequently proved to recruite hemocytes to enhance their encapsulation towards agarose in vitro. These results clearly suggested that CfLec-1 and CfLec-2 in Chlamys farreri could mediate immune responses after the recognition towards different PAMPs, and involved in the immune defense of scallop.CfLec-3 protein exhibited a broad tissue distribution including hepatopancreas, gill, kidney, mantle and muscle of the scallop. Expression of CfLec-3 mRNA in hemocytes was significantly up-regulated after stimulation of LPS, PGN orβ-glucan. The recombinant CfLec-3 (rCfLec-3) could bind LPS, PGN, yeast glucan,β-1,3-glucan, mannan and CpG ODN, but not LTA and ploy I:C by the method of PAMPs microarrays. Moreover, rCfLec-3 also bound to the surface of scallop hemocytes, and mediated the opsonization of hemocytes, including phagocytosis against Escherichia coli and encapsulation towards agarose beads. Three CRDs of CfLec-3 suffered an obvious functional differentiation, for CRD1 was mainly responsible for pathogen recognition, while CRD2 and CRD3 were in charge of opsonization, and such differentiation might result from obvious dissimilarity of their tertiary structures. Mutagenesis was performed to unravel the PAMPs binding mechanism. The proline (Pro) in Ca2+-binding site 2 was necessary for the PAMPs binding ability, for alteration of proline led to absolute deprivation of binding activity, meanwhile the asparagines (Asn) in Ca2+-binding site 2 determined the binding specificity towards mannan, suggesting PAMPs binding characteristic of molluscan C-type lectin displayed specificity on the amino acid level. Our study systematically dissected the functions C-type lectin performed in molluscan immune system, and provided new insight and evidence for structural basis of the interaction between C-type lectins and their ligands.The CfPGRP-S1 protein located in the mantle, gill, kidney and gonad of the scallops. Its mRNA expression in hemocytes was up-regulated extremely after PGN stimulation, while moderately after the stimulations of LPS andβ-glucan. The recombinant protein of CfPGRP-S1 (designated as rCfPGRP-S1) exhibited high affinity to PGN and moderate affinity to LPS, but it did not bindβ-glucan. Meanwhile, rCfPGRP-S1 also exhibited strong agglutination activity to Gram-positive bacteria Micrococcus luteus and Bacillus subtilis and weak activity to Gram-negative bacteria Escherichia coli. More importantly, rCfPGRP-S1 functioned as a bactericidal amidase to degrade PGN and strongly inhibit the growth of E. coli and Staphyloccocus aureus in the presence of Zn2+. These results indicated that CfPGRP-S1 could not only serve as a pattern recognition receptor recognizing bacterial PGN and LPS, but also function as a scavenger involved in eliminating response against the invaders.The mRNA expression of CfLGBP in hemocytes was up-regulated extremely post stimulation of LPS andβ-glucan, and also exhibited a moderate up-regulation after PGN injection. Further PAMPs binding assay with the polyclonal antibody specific for CfLGBP proved that the recombinant CfLGBP (designated as rCfLGBP) could bind not only LPS andβ-glucan, but also PGN in vitro. More importantly, rCfLGBP exhibited obvious agglutination activity towards Gram-negative bacteria E. coli, Gram-positive bacteria B. subtilis and fungi P. pastoris. Taking the results of immunofluorescence assay into account, which displayed CfLGBP was expressed specifically in the immune cells (hemocytes) and vulnerable organ (gill and mantle), we believed that LGBP in C. farreri, serving as a multi-functional PRR, not only involved in the immune response against Gram-negative and fungi as LGBP in other invertebrates, but also played significant role in the event of anti-Gram-positive bacteria infection.In summary, this study systematically studied the mechanism of immune response mediated by key pattern recognition receptors in Zhikong scallop. These data provided useful evidences for the mechanism studies of invertebrate immune response, as well as developed the knowledge of marine invertebrate s'immunology.