Study On The Immune Response And Its Mechanism Mediated By CgDM9CPs In The Oyster Crassostrea Gigas

Invertebrates lack adaptive immunity based on molecules such as immunoglobulin（Ig）and T cell receptor（TCR）,and only rely on innate immunity to defend against invading pathogens.Innate immunity plays a very important role in the immune defense of invertebrates.The immune recognition initiated by the pattern recognition receptor（PRR）is considered as the first step of the innate immune response.Recently,various PRRs,such as Toll-like receptors,C-type lectins,peptidoglycan recognition proteins and scavenger receptors,have been reported to induce immune defense in invertebrates,by recognizing conserved structures of different pathogens,called pathogen-associated molecular patterns（PAMPs）,which can subsequently trigger the activation of downstream signaling pathways,and induce effector expression or phagocytosis.Except for the canonical carbohydrate’s recognition proteins,some novel molecules were discovered in invertebrates to play roles in PAMPs recognition.DM9-containing protein（DM9CP）is an important class of recognition molecules discovered recently.The mechanism of the immune response induced by DM9CP in invertebrates is unknown.In the present study,eight members of the CgDM9CPs family were identified in the oyster Crassostrea gigas.The binding characteristics of CgDM9CPs were analyzed.With bioinformatics,molecular biology,cell biology and immunology methods,the cellular immune response mechanism mediated by CgDM9CP-1/3 and the humoral immune response mechanism mediated by CgDM9CP-5 were also explored in the oyster C.gigas.1.The composition of the CgDM9CPs protein family,their expression characteristics,and ligand-binding activity were clarified.Through homology alignment in the NCBI database,eight proteins containing DM9 domain were found in the oyster genome,and the existence of these eight genes was verified by gene cloning and sequencing.Eight CgDM9CP protein sequences show 65%-80%similarity to each other and there was a potential Ca²⁺binding site"VPN"motif in the protein sequence of CgDM9CP-3.The eight DM9CPs contained two tandem DM9 domains by SMART database analysis.It was found that the three-dimensional structures of CgDM9CP proteins were homodimers through SWISS-MODEL three-dimensional homology modeling.The distribution characteristics of CgDM9CP-1～CgDM9CP-6 in different tissues were detected by heat map analysis combined with real-time PCR.The relative expression of CgDM9CP-5 was higher in gills.Prokaryotic expression of r CgDM9CP-1-r CgDM9CP-6 to prepare CgDM9CP-1-CgDM9CP-6specific polyclonal antibody.The results of Western blotting showed that CgDM9CP-1 was distributed in serum,and CgDM9CP-5 was distributed in gill mucus.The binding activity of CgDM9CP-1-CgDM9CP-6 to different PAMPs was detected by enzyme-linked adsorption reaction in vitro.It was found that r CgDM9CP-1-r CgDM9CP-6 could bind to LPS,PGN,Mannose,Poly（I:C）,and all member of r CgDM9CP-1-r CgDM9CP-6 showed strongest binding activity to mannose.2.The mechanism of cellular immune response mediated by CgDM9CP-1/3 were revealedAfter stimulation by Vibrio splendidus,the expression of CgDM9CP-1 in serum increased and the phagocytic index increased.After blocking CgDM9CP-1 in serum with specific antibodies,the phagocytic index decreased.After further incubation with membrane protein CgIntegrin antibody,the phagocytosis index decreased significantly,so it is speculated that CgDM9CP-1promotes haemocytes phagocytosis in an integrin-dependent manner.It was found that r CgDM9CP-1 can bind to r CgIntegrin（KD=2.45×10-6 M）detected by Bio-Layer Interferometry.CgDM9CP-1 co-localized with CgIntegrin on haemocytes membrane by immunofluorescence assay.The expression of CgRho A in haemocytes increased after V.splendidus stimulation.After knockdown of CgDM9CP-1 or CgIntegrin by RNAi,the expression levels of CgRho A and Cgcathepsin L1 were decreased,the phagocytic index also decreased.After knockdown of CgRho A,the expression of Cgcathepsin L1 and the phagocytic index were reduced.In conclusion,CgDM9CP-1 may bind to the membrane protein CgIntegrin,regulate the expression of CgRho A and Cgcathepsin L1,and finally promote phagocytosis.A protein CgRab1 that can interact with CgDM9CP-1 intracellularly was identified.r CgRab1could bind to GTP by enzyme-linked adsorption reaction.The expression of CgRab1 increased in haemocytes after V.splendidus stimulation.After knockdown of CgRab1 expression by RNAi,the expression of Cgcathepsin L1 decreased,the phagocytic rate and phagocytic index also decreased.Therefore,CgDM9CP-1 could bind to CgRab1 intracellularly,regulate the expression of Cgcathepsin L1 and participate in the regulation of phagocytosis.CgDM9CP-3 with the function of enhancing cellular encapsulation was identified.CgDM9CP-3 has a"VPN"motif similar to the C-type lectin Ca²⁺binding site.r CgDM9CP-3 has binding activity to Mannose,LPS,PGN,Pichia Yarrowia,Escherichia coli,V.splendidus and Aeromonas hydrophila in a Ca²⁺-dependent manner by ELISA assay.In vitro encapsulation experiments showed that r CgDM9CP-3 could enhance cellular encapsulation in a Ca²⁺-dependent manner.These results showed that CgDM9CP-3 exhibit binding capability to mannose,LPS,PGN as well as various microbes in a Ca²⁺-dependent manner,and enhanced haemocytes encapsulation.3.The mechanism of humoral immune response mediated by CgDM9CP-5 was elucidatedCgDM9CP-5 showed the highest expression in the gills and was distributed in the gill mucus by RT-PCR and Western blotting experiments.The expression of CgDM9CP-5 in gills increased after stimulation by V.splendidus.r CgDM9CP-5 was obtained by prokaryotic recombination,and it was found that r CgDM9CP-5 have binding activity to P.pastoris,E.coli,V.splendidus and Staphylococcus aureus.r CgDM9CP-5 could bind r CgIntegrin by Bio-Layer Interferometry.After knockdown of CgDM9CP-1 or CgIntegrin by RNAi,the phosphorylation levels of JNK and P38kinases were reduced,and the expression level of CgIL-17s（CgIL-17-3,4,5 and 6）,Cg-Defh1,Cg-Defh2,CgMolluscidin were also decreased.Based on the above results,CgDM9CP-5 may mediate the activation of the MAPK pathway and regulate the expression of inflammatory factors and antimicrobial peptides by binding to the membrane protein CgIntegrin.In conclusion,each member of the CgDM9CPs from oyster C.gigas has 2 DM9 domains.r CgDM9CP-1-r CgDM9CP-6 could bind to LPS,PGN,Mannose,Poly（I:C）,and each of them showed strongest binding activity to mannose.CgDM9CP-1 might bind to the membrane protein CgIntegrin,regulate the expression of CgRho A and Cgcathepsin L1,and finally promote phagocytosis.CgDM9CP-3 exhibited binding capability to mannose,LPS,PGN as well as various microbes in a Ca²⁺-dependent manner,and enhanced haemocytes encapsulation.CgDM9CP-5mediated the activation of the MAPK pathway and regulate the expression of inflammatory factors and antimicrobial peptides by binding to the membrane protein CgIntegrin.These findings would expand our understanding of the immune function of DM9CPs,and provided new insight into the complex process of innate immune response in Mollusca.