Discovery Of A Novel Pattern Recognition Receptor SVWC Family Of Macrobrachium Nipponense And Its Immune Mechanism

Pattern recognition receptor（PRR）is considered to be an important component of invertebrate innate immunity,which can accurately recognize pathogen-associated molecular patterns（PAMPs）of invading microorganisms,and trigger cellular and humoral immune responses to eliminate pathogens.In the past 30 years,due to the importance of economic value,research on the mechanism of crustacean disease resistance has made great progress.Homologous clones have been obtained for many PRR family members,but there is a lack of in-depth analysis and comparative studies on their functions.In this study,we used M.nipponense,an important freshwater economic crustacean,to search for PRR genes from the transcriptome by bioinformatics methods,and analyze the molecular characteristics of the proteins encoded by PRR genes,and identify the functions of some members.In addition,a new PRR family,SVWC,was identified by functional analysis,and the microbial recognition mechanism and immune regulation function of this family member were analyzed in detail.The main findings are as follows:1.Using bioinformatics,10 PRR families with 264 members were screened from the transcriptome of Macrobrarium nipponense,including 8 Mn BGRPs,147 Mn CTLs,1Mn Galectin,40 Mn FREPs,7 MnSRs,17 Mn TEPs,7 Mn TLRs,21 MnSPHs,12 Mn TRBPs and 4 Mn DM9-CPs.2.Using bioinformatics methods,fourMnSVWCs were screened and identified from the transcriptome of M.nipponense.The tissue distribution and after A.veronii stimulation the temporal expression profile of 4 MnSVWCs were analyzed by real-time quantitative PCR（q RT-PCR）.The expression of MnSVWC1-4 was the highest in gills,hemocytes,muscles and gills,respectively.After Aeromonas veronii stimulation,the transcription of MnSVWC1 and MnSVWC2 in gills increased,the expression of MnSVWC3 was down-regulated,and MnSVWC4 did not respond.In hemocytes,the expression of MnSVWC1 and MnSVWC4 was down-regulated,and the transcription of MnSVWC2 and MnSVWC3 was increased.In nerves,the expression of MnSVWC1-4 was up-regulated.Through prokaryotic expression system and protein purification and renaturation techniques,rMnSVWCs were obtained.In vitro,rMnSVWCs exhibited pronounced binding(rMnSVWC2-4 was Ca²⁺dependent binding)and Ca²⁺-dependent agglutinating abilities against diverse microbes,including Gram-negative bacteria（Escherichia coli and A.veronii）,Gram-positive bacteria（Staphylococcus aureus and Bacillus subtilis）,and yeast（Pichia pastoris）.ELISA assays revealed that rMnSVWCs recognize a broad range of various pathogen-associated molecular patterns（PAMPs）,has high affinity to LPS,Lys-type PGN,Dap-type,PGN,Gal,MAN and BG.Compared with MnSVWC1,MnSVWC1^P57A-mut with an impaired Glu-Pro-Asn（EPN）motif displayed reduced affinity to all these PAMPs to varying extent.Through RNAi,q RT-PCR,monoclonal antibody preparation and immunofluorescence techniques,the antimicrobial mechanism of rMnSVWC1 was further studied.MnSVWC knockdown in prawn reduced the ability to clear invading bacteria,but did not block the activities of the Toll pathway or the arthropod immune deficiency（IMD）pathway,or the expression of antimicrobial peptide genes,or PO activity.MnSVWC1 bound to the surface of hemocytes and promoted their phagocytic activity and clearance of invasive bacteria,or directly play a bacteriostatic function.These results indicate that MnSVWC1 can act as an extracellular pattern-recognition receptor in M.nipponense to mediate cellular immune responses by recognizing PAMPs,agglutinating invasive microbes,and promoting phagocytosis in hemocytes,or exert antimicrobial peptide function to directly inhibit bacterial growth.3.In the present study,quantitative real-time PCR（q RT-PCR）was used to analyze the temporal expression profiles of fourMnSVWCs after White Spot Syndrome Virus（WSSV）stimulation.MnSVWC1-4 were increased after WSSV challenge in gill,hemocytes and nerve cord.In vitro,western blot and ELISA assays revealed that recombinant MnSVWC could combine with WSSV.After immunoprecipitation（IP）assay,we further found target proteins of MnSVWC on WSSV（VP26 and VP28）.Moreover,MnSVWC1 acts antiviral function by activating the JAK-STAT pathway to secrete Mn Viperin and promoting hemocytes phagocytic activity.4.A new fibrinogen-related protein gene was identified from the Macrobrachium nipponense transcriptome,named Mnfico3.The complete c DNA sequence of Mnfico3 was1133 bp long,containing an open reading frame of 765 bp coding forMnfico3,a protein consisting of 254 amino acids.The Mnfico3 protein contained a putative N-terminal signal peptide and a fibrinogen-related protein domain present at the C-terminal.Phylogenetic analysis indicated that Mnfico3 had a closer evolutionary relationship with vertebrate ficolins than with its invertebrate homologues.Tissue distribution analysis indicated that Mnfico3 was predominantly expressed in muscle,in which its transcription was increased following bacterial challenge by A.veronii.Function analysis using recombinant protein revealed that rMnfico3 had broad-spectrum binding capacity to a variety of microorganisms and pathogen-associated molecular pattern（PAMP）ligands.Furthermore,rMnfico3 exhibited Ca²⁺-dependent agglutinating activity against microbes in vitro,and ability to attach to the hemocyte surface which promoted phagocytosis and subsequent clearance of invasive bacteria in vivo.Silencing rMnfico3 in prawn through RNAi did not alter the expression of antimicrobial peptide genes（ALF and Crustin）and PO activity.These results manifested that Mnfico3 functioned as a potential pattern recognition receptor（PPR）to mediate cellular immune response by recognizing PAMPs,agglutinating invasive microbes,and promoting phagocytosis of hemocytes.