Gene Cloning And Functional Study Of Important Molecules Of Innate Immunity In Chinese Mitten Crab (Eriocheir Sinensis)

Chinese mitten crab Eriocheir sinensis is an economically important species and has been cultured commercially in China and other Asian countries.With the development of intensive culture and environmental deterioration,this aquatic production frequently incurred serious infectious diseases caused by viruses,fungi,bacteria,spiroplasma and parasites,resulting in decreased growth in crab production and vast economic losses.As an invertebrate,crustaceans(including crabs)lack a true adaptive immune response system.However,living in an aquatic environment rich in microorganisms,crabs have developed effective systems for detecting and eliminating noxious microorganisms,which depend entirely on a non-specific innate immune response.Therefore,studies on crustacean innate immunity are needed to provide new insights into the control of infectious diseases and the development of sustainable crab farming.Lectins are a type of important pattern recognition proteins(PRPs)that recognize specific sugar conjugates and promote bacteria agglutination by binding the sugar on the bacterial surface.L-type lectins are characterized by the luminal carbohydrate recognition domains(LTLD),and recently,some L-type lectins have been reported to participate in the immune responses.Calnexin(Cnx)and calreticulin(Crt),which are important chaperones localized in the lumen of the endoplasmic reticulum(ER),are involved in translocation,protein folding,and quality control of newly synthesized polypeptides;these proteins,also as lectin chaperones,affect biological processes,especially the innate immune system.Toll signaling pathway could have a significant function in inducing the innate immune response to microbial infection through the transcriptional induction of a battery of gene(e.g.,Toll,Myd88,Pelle,Tube,and Dorsal)encoding antimicrobial peptides.MicroRNAs(miRNAs)have roles in the post-transcriptional regulation of gene expression and various biological processes,such as proliferation,cell differentiation,apoptosis,tumorigenesis,and immune defense.Typically,targeted mRNA leads to translation repression and/or mRNA degradation.More miRNAs have been shown to participate in innate and adaptive immune response during virus infection by regulating the viral or host gene expression.Understanding the innate immunity of crab may provide a basis for development of better strategies for disease management in crab culture.In this work,two novel L-type lectins(designated as EsERGIC-53 and EsVIP36),two lectin-like chaperones(EsCnx and EsCrt),and three key molecules of Toll signaling pathway(EsMyd88,EsPelle and EsTube)were identified from E.sinensis,and their potential functions in innate immunity were investigated.Furthermore,in an attempt to explore the involvement of miRNA in host-virus infection,the crab microRNA-7(miR-7)suppressed the host Myd88-ILF2-(IL-16L)signaling pathway was characterized in this study.Main results of this dissertation included the following 4 parts:1.Cloning and characterization of two different L-type lectin genes from the Chinese mitten crab Eriocheir sinensisThis study identified two novel L-type lectins,designated as EsERGIC-53 and EsVIP36,from the Chinese mitten crab Eriocheir sinensis.The complete nucleotide sequence of ERGIC-53 cDNA was 1955 bp,which contained a 1506 bp open reading frame(ORF)encoding a putative protein of 501 deduced amino acids.The full-length cDNA of VIP36 was 3474 bp with a 984 bp ORF encoding a 327-amino acid peptide.The deduced ERGIC-53 and VIP36 proteins contained a putative signal peptide and an L-type lectin-like domain.Phylogenetic analysis showed that ERGIC-53 and VIP36 belonged to different clades of L-type lectin genes.Real-time PCR showed that ERGIC-53 and VIP36 were expressed in all tested tissues.Quantitative real-time reverse transcription PCR analysis exhibited that ERGIC-53 and VIP36 mRNA transcriptions in hepatopancreas were significantly expressed at various time points after infection with lipopolysaccharide(LPS),peptidoglycan(PGN),Staphylococcus aureus,Vibrio parahaemolyticus,and Aeromonas hydrophila.A bacterium-binding experiment showed that both ERGIC-53 and VIP36 could bind to different microbes.Sugar binding assay revealed that these lectins could also bind to the glycoconjugates of bacteria surface,such as LPS,PGN,D-Mannose,and N-Acetyl-D-mannosamine.Moreover,these two L-type lectins agglutinated bacteria in a calcium-dependent manner,and the results of bacteria clearance experiment showed that both ERGIC-53 and VIP36 facilitated the clearance of injected bacteria V.parahaemolyticus in the crab.Our results suggested that ERGIC-53 and VIP36 functioned as pattern recognition receptors in the immune system of E.sinensis.2.Two endoplasmic reticulum proteins(calnexin and calreticulin)are involved in innate immunity in Chinese mitten crab(Eriocheir sinensis)Cnx and Crt identified from Chinese mitten crab(Eriocheir sinensis)are designated as EsCnx and EsCrt,respectively.The complete nucleotide sequence of EsCnx cDNA was 3175 bp,containing a 1797 bp ORF encoding a putative protein of 598 deduced amino acids.The full-length cDNA of EsCrt was 1741 bp with a 1221 bp ORF encoding a 406-amino acid peptide.EsCnx and EsCrt are expressed in the hemocyte,hepatopancrea,gill,and intestine at the mRNA and protein level.Immunofluorescence analysis indicated that EsCnx and EsCRT are located in the ER.Moreover,the mRNA and protein expression levels of EsCnx and EsCrt were altered by challenge with lipopolysaccharides(LPS),peptidoglycans(PGN),Staphyloccocus aureus,and Vibrio parahaemolyticus.Recombinant EsCnx and EsCrt(rEsCnx and rEsCrt,respectively)proteins can bind to various Gram-positive and Gram-negative bacteria,as well as to different polysaccharides(LPS and PGN).rEsCnx and rEsCrt assisted in the clearance of V parahaemolyticus in vivo,and the clearance efficiency was impaired after silencing of EsCnx and EsCrt.Our results suggest that the two ER proteins are involved in anti-bacterial immunity in E.sinensis.3.Identification,characterization,and functional studies of one Myd88 and one Pelle gene in the Chinese mitten crab,Eriocheir sinensisIn the present study,one myeloid differentiation factor 88 Myd88 gene(named EsMyd88)and one Pelle gene(named EsPelle)were identified from the Chinese mitten crab Eriocheir sinensis.The cDNA of EsMyd88 was 2210 bp long with a 1416 bp ORF that encoded a protein with 472 amino acids.Predicted EsMyd88 protein had a death domain at the N-terminal and a TIR domain at the C-terminal.The full-length cDNA of EsPelle is 3797 bp with a 3156 bp-long ORF that encodes a 1051 amino acid protein.EsPelle protein contains a death domain at the N-terminal and a serine/threonine kinase domain at the C-terminal.EsMyd88 and EsPelle were detected in all the examined tissues of healthy crabs,when normal crabs were challenged with lipopolysaccharide,peptidoglycan,Staphylococcus aureus,Vibrio parahaemolyticus,or Aeromonas hydrophila,the expression levels of EsMyd88 and EsPelle significantly increased either in the hepatopancreas or hemocytes.Results of the pull-down assay showed that EsMyd88 could bind to downstream cytosolic adaptor EsTube.Overexpression of EsMyd88 and EsPelle in Drosophila Schneider 2 cells led to the activation of antimicrobial peptide genes.RNA interference assay showed that both EsMyd88 and EsPelle were involved in regulating the transcription of anti-lipopolysaccharide factor,Crustin and lysozyme in crab challenged with V parahaemolyticus.All the above mentioned results indicated that EsMyd88 and EsPelle have a key function in antibacterial innate immune defense in E.sinensis.4.Eriocheir sinensis microRNA(miR-7)target crab myeloid differentiation factor 88(Myd88)to enhance white spot syndrome virus(WSSV)replicationIn this study,the function of microRNA-7(miR-7)in host-virus interaction was investigated.Replication of White spot syndrome virus(WSSV)was enhanced with the overexpression of miR-7 and inhibited with the downregulation of miR-7 by using anti-miRNA oligonucleotide AMO-miR-7.The target gene of miR-7 was predicted using bioinformatics methods.Results showed that crab myeloid differentiation factor 88(Myd88)could be targeted by miR-7.When the expression of Myd88 was knocked down by sequence-specific siRNA,WSSV copies in crabs were significantly increased.Further findings revealed that knockdown of Myd88,Tube,or Pelle inhibited the expressions of interleukin enhancer-binding factor 2 homolog(ILF2)and interleukin-16-like gene(IL-16L).While ILF2 was silenced,IL-16L expression was inhibited.The overexpression of miR-7 inhibited the expressions of ILF2 and IL-16L.Moreover,when ILF2 or IL-16L was silenced,WSSV copies in crabs were increased.Thus,the upregulated expression of miR-7 during WSSV challenge suppressed the host Myd88-ILF2-(IL-16L)signaling pathway in crabs and enhanced WSSV replication.Our study indicated that WSSV utilized crab miR-7 to enhance virus replication during infection.