| Marsupenaeus japonicas is one of the important economic shrimp species,and aquaculture of the shrimp could bring huge economic profit in shrimp industry.However,the shrimp is vulnerable to some pathogenic microorganisms,including white spot syndrome virus(WSSV).yellow head virus,infectious muscle necrosis.Vibrio anguillarum and V.parahaemolyticus.The vertebrates could against pathogenic microorganisms by innate and adaptive immunity,but the invertebrates resist pathogen invasion only by innate immunity.In this paper,we studied the specific celluar and molecular mechanism in shrimp infected by white spot syndrome virus and V.anguillarum.and this study provided new ideas and methods for shrimp disease control.1.Polymeric immunoglobulin receptor-like protein is the new receptor of WSSV in M.japonicusThe polymeric immunoglobulin receptor(pIgR)acts as the receptor for pIg and transports pIgA/pIgM across intestinal epithelial cells(IECs)in vertebrates.As a type? transmembrane glycoprotein,pIgR is widely expressed in epithelial cells.Some studies on plgR have found that certain microorganisms hijack pIgR to their own benefit during the invasio n of host cells.In the present study,we identified an IgSF cell adhesion molecule that was similar to poly immunoglobin receptor(pIgR)of vertebrates from Marsupenaeus japonicus.and designed it as Marsupenaeus japonicus plgR like protein(MjpIgR).MjpIgR mRNA is expressed in hemocytes and in all other tested organs,and its expression was significantly induced by WSSV infection at the mRNA and protein level.RNA interference and antibody blocking assays inhibited WSSV proliferation in shrimp,and had a much higher survival rate compared with that of the control groups.AjpIgR-overexpression group promoted the proliferation of WSSV in shrimp.To confirm whether MjpIgR is the entry receptor forWSSV,binding assays was performed.Pulldown assays suggested that MjpIgR could interact with WSSV through VP24 as a cellular receptor of WSSV,and MjpIgR can independently render non-permissive cells(HEK 293T)susceptible to WSSV infection.The results suggest MjpIgR could hijacked for WSSV invasion as a new receptor.MjpIgR was oligomerized and internalized following WSSV infection and the internalization was associated with endocytosis of WSSV.Immunocytochemical assay was performed to detect the co-localization of MjpIgR with WSSV.Endocytosis of WSSV required the internalization of MjpIgR in shrimp hemocytes by flow cytometry.Further study found that the intracellular domain interacts with calmodulin(MjCaM)and several studies have reported that calmodulin could interact with clathrin.he viral internalization facilitating ability of MjpIgR could be blocked using chlorpromazine,an inhibitor of clathrin dependent endocytosis.indicating that MjpIgR-mediated WSSV endocytosis was clathrin dependent.The adaptor protein(AP-2)also associated with clathrin-mediated endocytosis in shrimp.All the results indicated that WSSV enters shrimp cells via the plgR-CaM-Clathrin endocytosis pathway.This is the first report of a WSSV IgSF receptor.WSSV has evolved multi-step attachment processes,and a requirement for more than one receptor in its infection,such as ?-integrin.This is consistent with other virus in a variety of host infection pathway2.The dyslexia-associated protein KIAA0319L restricts White Spot Syndrome Virus replication in shrimpThe dyslexia-associated proteins KIAA0319 was reported as a susceptibility gene for dyslexia.Polycystic kidney disease(PKD)domains in the protein were found involved in cell adhesion.The KIAA0319 is a transmembrane protein with signal peptide(SP).seven-cysteine motif(MANSC).five PKD domains and a transmembrane(TM)domain.It was reported that KIAA0319 could interact with adaptor 2 and be internalized in the classical clathrin-mediated endocytosis pathway,In the present study,we identified a KIAA0319L(The dyslexia-associated protein KIAA0319L,Mj KIAA0319L)in shrimp and found that its relative expression could be induced by WSSV stimulation.In order to explore the function of MjKIAA0319L in shrimp,we performed an RNA interference assay.After knockdown of MjKI4A0319L expression in shrimp.WSSV replication was enhanced and the survival rate decreased compared to the control group.Therefore.MjKIAA0319L in shrimp restricts White Spot Syndrome Virus replication.MjKIAA0319L was internalized following WSSV infection and its intracellular domain interacts with AP-2.However,the specific mechanism still needs further research.3.PIAS negatively regulates the JAK/STAT pathway and involved in innate immunityProtein inhibitor of activated STAT(PIAS)proteins are activation-suppressing proteins for signal transducer and activator of transcription(STAT),which involves gene transcriptional regulation.The inhibitory mechanism of PIAS proteins in the Janus kinase(JAK)/STAT signaling pathway has been well studied in mammals and Drosophila.However,the roles of PIAS in crustaceans are unclear.In the present study,we identified PIAS in kuruma shrimp Marsupenaeus japonicus and found that its relative expression could be induced by Vibrio anguillarum stimulation.To explore the function of PIAS in shrimp infected with V.anguillarum.we performed an RNA interference assay.After knockdown of PIAS expression in shrimp subjected to V.anguillarum infection,bacterial clearance was enhanced and the survival rate increased compared with those in the control shrimp(dsGFP injection).Simultaneously,the expression levels of antimicrobial peptides(AMPS),including anti-lipopolysaccharide factor(ALF)A1,C1,C2,and Crustin I,increased.Further study revealed that knockdown of PIAS also enhanced STAT phosphorylation and translocation.Pulldown assay indicated that PIAS interacts with activated STAT in shrimp.In conclusion,PIAS negatively regulates JAK/STAT signaling by inhibiting the phosphorylation and translocation of STAT through the interaction between PIAS and STAT.which leads to the reduction of AMP expression in shrimp.Our results revealed a new mechanism of PIAS-mediated gene regulation of the STAT signal pathway. |
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