| 1.Functions of Rho GTPases in innate immunity of kuruma shrimpIn the classical immunology theories,there are two forms in the host immune response system,innate immune response and adaptive immune response.In vertebrates,the hosts protect themselves from pathogen infection in both ways.However,since invertebrates lack adaptive immune system,innate immunity response becomes the key protective screen of invertebrates in fighting against pathogen invasion.There is a complicated signaling pathway network in the innate immune system of invertebrates to regulate the immune responses.In this network,a series of key molecules play a crucial role in regulating different signaling pathways though their interaction with downstream effectors.Function as molecular switches in various pathways,hundreds of members of the Ras GTPase superfamily play important roles in immune responses.However,the studies of Rho GTPases in innate immunity are in the minority.Thus,by using kuruma shrimp Marsupenaeus japonicus as our experimental models,we found Cdc42 and RhoA in Rho GTPase family can regulate antiviral and antibacterial innate immunity in shrimp through different molecular mechanisms,respectively.1)Interaction of the Small GTPase Cdc42 with Arginine Kinase Restricts White Spot Syndrome Virus in ShrimpMany types of small GTPases are widely expressed in eukaryotes and have different functions.As a crucial member of the Rho GTPase family,Cdc42 serves a number of functions,such as regulating cell growth,migration and cell movement.Several RNA viruses employ Cdc42 hijacking tactics in their target cell entry processes.However,the function of Cdc42 in shrimp antiviral immunity is not clear.In this study,we identified a Cdc42 gene in the kuruma shrimp Marsupenaeus japonicus and named it MjCdc42.MjCdc42 was upregulated in shrimp challenged by white spot syndrome virus(WSSV).The knockdown of MjCdc42 and injection of Cdc42 inhibitors increased the proliferation of WSSV.Further experiments determined that MjCdc42 interacted with an arginine kinase(MjAK).By RNAi assay and analyzing the binding activity and enzyme activity of MjAK and its mutant ?MjAK,we found that MjAK could enhance the replication of WSSV in shrimp.MjAK interacted with the envelope protein VP26 of WSSV.An inhibitor of AK activity,quercetin,could impair the function of MjAK in WSSV replication.Further study demonstrated that the binding of MjCdc42 and MjAK depends on Cys271 of MjAK and suppresses the WSSV replication-promoting effect of MjAK.By interacting with the active site of MjAK and suppressing its enzyme activity,MjCdc42 inhibits WSSV replication in shrimp.Our results demonstrated a new function of Cdc42 in the cellular defense against viral infection in addition to regulating actin and phagocytosis,which have been reported in previous studies.2)A Small GTPase RhoA Inhibits Bacteria Infection by promoting Phagocytosis in kuruma shrimp Marsupenaeus japonicusRas GTPase superfamily,including more than 100 members,plays a vital role in a series of cellular processes,such as cytoskeleton recombination,gene expression,signaling pathway regulation.A mass of small GTPases are continuously reported to take part in the innate immunity in invertebrates.However,the studies about Rho family relating to innate immunity are relatively in the minority.Since Cdc42 and Rac are revealed to protect the host cells against pathogen infection,whether RhoA has the same function remains unknown.We identified a RhoA GTPase in shrimp Marsupenaeus japonicus and named it MjRhoA.Expression patterns showed MjRhoA is significantly upregulated in hemocytes and heart,then knockdown of MjRhoA showed decreased bacterial clearance ability,increase of the total bacterial number in vivo and high mortality of shrimp.These results confirmed the antibacterial function of MjRhoA in shrimp.Interestingly,we found the subcellular localization of MjRhoA changed from cytoplasm to cell membrane in hemocytes of shrimp after challenged by Vibrio anguillarum;the expression level of MjRhoA also affected the expression of ?-intergrin mediated phagocytosis markers,ROCK2 and Arp2/3.Further study showed that?-intergrin regulates the expression level and subcellular localization of MjRhoA.Thus,MjRhoA inhibited bacteria infection by promoting integrin mediated phagocytosis.Our study confirmed the function of RhoA in the antibacterial innate immunity and enriched knowledge of small GTPases functions in cellular activities.2.The induction and mechanisms of trained innate immunity against WSSV in shrimpThe adaptive immune response has immune memory but the innate immune response doesn't have.However,more and more studies confirmed immune memory also exists in the innate immunity recently.This novel phenomenon is termed "trained innate immunity".Trained innate immunity is widespread in vertebrates.Since invertebrates lack adaptive immune response,innate immunity serves as the key defense of invertebrates against pathogens.Although there are some studies showing that immune memory also exists in the innate immunity of invertebrates,the molecular mechanism is ambiguous.In our study,we found UV inactivated WSSV and a kind of WSSV envelop protein,VP28,could induce trained innate immunity in kuruma shrimp.Training by UV-WSSV and VP28 enhanced the survival rate of shrimp and inhibited the WSSV infection;transcriptome sequencing showed a mass of genes were regulated in trained immunity induced by UV-WSSV and VP28,including phagocytosis related genes,autophagy related genes,genes in toll signaling pathway and genes in Ras signaling pathway.Analyzing by molecular biology and bioinformation,we confirmed UV-WSSV and the single component of WSSV,VP28,could affect the host immune response and induce trained immunity by regulating various signaling pathways.Our study found one single component of WSSV can induce trained innate immunity in invertebrates.Our study confirmed the immune memory in invertebrates and enriched the knowledge of innate immunity. |
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