Research On The Immune Mechanism Of Against White Spot Syndrome Virus And Vibrio Alginolyticus By PcLT,a C-type Lectin From Procambarus Clarkii

Crustaceans are thought to lack adaptive immunity and rely completely on an innate immune system to defend themselves against pathogens, which includes a set of cellular and humoral immune reactions. C-type lectins not only act as PRRs to distinguishes self and nonself, but also function as an immune modulator to participate lots of immune reactions.In this study, we studied the functions of C-type lectin PcLT from the the freshwater crayfish, Procambarus clarkii, including basic structure?activity and the function in against Vibrio alginolyticus and White spot syndrome virus(WSSV). We also fused the PcLT with protein transduction domains(PTD) to obtain oral protein subunit vaccine to protect shrimp from White spot syndrome(WSS).In this work,RT-PCR and qRT-PCR analyses demonstrated PcLT was specifically expressed in the hepatopancreas and the mRNA was markedly upregulated by V. alginolyticus and WSSV challenge, although a slight difference in timing was observed. In addition, PcLT protein could only be detected in the hepatopancreas of healthy shrimp. However,after infection with V. alginolyticus or WSSV, PcLT protein was detected in the hemolymph, suggesting that PcLT could be a secreted, pathogen-inducible protein to defense the pathogens. Recombinant mature PcLT expressed in E. coli BL21(DE3) could agglutinate all of the microbes tested, including gram-positive and gram-negative bacteria and yeast, when calcium ion was present. Agglutination activity could be inhibited by D-mannose, possibly due to the modified EPN motif.recombinant PcLT (rPcLT) cannot acted as an effector to kill Vibrio alginolyticus?Staphylococcus aureus and Escherichia coli cells,whether calcium ion was present or not. rPcLT was able to bind to Vibrio alginolyticus?Staphylococcus aureus and WSSV in calcium ion-independent manner.The study revealed upregulation of the mRNA expression and activity of immunological markers, peroxinectin, phenoloxidase, and superoxide dismutase in hemolymph in response to rPcLT. Moreover, rPcLT also enhanced the phagocytic activity of hemocytes, indicating that PcLT may play an important role in modulating humoral and cellular reactions. There was a report revealed a dramatic reduction in the activity of PO and SOD in WSSV-infected shrimp. Therefore, we inferred that PcLT might took part in WSSV defense by regulating humoral immune reactions. rPcLT could not bind to the receptor on the suface of hemocyte like an opsonin, In this work, peroxinectin mRNA was upregulated by injection of PcLT, and, simultaneously, PcLT enhanced the phagocytic activity of hemocytes, suggesting that PcLT-mediated phagocytosis was related to peroxinectin signaling.Our results showed that V. alginolyticus was rapidly eliminated in vivo and that this elimination could be accelerated by pre-incubation with rPcLT. The vivo antivirus assays also showed that pre-incubation of WSSV with rPcLT significantly increased the survival rate of shrimp.Combine the expression pattern after pathogens stimulation with the mechanism of bacterial and viral infections, We hypothesize that these mechanisms are likely related to the different infection mechanisms of bacteria and viruses, specifically, the extracellular nature of bacterial infections versus the intracellular nature of viral infections. White spot syndrome(WSS) is generally regarded as a syndrome mainly due to WSSV decreasing of cellular and humoral immunity functions,which caused the shrimp susceptible to other pathogenic microorganisms, such as Vibrio species.However, rPcLT provided protection against Vibrio and WSSV,which play an important role in preventing WSS.Recently, Protein transduction domains (PTDs), such as the HIV1-TAT peptide, have been previously used to promote the uptake of proteins into a range of cell types. In order to realize the oral administration of protein subunit vaccine by PTD-mediated delivery,we designed the new mutant PTD based on the TAT peptide,then expressed the fusion preotein PTD-PcLT-eGFP in E.coli. Immunofluorescence showed that strong green-fluorescent signals of EGFP were detected in the inner layer of the mid gut from crayfish fed with PTD-PcLT-eGFP, but not in the mid gut in groups treated with either PcLT-eGFP or PBS, indicating that PTD-PcLT-eGFP can travel across intestine.Further word needed to confirm whether PTD-PcLT-eGFP enter the hemolymph or not and increase the oral efficiency of PTD-fusion protein.