Immune Functional Analyses Of SpCrus2 In The Mud Crab,Scylla Paramamosain

Scylla paramamosain is commonly called mud crab,which belongs to the Portunidae.Its high nutritional,medicinal and economic value make it an important maricultural crab in China.In the past dacades,the water pollution,bacterial and viral infections and other factors has caused great losses on the mud crab aquaculture.Therefore,how to improve their own immunity to defend against serious diseases has become an urgent problem required to be solved in the crab aquaculture.Antimicrobial peptides?AMPs?is one class of small molecular peptide widely spreading in organism.It has a broad-spectrum antimicrobial activity.AMPs have been proved as important effectors of the innate immune system.Crustins are one important type of AMPs.Studies on the innate immune related genes,not only help to clarify the antibacterial and antiviral immune mechanisms of crustaceans,providing theoretical basis and technical support for the prevention and control of crab diseases,but also contribute to the improvement of the innate immune theory in invertebrates.Studies on the antibacterial activity and mechanism of SpCrus2.In this study,a novel crustin,SpCrus2,was characterized in mud crab.The bioinformatic analyses revealed that SpCrus2 cDNA sequence is 620-bp long with a 495-bp open reading frame encoding a164-amino acid protein.In the deduced protein,a 17-amino acid signal peptide,a glycine-rich hydrophobic region?GRR?,and a cysteine-rich region?CRR?containing a whey acidic protein domain were predicted.SpCrus2 shares high similarity with most type II crustins?types IIa and IIb crustins?in shrimps but has a novel distribution pattern of cysteine residues that is distinct from most crustins.SpCrus2 and Pacifastacus leniusculus Pl Crus3 share high similarity and the same distribution pattern of cysteine residues.Thus,we proposed them as type IIc crustins.In order to explore whether this novel cysteine residues distribution pattern has its unique features compared with the classical crustins distribution pattern,we harvested SpCrus2 mutants,which is named CRR-M,by mutating two amino acids?V106 to C,C151 to F?in SpCrus2.These mutations make the novel cysteine residues distribution pattern of CRR become into the classical distribution pattern of CRR-M.Tissue distribution and expression patterns analysis shows that SpCrus2 is mainly distributed in the gills and can be up-regulated after challenge with Vibrio parahemolyticus or Staphylococcus aureus.To investigate the biological functions of SpCrus2 and the underlying mechanisms,SpCrus2,GRR,CRR,and CRR-M were all overexpressed and purified.Then we carried out a series of functional experiments:bacteriostasisexperiment,microorganism-bindingexperiments,microbial polysaccharide-binding experiments and agglutination experiments.The results of comprehensive bacteriostasis and microbial binding experiments shows that SpCrus2 and its two functional domains GRR and CRR have broad and different antibacterial and binding activities on those microbes used in the experiments.Besides,CRR has stronger binding activity than CRR-M.The results of microbial polysaccharide-binding experiment revealed that SpCrus2 exhibits strong binding activity to LTA,LPS,and?-glucan in a concentration-dependent manner but a weaker binding activity to PGN.Besides,we can speculate that GRR may be the major structural motif that is responsible for the binding activity of SpCrus2 to bacteria,both GRR and CRR are required for the whole protein to exert its binding ability.The results of agglutination test showed that SpCrus2 could induce the agglutination of Gram-positive bacteria under the action of Ca²⁺.To sum up,SpCrus2 has a certain degree of antibacterial activity against Gram-positive bacteria,Gram-negative bacteria and fungi.SpCrus2 GRR itself,as a glycine-rich amphiphilic peptide,exhibited evident antibacterial ability against Gram-negative bacteria,whereas CRR possessed potent antibacterial activity against Gram-positive bacteria.Either GRR or CRR exhibited weaker antibacterial activity than the whole protein of SpCrus2,indicating that GRR and CRR synergized to exert their potential antibacterial functions.In addition,CRR exhibited slightly stronger antimicrobial activity than CRR-M,suggesting that SpCrus2 containing this novel cysteine distribution pattern may exhibit stronger antimicrobial activity than most type II crustins with the conventional distribution pattern of cysteine residues.The likely antimicrobial ability of SpCrus2 may result from its microbial polysaccharide-binding and agglutination activities.Studies on the antiviral activity and mechanism of SpCrus2.In addition to its broad antibacterial activities,AMPs also have antiviral and parasitic activities.Therefore,we set up some experiments to check whether SpCrus2 was involved in antiviral immunity.We used WSSV to challenge the mud crads,and then gills at different time points were collected and analyzed the expression patterns of SpCrus2 in gills.The results showed that the SpCrus2 expression was up-regulated in the mud crab.To confirm the antiviral activity of SpCrus2,WSSV was incubated with the recombinant SpCrus2 protein or GST tag protein and then it was injected into the mud crab.The amount of WSSV was measured at 48 h after injection in gills by real-time quantitative PCR,and the results showed that SpCrus2 can significantly inhibited the proliferation of WSSV.In addition,four proteins?VP15,VP19,VP26 and VP28?of WSSV were also overexpressed and purified,and the likely antiviral mechanism of SpCrus2 was explored by GST pull down.The results showed that SpCrus2 could bind to VP26 and VP28,but not VP15 and VP19,suggesting that the remarkable antivirial activity of SpCrus2 might be due to its binding capacity to VP26 and VP28.Overall,this study characterized the first type II crustin,SpCrus2,in crabs,and offered new insights into understanding the sequence and function diversity of crustins and their immune functions in crustaceans.It also provides an effective theoretical basis for disease resistance breeding of Scylla Paramamosain.