Functional Analyses Of Crustins In The Innate Immune System Of Mud Crab,Scylla Paramamosain

The mud crab(Scylla paramamosain)is one of the commercially important crabs.It is widely distributed in the East China Sea and the South China Sea in China.With the development of the intensive aquaculture and the enlargement of the cultivation scale of enocomic crustaceans,the frequent outbreaks of viruses and vibriosis have caused decreased production and catastrophic losses in the past decades.The mud crab posses an innate immune system which can effectively defend the intruding pathogens,making it a good model for studying crustacean immunity and for developing novel strategies against crustacean diseases.Crustins are crucial effectors in crustacean immune system.In this study,we identified three novel Crustins,named SpCrus3,SpCrus4 and SpCrus5.According to the current reports,crustins were divided into 5 categories.Type I Crustin comprises a signal peptide sequence,a typical cysteine-rich region at the N-terminus and a conserved WAP domain at the C-terminus.The obtained SpCrus3 and SpCrus4 belong to Type I Crustins.The cDNA sequences of these two genes shared the common 5' terminus.Both SpCrus3 and SpCrus4 were highly expressed in gill.After challenges with Vibrio parahemolyticus or Staphylococcus aureus,SpCrus4 was up-regulated,whereas SpCrus3 was down-regulated.SpCrus3 and SpCrus4 shared high identity of 66.06%,but SpCrus4 exhibited stronger antimicrobial activity than that of SpCrus3.Microorganism-binding assay results revealed that both SpCrus3 and SpCrus4 exhibited binding ability to all tested microorganisms.Furthermore,the polysaccharide-binding assay showed that these two proteins exhibited strong binding activity to bacterial polysaccharides,including lipopolysaccharide(LPS),lipoteichoic acid(LTA),and peptidoglycan(PGN).SpCrus3 and SpCrus4 exhibited stronger binding activity to LPS or LTA than to PGN.Moreover,SpCrus4 showed stronger binding activity to LTA than that of SpCrus3,which may be responsible for the remarkable disparity in antimicrobial activity between these two proteins.In addition,SpCrus4 displayed stronger agglutination activity against several kinds of microorganisms than that of Sp Crus3,and this increased agglutination activity may also contribute to the strong antibacterial activity of SpCrus4.On the basis of all these results,a possible antibacterial mode exerted by SpCrus3 and SpCrus4 was proposed as follows: SpCrus3 was highly expressed in normal crabs to maintain low-level antibacterial activity without bacterial challenges;however,when crabs were challenged with bacteria,large amount of SpCrus4 was generated to exhibit strong antibacterial activity against bacterial invasion.Type II Crustins contained a signal peptide sequence,a glycine-rich region,a typical cysteine-rich region at the N-terminus and a conserved WAP domain at the C-terminus.Type II and type I Crustins are distinguished by glycine-rich region(GRR),which is a major marker motif of type II Crustins.SpCrus5 contained a typical cysteine-rich region at the N-terminus,a conserved WAP domain in the center,and a special GRR at the C-terminus,which differs from the GRRs in typical type II Crustins found between signal peptides and cysteine-rich regions.SpCrus5 shared high similarities with most type II Crustins,and it was more closely related to type II Crustins than to other retrieved Crustins.SpCrus5 was predominantly expressed in gills and remarkably upregulated after the crab was challenged with V.parahemolyticus or S.aureus,suggesting that SpCrus5 might participate in antibacterial immune response.To further elucidate whether this C-terminal GRR affects the function of SpCrus5,we expressed a GRR deletion mutant protein(SpCrus5-?GRR)by deleting the GRR.Liquid growth inhibition assays demonstrated that the antimicrobial activity of SpCrus5 was stronger than that of SpCrus5-?GRR,and the antibacterial spectrum of the former toward Gram-negative bacteria was broader than that of the latter.Binding assays revealed that the microorganism-binding ability and polysaccharide-binding activity of SpCrus5 were stronger than those of SpCrus5-?GRR.SpCrus5 or SpCrus5-?GRR agglutinated all of the tested Gram-positive bacteria.Therefore,the antibacterial activities of SpCrus5 were stronger and broader than those of SpCrus5-?GRR.Microorganism-and polysaccharide-binding abilities might contribute to the antimicrobial activity of SpCrus5.These results suggested that the C-terminal GRR was essential to induce an efficient antibacterial activity of SpCrus5.Although SpCrus5 belongs to type I Crustin based on the classification criteria,SpCrus5 was highly identical with most type II Crustins and showed type II Crustin activities,indicating that SpCrus5 was more likely an atypical type II Crustin than a type I Crustin.In summary,expression patterns and immune functions of three Crustins in mud crab were studied.It was found that SpCrus3,SpCrus4 and SpCrus5 are important antibacterial proteins in the immune system and they participated in the immune response in different ways.This study provides new insights into the Crustins sequence and functional diversity,antimicrobial mechanisms,and how different Crustins collaborate to provide immune protection.