Characterization Of Scylla Paramamosain Neuropeptide FⅡ And Antimicrobial Activity Of Its Functional Domain Sp-NPFin

Scylla paramamosain is one of the important mariculture species along the southeast coast of China with high economic and nutritional value.However,in recent years,with the continuous expansion of the artificial breeding scale,problems such as seawater pollution and pathogenic microbial infection have become increasingly serious,which have caused huge losses to the aquaculture industry.Neuropeptides(NPs)are small proteinaceous substances produced and released by neurons through the regulated secretory route and act on neural substrates.They are widely distributed,low in content but highly efficient,and participate in regulating various physiological processes.NPs have recently been shown to be pleiotropic molecules with their important role between nervous and immune systems.NPs and antimicrobial peptide share several similar structural and physical characteristics including low molecular weight(<10 kDa),cationic and amphiphilic design,which makes it possible for NPs to interact with negatively charged components of microbial cell membranes.NPs have been considered as potential candidates of antibacterial peptides,however,there are few studies focused on their potential roles in the immune defense of invertebrates,especially in S.paramamosain.Based on the transcriptome database established by our laboratory,we obtained a gene NPFII which may be related to innate immunity of S.paramamosain.Further studies focus on its characteristics and function had been carried out,the results are as follows:1.Cloning of full-length of NPFⅡ cDNA.The cDNA sequence of NPFⅡobtained by RACE and sequence splicing technology and its basic structure was revealed.The full-length of NPFⅡ cDNA was 551 bp with a 375 bp open reading frame(ORF)encoding a protein consist of 124 amino acids.The signal peptide cleavage site was located between the 26th(glycine)and 27th(lysine)amino acid residues.The mature peptide of NPFⅡ contained 98 amino acids with a conservative PAH domain,the molecular weight and theoretical isoelectric point of which were 11.1 kDa and 9.75,respectively.Secondary structure and three-dimension structure prediction results showed that NPFⅡ mature peptide would mainly formed an alpha helical structure.2.Gene expression profile of NPFⅡ during different development stages and in different tissues of S.paramamosain.NPFⅡ was expressed throughout early development stages of S.paramamosain and highest expression level was observed in zoea stage Ⅱ.In addition,there were gender difference and tissue specificity in NPFⅡgene expression.NPFⅡ was specifically expressed in males and could be detected in all collected tissues,while the highest expression levels were presented in anterior vas deferens and seminal vesicle.And there was almost no expression in females.3.Gene expression pattern of NPFⅡ induced by LPS challenge and Vibrio alginnolyficus infection.S.paramamosain transcriptome analysis found that,in zoea stage I,the expression of NPFⅡ was significantly decreased at 3 h after LPS challenge while significantly induced at 12 h after V.alginnolyficus infection;at megalops stage,the expression of NPFⅡ was significantly higher at 24 h after LPS challenge but showed no changes after V.alginnolyficus infection;at juvenile stage,the expression of NPFII showed no significant changes under neither LPS challenge nor V.alginnolyjicus infection.By qPCR detection,we found that expression level of NPFⅡwas significantly increased in testis at 3 h and 48 h after LPS challenge,similar tendency was discovered in hepatopancreas at 12 h after LPS challenge.At 6 h after V.alginnolyficus infection,NPFⅡ gene expression in the seminal vesicle increased significantly while no significant change was found in hepatopancreas.All results indicated that the NPFⅡ may be involved in the immune response of the S.paramamosain.To date,no reports have been published on the immune-related functions of NPFⅡ from marine crustacean.4.Expression and purification of the recombinant NPFⅡ mature peptide.We constructed the recombinant expression plasmid and expressed the recombinant protein of NPFⅡ mature peptide successfully using prokaryotic expression system.Studies found that it could recognize and bind to various components of the bacterial cell wall,such as LPS,LTA,PGN and glucan.Moreover,it could bind with a variety of bacteria like Pseudomonas aeruginosa,Vibrio harveyi,Vibrio fluvialis,V.alginnolyficus,Cryptococcus neoformans,Pichia pastoris GS115,and had agglutination effects on V.harveyi,V fluvialis and V.alginolyticus.5.Identification of a high-activity functional domain of NPFⅡ.We identified a high-activity functional domain of NPFⅡ(from the 29th to 73rd residues of NPFⅡmature peptide)according to functional domain prediction and secondary structure prediction,which was then synthesized and named Sp-NPFin.Sp-NPFin had a molecular weight of 5.12 kDa,a net charge of+7,hydrophobicity of 28%,and formed two helical structures.6.Illuminated the antibacterial properties of Sp-NPFin.Sp-NPFin exerted strong antimicrobial activity against various bacteria including Staphylococcus aureus,Bacillus subtilis,Corynebacterium glutamicum,Escherichia coli,P.aeruginosa,Pseudomonas stutzeri,Shigella flexneri and Pseudomonas fluorescens.It also had strong antifungal activity against several fungi like Fusarium oxysporum,Aspergillus niger and Fusarium solani,but it only showed bacteriostatic action but no bactericidal activity on Fusarium graminearurm,Aspergillus fumigatus and Aspergillus ochraceus.Sp-NPFin displayed good thermal stability but could be easily affected by cations.The killing kinetic assays showed that Sp-NPFin could kill all the bacteria at 120 min after incubated with P.aeruginosa and at 30 min with S.aureus.In addition,Sp-NPFin showed no cytotoxicity against hemocytes of S.paramamosain and mammalian cells(HEK-293T and L02),and showed no significant antitumor activity against cancer cells(HepG2 and NCI-H460).The antibacterial activity evaluation results showed that it possessed broad-spectrum and effective antibacterial activity.7.Investigation of antimicrobial mechanism of Sp-NPFin.The effects of Sp-NPFin on the cell integrity and ultrastructure of P.aeruginosa,S.aureus,F.oxysporum and A.niger were observed by optical microscope,scanning electron microscopy(SEM)and transmission electron microscopy(TEM).We found that Sp-NPFin could induce morphological changes of four tested microorganisms.It could also inhibit the spore germination and destruct the cell structure of F.oxysporum and A.niger.Binding assay showed that Sp-NPFin could recognize and bind to various components of the surface of microorganisms,such as LPS,LTA,PGN and glucan.Further more,we found Sp-NPFin was localized on the surface of microorganisms though confocal laser scanning microscopy(CLSM).Results showed that Sp-NPFin may bind to the microbial surface by electrostatic force,interact with the cell membrane and change its permeability,leading to content leakage,cell lysis and even death.The antimicrobial mechanism of SP-NPFin remains to be further explored.Our studies showed that Sp-NPFin had spectral antibacterial and antifungal activity,and had good potential application value in the field of antibacterial drug development and application.