Characterization And Antifungal Mechanisms Of A Novel Antimicrobial Peptide From Scylla Paramamosain

Scylla paramamosain is an important farming breed along southeastern coast.However,its susceptibility to microorganism infection might lead to poorer survival in the process of breeding.Antimicrobial peptides(AMPs)are important immune factors of innate immune response,which could defense against invasion exogenous pathogens.So far,most of the AMPs isolated and identified from S.paramamosain show antibacterial activities,but little of them exert antifungal activity.The mildew of feed is also one of the important factors restricting the healthy development of aquaculture industry.Chemical fungicide as feed additive although can effectively prevent feed mildew,but chemical fungicide may affect the palatability of feed.Besides,chemical fungicide remaining in the feed may also affect the health of the animals.Therefore,it is of great scientific significance and application value to obtain antibacterial peptides with strong antifungal activity.Based on S.paramamosain transcriptome analysis,a potential AMP was obtained.Following studies on its expression pattern and antimicrobial activity indicated that it was a novel potent antimicrobial peptide,named Sp-AMP.We further investigated antimicrobial activity of Sp-AMP segments,one domain with most potent antimicrobial activity was screened and its in vitro antifungal mechanism was illustrated.The results of this research are as follows:1.Characterization of a novle gene Sp-AMP in S.paramamosain.The Sp-AMP gene expression kept a very low level at embryo,zoea,megalopa and juvenile crab stages.The results also showed that the injection of LPS or Vbrio alinolyticus could not significantly change the Sp-AMP gene expression level at zoea I,megalopa or juvenile crab stages.The Sp-AMP gene expression kept a high level in the ovary of adult female crabs indicated that this antimicrobial peptide was related to gonad.2.Determination of the cDNA sequence of Sp-AMP.The complete cDNA was determined using RACE technique.It contained 573 bp including 181 bp in the 5'untranslated region(UTR),an open reading frame(ORF)of 231 bp,and a 1611 bp in the 3' UTR.The open reading frame(ORF)was predicted to encode 76 amino acids comprising an N-terminal signal peptide and a mature peptide.The deduced cleavage site for the signal peptide was between positions Ser22 and Ala23.Two a-helices and a?-strand were identified in the mature peptide,among which al-helix comprised amino acids from Ile13 to Phe24,the a2-helix comprised amino acids from Pro31 to Arg39 and?-strand comprised amino acids from Asn48 toMet52.Functional domain similar with plant antimicrobial peptide(Antimicrobial21 domain)also identified in the mature peptide which comprised amino acids from Ile26 to Phe54.3.Gene expression pattern analysis of Sp-AMP in normal S.paramamosain.Sp-AMP was detected in all collected tissues of both male and female S.paramamosain while the highest expression level was present in the testis,posterior ejaculatory duct contained androgenic gland and muscle of normal male crab.The highest expression level was present in the ovary and muscle of normal female crab.The expression level of Sp-AMP is greatly higher in male than in female.4.Identification of a high-activity functional domain of Sp-AMP.Different fractions of Sp-AMP are synthesized according to protein secondary structural and functional domain prediction results of Sp-AMP.Antimicrobial activity test of each fraction showed that fraction Sp-AMP26-54 possess higher antimicrobial activity and a wider antimicrobial spectrum in comparison with Sp-AMP1-25 and Sp-AMP1-54.5.Illumination of antimicrobial activity of Sp-AMP26-54.Sp-AMP26-54 exerted strong antimicrobial activity against Micrococcus lysodeikticus,Staphylococcus aureus,Bacillus subtilis,Corynebacterium glutamicum,Escherichia coli,Psdeuomnoda fluorescens,Pseudomonas stutzeri,etc.with the a minimal inhibitory concentration(MIC)around 6 ?M and against Cryptococcus neoformans and Pichia pastoris GS115 with MIC values of 1.5-3 ?M.It also exerted good antifungal activity against Fusarium oxysporum,Fusarium graminearum,Aspergillus niger,Aspergillus ochraceus,Aspergillus fumigatus,etc.with MIC values below 24 ?M.The antimicrobial activity against S.aureus and C.neoformans of Sp-AMP26-54 were maintained after heat treatment while its killing C.neoformans activity is much more sensitive to ionic strength.The killing kinetics of Sp-AMP26-54 showed that it killed 50%bacteria after 5 min incubation and destroyed all the bacteria at around 180 min.As for fungi,Sp-AMP26-54 killed 50%fungi after the incubation of 10 min and destroyed all the bacteria at around 360 min.In addition,Sp-AMP26-54 has no significant cytotoxicity for AML12 and L02 cells and no significant antitumor activity for HepG2 and Hela cells.6.Investigation of antifungal mechanisms of Sp-AMP26-54.Sp-AMP26-54 could inhibit spore germination of F.oxysporum,F.graminearum,A.niger,A.ochraceus,A.fumigatus,etc.and hypha growth of A.nige.After Sp-AMP26-54 treament,the scanning electron microscopy(SEM)images of A.niger,F.graminearum and A.ochraceus spore and C.neoformans showed clear rougher surface,even the emergence of collapsed architecture.Fluorescence activated cell sorting(FACS)assay indicated that Sp-AMP26-54 could change fungi membrane permeability.Confocal laser scanning microscopy(CLSM)image showed that fluorescence localization of Sp-AMP26-54 was present around cellular surface but was absent inside the cell when treated to C.neoformans.In all,Sp-AMP might exert its antifungal activity by targeting and changing permeability of cell membrane,then ultimately lead to death of the microbes.