Investigation Into The Expression And Antimicrobial Mechanisms Of Crustins Fromscylla Paramamosain

The mud crab（Scylla paramamosain）,is an economically important farming breed distributed throughout the coasts of southeast China.However,S.paramamosain constantly encounters a range of pathogenic organisms in the process of breeding.Antimicrobial peptides（AMPs）are important effective molecules of the innate immunity system of S.paramamosain.For traditional antibiotics,antimicrobial peptides are potential substitutes which possess broad spectrum antibacterial activity and difficult to develop bacteria resistance.Crustin family is one of antimicrobial peptides which has been completed extensive research in crustaceans.It exhibited broad antimicrobial spectrum and potent antimicrobial effect.Our laboratory targeted published SpCrus5（GenBank MF431586）and SpCrus6（GenBank MF431585）,Therefore,my work is that recombinant expression of SpCrus5 by Pichia pastoris system and synthesizing partial SpCrus6 fragment,to explore the antimicrobial spectrum and antimicrobial activity,finally to uncover the antimicrobial mechanisms.The results of this research are as follows.（1）The recombinant expression and antimicrobial activity of the hybrid antimicrobial peptideScy-SpCrus5:Constructedtherecombinantexpressionvectorplasmid pPIC9K-Scy-SpCrus5,transformed into the Pichia pastoris GS115 and induced them to express Scy-SpCrus5 peptides.The target peptides were purified by the method of Ni²⁺affinity chromatography to determined the antimicrobial assays in vitro.The results of antimicrobial assay showed the hybrid antimicrobial peptide Scy-SpCrus5 has no antimicrobial activity for selected eight bacteria within the limited concentration（15μM）.（2）Identification the antimicrobial mechanism of hybrid antimicrobial peptide Scy-SpCrus5.Confocal laser scanning microscopy（CLSM）images showed the fluorescence localization of Scy-SpCrus5 was present around the cellular surface but was absent inside the cell when treated to S.aureus.While Scygonadin was not only absent around the cellular surface,but also was absent inside the cell of the untreated group.Enzeme linked immunosorbent assay（ELISA）results further revealed that for the hybrid antimicrobial peptide Scy-SpCrus5,showed binding activity of LTA in a concentration-dependent manner,and was stronger than Scygonadin.（3）Antimicrobial assay and kinetics curve for the synthetic SpCrus6_29-47.Bioinformatics analysis predicted that the synthesized SpCrus6_29-47 fragments exhibit antimicrobial activity,then the results of antibacterial activity in vitro revealed that SpCrus6_29-47 exhibited potent antimicrobial activities against all tested Gram-positive bacteria（Corynebacterium glutamicum,Micrococcus luteus,Micrococcus lysodeikticus,Staphylococcus aureus）with MICs ranging from6²4μM.Furthermore,SpCrus6_29-479-47 could weakly inhibit the growth of selected Gram-negative bacteria（Psdeuomnoda fluorescens,Pseudomonas stutzeri）with MICs of varying from 24⁴8μM.While for both Escherichia coli and Shigella flexne,MICs were higher than 48μM.Killing kinetics results showed that when the bacteria were incubated with SpCrus6_29-47 in 5 min,half of bacteria could be killed,and when the incubating time was prolonged for around 120 min,all of bacteria was destroyed.（4）Investigation antimicrobial mechanisms of SpCrus6_29-47.After incubated with SpCrus6_29-479-47 for 45 min,Scanning electron microscope images of Staphylococcus aureus revealed that the surface of bacteria cells became clearly rough,even the emergence of more distorted architecture,meanwhile,a large amount of cellular debris was observed in the visual field.While for untreated bacteria cells,their cell surface was smooth and intact,and the change of cellular morphology was not observed at all.Equally,Transmission electron microscope images of treated S.aureus showed that SpCrus6_29-479-47 could induce cytoplasmic membrane ruptured and even disappeared completely accompanied with cellular debris was covered around the bacteria.While for the untreated bacteria,the bacteria cells had intact cytoplasmic membrane and their continuous double membrane structure was clearly visible.Flow cytometry analysis was used to test the SYTO 9/Propidium iodide（PI）uptake by the treated bacteria,and the results revealed the antimicrobial mechanism of SpCrus6_29-47 likely via changing the cytoplasmic membrane permeability of S.aureus.