The Studies On Antimicrobial Mechanism Of Antimicrobial Peptides Brevilacterin From Brevibacillus Laterosporus

Antimicrobial peptide（AMP）is a kind of peptide that has antimicrobial activity.Because AMP is safe,high efficient and hard to occur resistance to drug,there is a wide application prospect in medical,food and animal and plant disease and pest resistance,etc.Antimicrobial peptides,Brevilacterin,were produced by Brevibacillus laterosporus isolated from soil,and had a broad-spectrum antimicrobial activity,which could inhibit or kill gram-positive,gram-negative and fungus,but the antibacterial mechanism was not well understood.In this investigation,the antibacterial mechanism of Brevilacterin was studied using Staphylococcus aureus（S.aureus ATCC 25923）and Escherichia coli（E.coli ATCC 44752）as indicators.This investigation would provide a theoretical basis for the application and development of Brevilacterin.Antibacterial mechanism of Brevilacterin on S.aureus ATCC 25923 was investigated.The minimum inhibitory concentration（MIC）was measured by broth micro-dilution method,and the time-kill curve was drawn.Probe 3,5-dipropylthiadicarbocyanine iodide（Di SC₃（5））was used to investigate the change ofΔψand propidium iodide（PI）staining flow cytometry was used to study the membrane permeability.The cell ultrastructure was observed by transmission electron microscope（TEM）.The leakage of nucleic acid and protein was detected by UV Spectrophotometer,and the interaction of Brevilacterin on DNA was studied by electrophoretic mobility shift assay and UV-visible absorption titration method.The effect of Brevilacterin on protein was tested by SDS-PAGE.The result showed that the MIC of Brevilacterin against S.aureus ATCC 25923 was 12.9AU/m L.After treated with Brevilacterin,Δψof cell membrane was decreased,and the permeability of cell membrane was changed.Besides,the results on morphology and ultrastructure showed that the cell walls were coarse,the cell membranes were shrunken,and plasmolysis occurred.But the membrane integrity was not damaged and there was not obvious nucleic acid or protein leakage.Brevilacterin could bind with DNA,reduce the number of DNA and increase the ultraviolet absorption value of DNA.Brevilacterin could also affect the expression of mycoprotein.Antibacterial mechanism of Brevilacterin on E.coli ATCC 44752 was investigated.The MIC was measured,the time-kill curve was draw,and the change ofΔψwas detected.The outer membrane permeability of E.coli ATCC 44752 was investigated by N-Phenyl-1-naphthylamine（NPN）probe labeling experiment and the inner membrane permeation of E.coli 44752 was studied by o-nitrophenyl-β-d-galactoside（ONPG）inducible enzyme lactose indicator method.The ultrastructure of the cells was observed,the leakage of nucleic acid and protein was detected,and the interactions of Brevilacterin on DNA and protein were studied.The result showed that the MIC of Brevilacterin against E.coli ATCC 44752 was 205 AU/m L.After treated by Brevilacterin,Δψof cell membrane was decreased,and the outer and inner membrane permeability was increased.The results on morphological and ultrastructural observation showed that some cells were damaged,broken and empty.Besides,Serious DNA and protein leakage were found.Brevilacterin could bind with the DNA of E.coli ATCC 44752,heighten the ultraviolet absorption value of DNA or interfere with the DNA replication.Brevilacterin could also affect the decrease of mycoprotein.