Study On The Antibacterial Action Mechanism Of The Antibacterial Peptide Scolopin 2-NH₂

Antibacterial peptides(AMPs)are small cationic peptides from various species.They have innate immune function and can kill bacteria,fungi,viruses,protozoa and cancer cells.In recent years,the wide spread use of antibiotics caused that multi-resistant bacteria infection spread in clinical.So low or no drug-resistance action of antibacterial peptides has wide application prospection the aspects of prevention and treatment of drug-resistant bacteria infection.So AMPs would be developed a new type of broad-spectrum and efficient antimicrobial drugs.AMP-Scolopin-2(S-2)was isolated from centipede venoms of Scolopendra subspinipes mutilans,which was a cationic peptide,riched hydrophilic amino acid residues in C-terminal and riched hydrophobic amino acid residues in N-terminal,including 25 amino acids.We achieved Scolopin-2-NH2(S-2-N)by amidated modification.In the present study,we found that S-2-N had much stronger antibacterial activity than its mother peptide S-2,but the antibacterial mechanism of AMP-S-2-N/S-2 remained unknown.Therefore,this paper explores not only the antibacterial activity and physical and chemical properties of AMP-S-2-N/S-2,but also the antibacterial mechanism on cellular and molecular levels,which provides a certain theoretical basis for antimicrobial peptide eventually developing into a novel antimicrobial agent.Our research is divided into three parts:1.The antibacterial activity and physical and chemical properties of AMP-S-2-N/S-2 were explored.First of all,the agarose cavity method was used to detect antibacterial activity of AMP-S-2-N/S-2 on E.coli(K12D31),Staphylococcus aureus,salmonella and Bacillus subtilis.The results showed that both had obviously antibacterial effect and the antibacterial effect of S-2-N was stronger than the mother peptide.Meanwhile,E.coli(K12D31)ultimately was choosed as the antibacterial sensitive strain,which would be used to research the antibacterial mechanism.In addition,we studied physical and chemical properties of AMP-S-2-N/S-2,including the environment sensitivity of both peptides.The agarose cavity method revealed that AMP-S-2-N/S-2 had certain heat resistance,acid and alkali resistance and the ability to resist ionic strength and digestive enzyme.2.The antibacterial mechanism of AMP-S-2-N and S-2 were rsearched on cellular level.Primarily,confocal laser scanning microscopy was used to survey the location of FITC-S-2-N and FITC-S-2 in E.coli(K12D31)cells.Results demonstrated that both AMP-S-2-N and S-2 could rapidly enter into E.coli(K12D31)cells.Secondly,transmission electron microscopy assay indicated that S-2-N could disorganized E.coli(K12D31)cells in 2 h and led to the contents leaking.Lastly,cell membrane integrity and the efficiency in E.coli(K12D31)treated with S-2-N and S-2 were tested by flow cytometry.The results showed that both peptides could lead to a small degree of cell membrane damage in a short period of time(30 min),and the membrane efficiency of S-2-N was much higher.3.The antibacterial mechanism of AMP-S-2-N and S-2 were investigated on molecular level.Firstly,we examined the change of DNA and RNA content by microplate reader.The results found that the nucleic acid content decreased significantly after treated with AMP-S-2-N/S-2;Wha's more,different concentrations of S-2-N and S-2 could combine with DNA and RNA in different degrees by gel electrophoresis technology,which suggested that AMP-S-2-N/S-2 influenced E.coli(K12D31)metabolism;the changes of the DNA secondary structure were studied by Circular Dichroism spectroscopy.The results showed that the positive peaks and negative peaks of DNA have the phenomenon of reduce,suggesting AMP-S-2-N/S-2 affected the secondary structure of DNA;Finally,we analyzed the impact of AMP-S-2-N/S-2 on E.coli(K12D31)cell cycle by flow cytometry.We found the increase of the R phase cells number,which implied AMP-S-2-N/S-2 suppress E.coli(K12D31)cell cycle.The result of qRT-PCR proved that AMP-S-2-N/S-2 inhibited the expression level of related genes(dnaA,dnaB,dnaG and SSB)of the E.coli(K12D31)cell DNA replication and promoted the expression level of the E.coli(K12D31)cell DNA repair related genes(RecA and RecN).In conclusion,AMP-S-2 and S-2-N could destroy the E.coli(K12D31)cell membrane,combine with the DNA and RNA,influence the secondary structure of DNA,suppress cell cycle and influence the expression levels of replication and repair related genes of the cell DNA.These results would provide necessary experimental and theoretical basis for applying in clinical treatment of multi-resistant bacteria infection.