Preliminary Research Antibacterial Mechanism Of The Dybowskii Antibacterial Peptide Dybowskin-2CDYa

Dybowskii antimicrobial peptides Dybowskin-2CDYa(GenBank Index No.:EU827809)is a novel antimicrobial peptides separated from the northeast Rana(Rana dybowskii)frog skins in previous work of our laboratory,arginine-rich cationic Dybowskin-2CDYa contains 18 amino acid residues,the amino acid sequence SAVGRHGRRFGLRKHRKH,the relative molecular weight of 2154.2 daltons,determined the isoelectric point is 12.60,with ten positive charge.In the Lipid simulated environment performance of a-helical secondary structure.Laboratory preliminary work results showed that the antimicrobial peptides Dybowskin-2CDYa with low hemolytic activity,high antibacterial activity and broad-spectrum antimicrobial activity and other biological characteristics.These results indicate Dybowskin-2CDYa has the potential to be developed into new antibiotics,but its structural stability and antibacterial mechanism is not clear.On the basis of previous studies,we conducted a preliminary study to Dybowskin-2CDYa for its structural stability and antibacterial mechanism,in order to be a reasonable explanation for its antibacterial process,to provide a theoretical basis for Dybowskin-2CDYa applications.1.Using molecular dynamics(MD)simulation methods,to study the stability of predicted secondary structure of peptides Dybowskin-2CDYa,the results show that antimicrobial peptides Dybowskin-2CDYa can maintain its a-helical secondary structure in aqueous solution,laid the foundation for the subsequent study of structure and behavior of the inhibitory process.2.Using transmission and scanning electron microscopy analysis of the interaction of antimicrobial peptides Dybowskin-2CDYa with Staphylococcus aureus bacterial cell surface.FITC fluorescence dye can only enter the cell interior in the case of the destruction of the bacterial cell membrane,CTC can be combined with the normal bacterial membrane,and DAPI binds to double-stranded DNA,the results showed that bacterial surface appears convex and pore,the permeability of cells increasing,osmotic pressure is not stable,the interior of the cell substances leak out,causing the death of the bacteria;3.Using fluorescence microscopy and CLSM to further analyze the antibacterial mechanism of antimicrobial peptides Dybowskin-2CDYa,the results showed that the fluorescent dye FITC entered the interior of the Staphylococcus aureus cell when the cell exposuring to antimicrobial peptides Dybowskin-2CDYa,indicates that the peptide has effect of generating channels in the cell membrane,which is one of the reasons for changing the osmotic pressure of the bacterial cell;Compared with the control group,CTC,DAPI fluorescent dye result indicates that antimicrobial peptides Dybowskin-2CDYa also entered into the interior of the cell,but dose not inducing cells DNA cleavage.