Effect Of Amino Acid End-tagging On Therapeutic Potential Of Antibacterial Peptide

Antimicrobial peptides(AMPs)are a class of small molecular peptides that protect animals and plants from external microorganisms and remove mutant cells in the body.They have broad-spectrum bactericidal activity,a wide range of species,rapid antibacterial efficiency.Therefore,antimicrobial peptides are considered to be the most potential alternatives to antibiotic.However,many natural antimicrobial peptides have poor biological activity in vitro,high toxicity to mammalian cells,poor stability in salt ions and serum environments and easy degradation by proteases,which limited their clinical applications.These problems can be effectively solved by the terminal modification,and it has been prove that adding hydrophobic amino acids such as tryptophan or phenylalanine,which can effectively improve the antibacterial peptide activity and stability.Moreover,the addition of glycine at the N-terminus to form a cap structure can also affect the biological activity of antimicrobial peptides,facilitate the formation of alpha-helical hydrogen bonds,and prevent alpha-helical antimicrobial peptides from losing their helicity in pathogenic cells to improve the stability and activity of antimicrobial peptides.End-tagging with amino acid residues contributes to improve the cell selectivity of antimicrobial peptides(AMPs),but systematic studies have been lacking.Thus,this study aimed to systematically investigate how end-tagging with hydrophobic residues at the C-terminals and Gly capped at the N-terminus of W4(RWRWWWRWR)affects the bioactivity of W4 variants.After the end-tagging peptides were successfully synthesized,the secondary structure of the engineered peptides were tested by circular dichroism chromatography(CD)in various membrane simulation environments at first.Then,under normal test conditions,evaluate the biocompatibility of all engineered peptides including hemolytic activity and cytotoxicity.Next,the biological activity of the end-tagging peptides were evaluated,including the minimum inhibitory concentration,minimum bactericidal concentration,bactericidal power against various infectious bacteria,and all engineered peptides against E.coli 25922 and S.aureus 29213 antibacterial activity in the presence of physiological salt concentration and serum.Finally,through the fluorescence imaging,cell wall permeabilization,cytoplasmic membrane permeability,liposome leakage test,scanning electron microscopy(SEM),transmission electron microscopy(TEM)and other technological means and experimental methods to study GW4 A against E.coli 25922 and S.aureus 29213 antibacterial mechanism.Among all the hydrophobic residues,only Ala end-tagging improved the antibacterial activity of W4.Meanwhile,Gly capped at the N-terminus could promote the helical propensity of the end-tagged peptides in DPC micelles,increasing their antimicrobial activities.Of these peptides,GW4A(GRWRWWWRWRA)showed the best antibacterial activity against the 19 species of bacteria tested(GMMIC=1.86 ?M)with low toxicity,thus possessing a highest cell selectivity(TIall=137.69).It also had rapid sterilization,salt and serum resistance and LPS-neutralizing activity and GW4 A killed bacteria by destroying cell membrane integrity and causing cytoplasmic leakage.Overall,these findings suggested that systematic studies on terminal modification promoted the development of peptide design theory,and provide a potential method for optimization of effective antimicrobial peptides.