Screening Of High-efficiency And Low-toxicity Antimicrobial Peptides And Study On Antibacterial Mechanism

With the continuous increase of drug-resistant bacteria,humans urgently need to develop new antimicrobial drugs with high efficiency and low toxicity.Antimicrobial peptide L163,computationally designed by our laboratory,is active against multidrug-resistant(MDR)bacteria but is easily degraded in plasma and by trypsin.Structural modifications are speculated to enhance the biostability of antimicrobial peptide analogues toward protease degradation.To confirm this hypothesis,amino acid substitution,cyclization and acetylation were performed.The stability,antimicrobial activity and biosafety of L163 and its analogues were investigated.Compared with unmodified L163,acetylation enhanced the stability toward p H,plasma and trypsin digestion,while phenylalanine(Phe)substitution for leucine(Leu)and cysteine bridge(S-S)cyclization decreased the stability.Acetylation also exhibited enhanced antimicrobial activity against MDR Streptococcus Sc181,Listeria monocytogenes and Enterococcus E1478F;maintained the activity against MDR Staphylococcus aureus 9,Staphylococcus sciuri P254 and Staphylococcus aureus RN4220;and decreased the activity against Candida tropicalis,Candida albicans and Enterococcus faecalis Fbc35.But Phe substitution for Leu and S-S cyclization decreased the antimicrobial activity.During the biofilm formation process of the tested microbial strains,it was also observed that L163 and L163-Ac had anti-biofilm activity.The results indicated that,compared with Phe substitution for Leu and S-S cyclization,N-terminally acetylated L163(L163-Ac)is the best candidate.Taking Staphylococcus aureus 9 and Listeria monocytogenes as the research objects,explore the antimicrobial action mechanism of antimicrobial peptides L163 and L163-Ac.According to the results of scanning electron microscopy(SEM),L163 and L163-Ac caused the cell wall membrane of Listeria monocytogenes to shrink and rupture,but had little effect on the external morphological structure of the cell wall of Staphylococcus aureus 9.NPN and membrane potential experiment confirmed that L163 and L163-Ac increased the permeability of the inner and outer membrane of these two bacteria.Calcein leakage test detects the damaging effect of L163 and L163-Ac on the simulated membrane of Gram-positive bacteria.Intracellular mechanism studies have shown that compared with Listeria monocytogenes,L163 and L163-Ac induce the accumulation of more reactive oxygen species(ROS)in Staphylococcus aureus 9 cells.In-depth study found that L163 and L163-Ac can cause the fragmentation of Staphylococcus aureus 9 DNA without breaking the DNA of Listeria monocytogenes.The results of the in vitro interaction between the antimicrobial peptides L163 and L163-Ac and DNA show that small molecule peptides and DNA are combined by electrostatic action or groove action,but their binding action does not affect the DNA mobility.In summary,this study obtains two highly active antimicrobial peptides L163 and L163-Ac by screening and structural modification of the antimicrobial peptides designed in the laboratory.It is proved that the antimicrobial peptides L163 and L163-Ac both induced cell apoptosis.For Staphylococcus aureus 9 it was mainly through the accumulation of reactive oxygen species and DNA fragmentation,and for Listeria monocytogenes it was mainly through the reduction of membrane potential.