Study On The Antimicrobial Activity And Mechanism Of Two Types Of Antimicrobial Peptides

The discovery of antibiotics is a landmark event in human history.It is a great revolution in the medical world and has saved the lives of countless patients.Unfortunately,accompanying the birth of these antibiotics,a large number of drug-resistant strains of bacteria have emerged in recent years.Therefore,developing antimicrobial drugs with noval mechanisms and new treatment strategies is crucial.Antimicrobial peptides are essential for host defense and play an important role in the innate immune system.They show broad spectrum of antimicrobial activity against Gram-negative bacteria（Escherichia coli,Pseudomonas aeruginosa,Acinetobacter baumannii,etc.）,Gram-positive bacteria（S.aureus,Bacillus subtilis,etc.）and many drug-resistant bacteria identified by clinical isolation.Most antimicrobial peptides killed bacteria by destroying the bacterial cell membrane and causing the contents to leak.This mode of action is different from the mechanism of traditional antibiotics that kill bacteria by targeting the intracellular enzymes of bacteria.It is difficult for bacteria to repair the damaged membrane,so antimicrobial peptides seldom induce drug resistance.Therefore,antimicrobial peptides have been described as a new potential class of antimicrobial drugs and provoked great interest of research and development in the lab of institutes as well as pharmaceutics.MSI-367 is a typical amphiphilic alpha-spiral antimicrobial peptide,which contains only three natural amino acids of lysine（Lys）,phenylalanine（Phe）and alanine（Ala）.They consist of the peptide by thrice repeating the unit of KFAKKFA with an amide end,（KFAKKFA）₃-NH₂.MSI-367 was reported to have good antimicrobial activity,but it was limited in preclinical study phase due to the strong cytotoxicity in mammalian cells and hemolytic activity in host red blood cells.Substance P is an important member of mammalian tachykinin family with a sequence of RPKPQQFFGLM-NH₂.The hydrophobic amino acids in the sequence are arranged at the end of the-COOH,while the positively charged hydrophilic residual base is located at the end of its-NH₂.Such arrangement of amino acid makes substance P an amphiphilic peptide.This is typically characteristic of the structure of most antimicrobial peptides.In addition,it has been reported that substance P can bind its natural receptor Neurokinin-1（NK1R）to regulate the innate immune.In this paper,a series of peptide analogues were synthesized by coupling different fragments of substance P with MSI-367.All of the peptides were tested to determine their minimum inhibitory concentration（MIC）.Among them,M2X,M25,M213 and M214 showed forceful antibacterial activity（MIC ranging in 2-8μg/mL）against Escherichia coli（ATCC 25922）,Pseudomonas aeruginosa（ATCC 27853）,Acinetobacter baumannii（ATCC 19606）and nine clinical drug-resistant strains corresponding to these three gram-negative bacteria.In the hemolytic activity experiment,M2X and M25 showed about 20%of hemolysis rate at the concentration of 256μg/mL,while M213 and M214 showed no hemolytic activity at the same concentration.In mammalian cytotoxicity experiments,M2X and M25 were found to exhibit significant cytotoxicity（15%of cell survival rate at the concentration of 25μM）,while M213 and M214 were not significantly cytotoxic even at the concentration of 100μM.These results show that M213 and M214 performed a preferential selectivity of Gram-negative bacteria over eukaryotic cells,which may promise fewer side effects on mammal.The mechanism of antibacterial activity of the peptide analogues was investigated in E.coli.It was found that all peptide analogues killed bacteria by destroying the structure of bacterial cell membranes.M213 and M214 act on E.coli cell membranes in a milder manner while M2X and M25 were very rapid and violent.The assay of bacterial outer membrane permeability showed that these four peptide analogues affected the integrity of bacterial outer membranes at similar degree.In a summary,the M213 and M214 peptide which are derived from the structural modification of MSI-367 peptide,showed good antibacterial activity.They have low hemolytic activity and little cytotoxicity in mammalian cells.In contrast,M2X and M25 peptide,though having good antimicrobial activity,were found to exhibit strong cytotoxicity and hemolytic activity.Based on the analysis of the sequences of the peptide analogues,the differences between the two types of peptide analogues are mainly related to their hydrophobicity and physicochemical properties such as the number of net positive charges.This study provided an interesting approach to improve the cell-selectivity of antimicrobial peptide and lower its toxicity and side-effects.