De Novo Design,Screening And Biological Activity Research Of Antimicrobial Peptides With High Activity And Low Toxicity

The resistance of bacteria and fungi to antibiotics has become a problem that can not be ignored.It is urgent to develop novel antibiotics.Because of their special mechanism of action,antimicrobial peptides have potential to be developed novel antibiotics.However,natural antimicrobial peptides have some drawbacks,such as low activity and hemolysis,which limit their development and application.De novo design of polypeptides is an effective way to obtain novel antimicrobial peptides.In this study,100 peptide sequences were randomly selected from the peptide library designed with the software V1.0 developed in our lab,and synthesized with solid phase synthesis method.An antimicrobial peptide with high broad-spectrum antimicrobial activity was selected and named as SAMP1.More efficient and less toxic antimicrobial peptides SAMP1-A3 and SAMP1-A4 were obtained by optimizing the structure of the antimicrobial peptide SAMP1.The results of the antimicrobial activity of SAMP1-A3 and SAMP1-A4showed that their minimum inhibitory concentration?MIC?for C.tropicalis was 3.9?g·mL^-1,and the MIC for L.monocytogenes was 3.9?g·mL^-11 and 0.95?g·mL^-1,respectively.The hemolysis rate was 0%and 2.7%when the concentration of SAMP1-A3 and SAMP1-A4was 62.5?g·mL^-1,respectively.Cytotoxicity test showed that SAMP1-A3 and SAMP1-A4did not inhibit the growth of HUVEC cells even post-treatment for 24 hours.The growth of cancer cells SKOV3 was not affected at below 125?g·mL^-1.Therefore,SAMP1-A3 and SAMP1-A4 were antimicrobial peptides with strong antimicrobial activity and high biological safety.The results of stability showed that the antimicrobial activity of SAMP1-A3 and SAMP1-A4 remained stable after 30 minutes of heating at 80?,1 hour of treatment with pH 2.0-8.0 solutions,6 hours in 25%plasma,but unstable in trypsin abore 4?g·mL^-1.The results of circular dichroism showed that the proportion of alpha-helix of SAMP1-A3 and SAMP1-A4 increased in 50%TFE solution.So,they remain?-helix structure when they acted with cell membrane.The mechanism of action of SAMP1-A3 and SAMP1-A4 was studied using scanning electron microscopy,transmission electron microscopy and flow cytometry.The results of scanning electron microscopy?SEM?and flow cytometry showed that SAMP1-A3 and SAMP1-A4 destory cell membrane and led to the leakage of cell content in L.monocytogenes cells and C.tropicalis cells.The results of transmission electron microscopy?TEM?showed that SAMP1-A4 could destory intracellular organelle,damage cell membrane in L.monocytogenes.In summary,SAMP1-A3 and SAMP1-A4,with high efficiency,low toxicity and high stability were screened.It was proved that SAMP1-A3 and SAMP1-A4 played an antimicrobial role by destroying the pathogenic cell membrane and introcellular organelle structrue.