The Studies On The Rational Design, Prokaryotic Expression And Antibacterial Mechanisms Of Five Antimicrobial Peptides

The antimicrobial peptide A20L was successfully expressed by fusion with anubiquitin tag (Ub-tag) and human SUMO tags (SUMO1/2/3/4-tags). A20L exhibitedexcellent antimicrobial activity against various Gram-negative and Gram-positivebacteria. Based on the hemolytic activity against human red blood cells, A20Lshowed good specificity against bacteria.The antibacterial mechanism of action andmembrane specificity of A20L was further studied using membrane permeabilizationexperiments, and tryptophan fluorescence and quenching experiments in liposomes.The mechanism of action of A20L was proved as membrane disrupting mechanism.A20L showed stronger specificity on liposomes mimicking bacterial membrane thanthose mimicking eukaryotic cell membrane, which is consistent with the biologicalactivity studies. We believe that the fusion tag systems represent good alternatives tochemical synthesis for the industrial production of antimicrobial peptides with lowcosts and high yields.We developed alpha-helical segment of XPF-St7termed as XPF2. Using the XPF2as a framework, we increased the positive net charge of XPF2by amino acidsubstitutions, thus obtained two novel antimicrobial peptides XPF4and XPF6. XPF4and XPF6showed much better overall antimicrobial activity against bothGram-negative and Gram-positive bacteria than XPF2. The therapeutic index ofXPF4and XPF6was5.6-fold and6.7-fold of XPF2, respectively. Bacterial cellmembrane permeabilization and genomic DNA interaction assays were utilized toexplore the mechanism of action of XPF serial peptides. The results revealed that thetarget of these antimicrobial peptides was the bacterial cytoplasmic membrane.We report the application of ubiquitin fusion technology to the expression andpurification of AN5-1. Minimum inhibitory concentration (MIC) and measurementof hemolytic activity (MHC) were measured to confirm the biological activities ofthe expressed AN5-1. Bacterial cell membrane permeabilization was investigated toshow the interaction between the AN5-1and the bacterial cytoplasmic membrane. Furthermore, intracellular activities of the AN5-1were determined by genomic DNAinteraction assays. The results revealed AN5-1damaging bacterial membranes andbinding to bacterial genomic DNA to inhibit cellular functions, suggesting that it hasmultiple intracellular targets in bacteria. The application of ubiquitin fusiontechnology may be an excellent approach for industrial production to the expressionand purification of antimicrobial peptide. Furthermore, AN5-1was demonstrated asan antimicrobial peptide with great potentials, since bacterial resistance to AN5-1would be not expected, due to the dual mechanisms of AN5-1against bacteria.