Design And Bioactivity Of Novel Antimicrobial Peptides And Its Interaction With Phospholipid

With the rapid rise in the emergence of multidrug-resistant bacteria, there is an urgent need to develop novel antimicrobial therapies to these pathogens. Antimicrobial peptides (AMPs) are an integral part of the innate immune system that protects a host from the invasion of pathogenic bacteria. Antimicrobial peptides show broad-spectrum antimicrobial activity against Gram-negative bacteria, Gram positive bacteria, viruses, fungi, protozoa and cancer cell.The mechanism of antimicrobial peptides is different from traditional antibiotics, which makes it difficult for bacteria to develop resistance. Antibacterial peptides which are characterized by high and broad-spectrum antimicrobial activity and low antibiotic resistance have attracted wide attention, and they are regarded as a substitude for traditional antibiotics or synergistic agent. Thus, one of the hot spots of the antimicrobial peptide research is to design new antimicrobial peptides, study their structures and physicochemical properties in depth and screen out the potential peptides. The modified peptides may display even higher bioactivity compared to parent peptide. In order to solve the problems of antimicrobial peptides in their application, it is of significance to design new antimicrobial peptides with increased antibacterial activity, low cytotoxicity and low hemolytic activity.In this thesis, we chose some short sequences KFNFK, KFTFK, KFSFK and RRWWRF, FRWWHR as bioactive sequence to design short peptides with single bioactive sequence and linear and cyclic peptides with dual bioactive sequences. By studying the biological activity of these designed antimicrobial peptides, such as antibacterial activity, cytotoxicity and hemolytic acitity, the influence of peptide structure on the activity was investigated. The new short sequences KWTWK, RFTFR, RWTWR were designded based on the strategy of replacing Lysine with Arginine or substituting Tryptophan for Phenylalanine, and the new short peptides with single short sequences and linear peptides with two short sequences were further designed. Following the bioactivity investigation of these peptides, potential peptides which showed high antibacterial activity, low cytotoxicity and hemolysis were screened. In addition, the mechanism of these new antimicrobial peptides interacting with biomembrane was discussed based on the investigation of the interaction between peptides and mimic biomembrane, which provides a theoretical basis for peptide modification.In this thesis, we used broth microdilution method to determine the minimum inhibitory concentration of the three new designed classes of peptides against seven bacteria and screened out eight peptides with high antibacterial activity. The cytotoxicity of these eight peptides against normal cell was later investigated by MTT method and their hemolytic activity was determined by hemolysis of rabbit red blood cells. These experiments show that compared with cyclic peptides and short peptides, linear peptides are more antibacterial, low cell toxic and hemolytic which indicates an ideal bioactivity. Doubling the bioactive sequence in one peptide molecule strongly increases the antibacterial activity of peptide, which suggests that dual bioactive sequences design is helpful to increase antibacterial activity of the antimicrobial peptides. Through investigating the changes of Zeta potential of bacteria after applying antimicrobial peptides on bacteria, the affinity of antimicrobial peptides on bacterial cell membrane was reflected. The experiment results show that linear peptides change the Zeta potential of bacteria to a greater extent compared with cyclic peptides and short peptides. By investigating the particle size of liposomes after antimicrobial peptides interacted with DMPC, DMPG liposomes, the disruption effect of antimicrobial peptides on biomembrane was evaluated. The results show that biological phospholipid membrane is not disrupted by all peptides. The surface pressure-area (Ï€-A) curves of peptides1-combi-1, c-combi-1with DMPC, DMPG in mixed momolayer were measured by Langmuir technology. And then analyse the miscibility, thermodynamic stability and compressibility of the hyhrid stystem. Through analysing the stability of the mixed momolayers and the type of molecular forces to reflect the behavior after which antimicrobial peptide was inserted into the phospholipids membrane. The repulsion exists the stability of the hybrid membrane which antibacterial peptide inserted into phospholipid membrane to formed associate with the biological activity of the antibacterial peptide.