Optimal Design And Screening Of Potent Antimicrobial Peptides Based On Temporin-GHa And A Structure-activity Study

Infectious diseases have been one of the life-threatening health problems worldwide throughout human history.The discovery and development of antibiotics have had a transformative impact on humans against infectious diseases.However,bacteria had evolved various strategies to evade the killing of antibiotics as a result of the abuse of antibiotics,making the treatment of multi-drug resistant infections a serious challenge.Staphylococcus aureus is an important clinical pathogen that can cause a variety of superficial skin infections and systemic diseases（endocarditis and septic arthritis）.Epidemiological data in recent years showed the rapid increase in the number of methicillin-resistant S.aureus（MRSA）acquired in hospitals and communities.Therefore,exploring alternatives to antibiotics has become an urgent problem in the world.Due to the broad-spectrum antibacterial activity,low drug resistance and immunogenicity of antimicrobial peptides（AMPs）,they have become a very promising alternative to antibiotics for the treatment of bacterial infections.In the previous research,the natural AMP temporin-GHa（GHa）was cloned from the Hylarana guentheri.In order to improve the therapeutic potential of GHa,we combined template-based and database-aided design methods to design new AMPs.Bioinformatics statistics showed that the frequency of K in temporin AMPs is significantly higher than R and H.We used lysine to replace histidine at both ends of GHa.Three single-point or multi-point mutation peptides were derived—GHa K,GHa4K,and GHa11K.The frequency of R in the amino acid profile of anti-MRSA peptides and anti-biofilm peptides is very high.In order to design efficient anti-MRSA and anti-biofilm peptides,we continued to replace K with R to obtain GHaR,GHa4R and GHa11R.The derived peptide showed significantly enhanced and broad-spectrum antibacterial activity.The derived peptides had strongest activity against S.aureus,and the activity against MRSA of arginine mutants were stronger than lysine mutants.K mutants exhibited much stronger antibacterial activity against S.aureus than GHa.They could still inhibit the growth of S.aureus at sub-MIC and had a rapid sterilization rate.Fluorescence spectroscopy results indicated that both GHa and the derived peptides could interact with the bacterial membrane to exert their antibacterial activity and the membrane penetration rate caused by the derived peptides were faster.The MTT experiment confirmed that the anti-biofilm activity of derived peptides had increased by 4to 8 times compared with the parent peptide.The peptide’s hemolytic activity and toxicity to human cells are used as indicators to evaluate its safety.Experiments showed that,compared with the parent peptide,the therapeutic index（TI）and the cell selectivity index（CSI）of the derived peptides had increased 4-8 and 2-4 times,respectively.All peptides showed very low cytotoxicity to human normal cell line HL-7702.The results of circular dichroism showed that the derived peptides retained theα-helix structure.The anti-MRSA peptides GHaR and GHa11R obtained through targeted design were stable under the conditions of high temperature,acidity and alkalinity and cations(Na⁺and Ca²⁺),and had good protease resistance and serum stability.This provides an advantage for derived peptides in the treatment of MRSA infections.The results of growth inhibition curve and bactericidal kinetic curve showed that the derived peptides had more rapid and efficient antibacterial activity.Fluorescence spectroscopy and fluorescence microscopy observations consistently showed that GHaR can quickly destroy the structural integrity of bacterial membranes,while GHa and GHa11R just induced a small degree of cell membrane damage to MRSA.Scanning electron microscope（Scanning Electron Microscope,SEM）showed that GHaR can completely destroy the membrane structure of MRSA,while GHa and GHa11R had slight effect.Membrane potential measurement experiments showed that GHa and derived peptides can depolarize the MRSA membrane potential.In addition,gel retardation experiments showed that the derived peptides can bind to bacterial DNA.The resistance induction experiment found that MRSA was less resistant to derived peptides compared with vancomycin,which may be related to the multi-target mechanism of action of derived peptides.The measurement of anti-biofilm activity showed that the ability of the derived peptide to inhibit MRSA initial attachment,biofilm formation and eradication of mature biofilm was significantly higher than that the parent peptide.Confocal laser scanning microscope（CLSM）characterization experiments showed that GHaR effectively inhibited the formation of biofilm,while GHa and GHa11R were less effective.GHaR and GHa11R can completely destroy the mature biofilm under 8×MIC,but the mechanism is different.The determination of the content of water-insoluble extracellular polysaccharides found that GHa had a small inhibitory effect on water-insoluble EPS of MRSA.GHaR could significantly inhibit the synthesis of MRSA water-insoluble EPS and reduce the adhesion of bacteria,thereby inhibiting the formation of biofilm.The CCK-8 method evaluated peptide cytotoxicity.All peptides showed low cytotoxicity to human normal cell line HL-7702.The hemolytic activity results showed that GHaR and GHa11R significantly increased the CSI value,reflecting the broad treatment window and reducing the treatment risk.GHaR with the best activity was used as a template to design and obtained GHaR6R,GHaR7R,GHaR8R,GHaR9R and GHaR9W.The antimicrobial activity and hemolytic activity experiments showed that GHaR6R,GHaR8R,GHaR7R and GHaR9W retained strong antibacterial activity.Gha R6R,GHaR8R and GHaR9W had lower hemolytic activity and higher cell selectivity.Although the hemolytic activity of GHaR9R reduced,its activity also decreases.The optimally designed K mutants and targeted modified GHaR and GHa11R in this study are promising new antibacterial agents for the treatment of S.aureus and MRSA infections.With high-efficiency and low-toxicity,GHaR6R,GHaR8R and GHaR9W will also be the leading compounds with research value.This study provides a theoretical basis and strategy for designing highly effective and low-toxic AMPs...