Mechanism Of Anti-biofilm Activity Of α-Helical Peptides Against Streptococcus Mutans

In recent years,bacterial biofilms have attracted much attentions because they are difficult to be removed.Dental caries,as a typical oral biofilm disease,seriously affects the quality of people’s life and has become the focus of research.At present,anti-caries agents have some side effects,so it is urgent to develop new anti-biofilm drugs with high biological activity.Antimicrobial peptides are widely distributed in nature,have high antibacterial and anti-tumor activity,and are not easy to induce bacteria to develop drug resistance,so they become potential anti-biofilm agents.In this paper,G（IIKK）₃I-NH₂,which is developed by our research group,is taken as the main research object to investigate its effect on the biofilm of Streptococcus mutans,the main pathogenic bacteria of oral caries.And the mechanism of action is explored in detail from the different stages of bacterial biofilm formation.In addition,we screen the anti-biofilm activity ofα-helical peptides,and analyze the reasons for the difference in biological activity among these peptides.Finally,we screen the toxicity of theseα-helical peptides against human red blood cells,human oral epithelial cells and other normal mammalian cells.These researchs will lay the experimental and theoretical foundation for the design and development of effective anti-caries agents in the future.In the initial bacterial adhesion stage,G（IIKK）₃I-NH₂ inhibits bacterial adhesion in the following ways.（1）Cationic helical peptide quickly binds to bacterial surface through electrostatic interaction,and the charge shielding effect induces bacterial agglomeration,which is not conducive to bacterial adhesion.（2）The binding peptide can greatly reduce the hydrophobicity of bacterial surface and hinder the further adhesion of bacteria.（3）Through inhibiting the expression of the quorum sensing gene（lux S）and the glycosyltransferase gene（gtf B）,peptide inhibits the formation of biofilm by reducing the content of signal molecules and water-insoluble exopolysaccharides.In the mature biofilm stage,G（IIKK）₃I-NH₂ disperses biofilm by destroying e DNA,and has the bactericidal effect on the exfoliated bacteria and the bacteria on residual biofilm.（Chapter 4 of the paper）By studying the structure-activity relationship ofα-helical peptides,we find that the removal of nonpolar amino acids（Gly or Ile）at both ends of G（IIKK）₃I-NH₂ reduces the anti-biofilm activity of peptides.（1）In the initial adhesion stage,peptides inhibit biofilm formation by reducing the hydrophobicity of bacteria.The higherαhelicity of peptides,the higher anti-biofilm activity of peptides.（2）In the mature biofilm stage,the simulated biofilm system containing extracellular polymer matrix components（e DNA,α-1,6-glucan,water-insoluble glucan）is constructed to investigate the secondary structure and surface activity of peptides.The results also show that deleting the end groups reduces the ability of the peptide to clear biofilm.The main mechanism of peptides to remove the biofilm is that peptides adsorb to e DNA by electrostatic action and destroy the e DNA structure,thereby removing the biofilm.The higherαhelicity of peptide,the more favorable it is to interact with e DNA.In conclusion,the helicity of peptide is an important parameter for the construction of anti-biofilm peptides.In addition,cytotoxicity experiments show that G（IIKK）₃I-NH₂,（IIKK）₃I-NH₂ and G（IIKK）₃-NH₂not only have high anti-biofilm activity,but also have low toxicity to human red blood cells and human oral epithelial cells.They have the potential to be developed as new anti-caries agents.（Chapter 5 of the paper）...