| Objective: As the most common chronic infectious disease in the oral cavity,caries has the characteristics of high incidence and wide distribution.Streptococcus mutans（S.mutans）is the main pathogenic bacterium of caries,its pathogenicity not only includes acid production and acid resistance,but also can promote the formation of plaque biofilm through the synthesis of extracellular polysaccharide（EPS）.As the initiating factor of dental caries,plaque biofilm can promote microbial interaction and maintain the acidic characteristics of microenvironment.Therefore,the use of antibiotics to inhibit the activity of S.mutans and reduce the formation of plaque biofilm is one of the main strategies for the prevention and treatment of dental caries,but overuse of antibiotics will lead to the enhancement of bacterial resistance,which leads to the unsatisfactory effect of this method in the long-term treatment.As a natural antimicrobial agent,antimicrobial peptides（AMPs）play an antibacterial role mainly by destroying the cell membrane.This special antibacterial mechanism makes it difficult for bacteria to produce drug resistance.Because it makes up for the deficiency of antibiotics in terms of drug resistance and has good bacteriostatic effect,the application of AMPs in the prevention of dental caries has become a new idea of clinical treatment.Specifically targeted antimicrobial peptides（STAMPs）not only have low bacterial resistance,but also have good targeting ability,which can achieve the inhibition effect on pathogenic bacteria without affecting the normal oral flora.The purpose of this study is to design and synthesize a novel type of STAMPs for S.mutans,providing a new scheme for clinical prevention and treatment of dental caries.Methods:1.By consulting the literature to design specific targeted antimicrobial peptides,using CSPC16 as targeting region and antimicrobial peptide IG-13-1 as antibacterial region,a specific targeted antimicrobial peptide C16-IG-13-1 was designed,and the secondary structure of C16-IG-13-1 was predicted by PSIPRED protein structure prediction server.C16-IG-13-1 was synthesized by standard solid-state synthesis technique（Fmoc protection method）.2.With C16-IG-13-1 as the experimental group,IG-13-1 as the positive control group and CSPC16 as the negative control group,the minimal inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of S.mutans,Streptococcus sanguinis（S.sanguinis）and Actinomyces viscosus（A.viscosus）were detected by microbroth dilution method.3.The antibacterial rates of C16-IG-13-1 to S.mutans,S.sanguinis and A.viscosus at 1 min were calculated by plate colony counting.4.After adding C16-IG-13-1 to 1 min,5 min,10 min,20 min,30 min and 60 min,the colony counts of S.mutans,S.sanguinis and A.viscosus were recorded by plate colony counting method,and the time-killing curve was drawn.5.The cell morphology of S.mutans treated with C16-IG-13-1 was observed by scanning electron microscope（SEM）.6.The inhibitory effect of C16-IG-13-1 on the formation of S.mutans biofilm was determined by crystal violet staining,and the inhibitory effect of C16-IG-13-1 on S.mutans in mature S.mutans biofilm was observed by MTT staining.Results: 1.The secondary structure of C16-IG-13-1 was predicted to be α-helix by PSIPRED protein structure prediction server,and C16-IG-13-1,IG-13-1 and CSPC16 were successfully synthesized by standard solid phase synthesis technique（Fmoc protection method）.2.The MIC and MBC of C16-IG-13-1,IG-13-1 and CSPC16 to S.mutans,S.sanguinis and A.viscosus were determined successfully.The MIC of C16-IG-13-1 to S.mutans,S.sanguinis and A.viscosus is 5 μM,10 μM and 20 μM,respectively,and the MBC is 10 μM,20 μM and 40 μM,respectively.3.At 1 min of treatment,the antibacterial rate of C16-IG-13-1 against S.mutans,S.sanguinis and A.viscosus was 98.28%,38.16% and 8.62%,respectively（P<0.001）.4.According to the time-killing curve,it was observed that all S.mutans died when 10 min was treated with C16-IG-13-1,and a large number of bacteria survived when S.sanguinis and A.viscosus were treated with 60 min.5.SEM could observe that the S.mutans cells in the control group were intact and smooth,without cell fragmentation or cell lysis,while in the C16-IG-13-1 treatment group,the surface of S.mutans cells was rough,cell rupture and a large number of cell fragments could be seen.6.The results of crystal violet staining showed that C16-IG-13-1 could significantly reduce the formation of S.mutans biofilm,and the results of MTT assay showed that C16-IG-13-1 could destroy the mature S.mutans biofilm.Conclusion: 1.In this study,a novel antimicrobial peptide C16-IG-13-1 was successfully synthesized by standard solid-phase synthesis technique（Fmoc protection method）.2.Through the determination of antibacterial activity and targeting specificity of C16-IG-13-1,it is proved that C16-IG-13-1 has significant antibacterial activity and targeting specificity against S.mutans.3.C16-IG-13-1 can exert antibacterial activity by destroying the cell membrane of S.mutans.4.C16-IG-13-1 can significantly inhibit the formation of S.mutans biofilm and destroy the mature S.mutans biofilm. |
