Antibacterial Properties Of Small-size Peptide Derived From Penetratin Against Oral Streptococci

Objective: The aim of this study was to evaluate the antibacterial activity,mechanism and biocompatibility of a derived peptide against the bacteria at the initial stage of oral cavity,and to obtain an effective antibacterial strategy for controlling biofilm formation.Oral biofilm is a complex micro ecosystem formed by a variety of microorganisms,which plays an important role in a variety of oral diseases.The attachment of Streptococcus in the first step in pathogenic biofilm formation affects the structure and properties of biofilm,and the disturbance of the attachment bacteria in the first step can effectively control the formation of biofilm.Oral Streptococcus,such as Streptococcus oralis(S.oralis),Streptococcus sanguinis(S.sanguinis)and Streptococcus gordonii(S.gordonii)is the early attachment bacteria of dental plaque formation.Penetratin is a kind of multifunctional peptide with cell penetration and antibacterial properties.In this study,we synthesized the short peptide RR9(Arg-Gln-Ile-Arg-Arg-Trp-Trp-Gln-Arg-NH2)derived from penetratin to explore its antibacterial effect and its mechanism on Streptococcus oral.In order to evaluate the biocompatibility of RR9,the ability of RR9 to proliferate human gingival fibroblasts(HGFs)was tested.Method: The structural properties of RR9 were verified by protein analysis software and Circular Dichrosim(CD).The bioactivity of short peptide RR9 against S.oralis,S.sanguinis,S.gordonii was evaluated by detecting Minimal Bactericidal Concentration(MIC),Minimal Bactericidal Concentration(MBC)and time lethal curve in vitro.The effect of RR9 on the formation of oral Streptococcus biofilm was determined by crystal violet quantitative method(biofilm susceptibility test)and laser scanning confocal microscopy(CLSM).At the same time,we observed the morphological changes of the bacteria at the initial stage of oral cavity by scanning electron microscopy(SEM)and transmission electron microscopy(TEM),and explored its antibacterial mechanism by real-time quantitative PCR(q RT-PCR).The cytotoxicity of RR9 on human gingival fibroblasts was studied by cell culture in vitro,CCK-8 and AO / EB fluorescence staining.Result:Software prediction and CD results showed that short peptide RR9 derived from penetratin was non-structural short peptide.MIC,MBC and biofilm susceptibility test results showed that RR9 had significant antibacterial activity against S.oralis,S.sanguinis and S.gordonii,and there was no significant difference in antibacterial activity against three kinds of Streptococcus(P>0.05).The Time-kill curve showed that in RR9 treatment group,the number of bacteria surviving decreased significantly after 4 hours,and reached almost 100% killing within 48 hours.The results of CLSM staining showed that the number of red dead bacteria in RR9 treatment group increased significantly,while SEM and TEM showed that compared with the control group,the morphology of bacteria in RR9 treatment group changed significantly.RR9 down regulated the expression of ssp A in S.gordonii.The results of CCK-8 and fluorescent staining showed that there was no significant difference in cell proliferation between the RR9 treated group and the control group,that is,RR9 had no significant cytotoxicity to HGFs.Conclusion: The short peptide RR9 has strong antibacterial and anti-biofilm effect on S.oralis,S.sanguinis and S.gordonii,and it has low cytotoxicity on human gingival fibroblasts,which indicating that RR9 has potential clinical application value in oral diseases.The peptide derived from penetratin is an effective template for the development of new antibacterial drugs.