Antimicrobial Effects Of Peptides From Human Beta-defensin-3 On Planktonic State And Biofilm State Of Streptococci

Background:Infectious diseases are the underlying cause of death worldwide at present.Primary etiological agent of infection is microorganisms.Three quarters of all microbial infections found in humans are involved in microbial biofilm,which are strongly associated with the etiology of dental caries,periodontal diseases,pulpal diseases,apical diseases and peri-implantitis.Caries experience in adults remains high,and periodontal disease remains high in both developing and developed countries.Primary etiological agent of oral biofilm-diseases is microbe.Formation of dental plaque is a multi-step process starting with the initial colonizers adhesion,in which includes Streptococcus gordonii(S.gordonii),Streptococcus oralis(S.oralis)and Streptococcus sanguinis(S.sanguinis),which account for 60 to 90% of the bacteria that colonize on the teeth during the first 4 h.As the attachment of primary colonizers on various surfaces in the oral cavity,middle/late colonizers are attached in succession for the formation of biofilm.And the process plays a causative role in dental caries,periodontitis and peri-implantitis.Therefore,we try to use Human beta-defensing-3(hBD3)according to the existing research.To maximize the retention of antimicrobial activity and the biological stability of hBD3,we synthesized the peptide fragments from hBD3 to inhibit the bioactivity and growth of streptococci and prevent the formation of oral biofilm.We use host defensin peptide to avoid specific immune response and overcome drug resistance,which prevents peptide losing activity in high-salt condition.This study may provide new thought for generated chimeric peptide,which provides new avenues to treating oral biofilm associated infection diseases.Objective:hBD3,one of “natural antibiotics”,acts as a first line of defense against both Gram-positive and Gram-negative bacteria infection.Streptococci,which are the initial colonizers of oral plaque,are the significant cause for oral biofilm associated diseases.We designed and synthesized three fragments(hBD3-1,hBD3-2,hBD3-3)from the hBD3 protein starting at the N-terminus and evaluated the antibacterialefficacy on oral streptococci.Methods:In this study,we synthesized hBD3-1,hBD3-2,hBD3-3 and examined the inhibition ability of the peptides on the bioactivity of S.oralis,S.sanguinis and S.gordonii assessed by minimal inhibitory concentration(MIC),minimum bactericidal concentration(MBC)and biofilm formation test.And the secondary structures of the three fragments were investigated by using Raman and Circular Dichroism spectroscopy.The morphology of bacteria was observed by confocal laser scanning microscopy(CLSM)and scanning electron microscopy(SEM).Results:Three fragments of hBD3 had antimicrobial activity on streptococci,and planktonic state of S.oralis and biofilm state of S.sanguinis had much more sensitive to hBD3-3(P<0.05).Raman spectroscopy analysis showed that the three fragments shared amide? and ?-sheet structure,but tyrosine were not found in hBD3-2 and hBD3-3.CD spectroscopy analysis indicated antimicrobial activity of the three fragments of hBD3 remained stable in the oral cavity.CLSM determined higher live/dead ratios in hBD3-3 group than in control groups.SEM showed the decrease of biomass and bacterial morphology destruction.Conclusion:hBD3-3,one fragment of 45 amino acid residues,possessed the potential capacity for depressing the growth of bacteria,especially first colonizers during the development of oral biofilm.Thereby it prevents and reduces the occurrence of biofilm-related infections and maintenance of oral health.