The Antibacterial And Antibiofilm Activities Of Temporin-GHa And Its Derived Peptides Against Streptococcus Mutans

At present,the unreasonable use of antibiotics is the main reason for the increase of drug-resistant bacteria.If we don’t control the misusage of antibiaotics,and accelerate the development of new and effective antibacterial drugs,the health of human will be seriously threatened.The mechanism of antimicrobial peptides（AMPs）on bacteria is different from traditional antibiotics,which makes it become a promising candidate of antimicrobial reagent.It has been reported that some AMPs can not only inhibit and kill planktonic bacteria,but also exhibit antibiofilm activity.They inhibit the formation of bacterial biofilms and eradicate mature biofilms.Dental caries is a common oral disease caused by a variety of cariogenic microorganisms,including lactic acid bacteria,Streptococcus and so on.Streptococcus mutans is one of the main pathogens of dental caries.When sugar is consumed,S.mutans convert the sugar into acid,which will corrode teeth and form cavities.In addition,S.mutans also has a strong ability to form biofilms,which can protect pathogenic bacteria from damage by drugs.Despite the dental experts doing their best to treat oral diseases,oral infections are still very common.Therefore,the discovery of new anti-caries drugs is urgent.In this study,temporin-GHa from Hylarana guentheri was used as a template to design temporin-GHaR（GHaR）and temporin-GHa11R（GHa11R）.The structures of GHa,GHaR and GHa11R were determined by circular dichroism（CD）.They all showed a random coil structure in physiological environment,but anα-helix state in a simulated membrane environment.The minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of GHaR and GHa11R against S.mutans were better than that of the parent peptide GHa.The derived peptides had good bactericidal efficiency at 2×MIC（the bacteria are completely killed within 15 min）.They maintained stability under different conditions(heating temperature,p H,Na⁺,K⁺and Mg²⁺,storage temperature and time,and saliva).Membrane permeation experiment,scanning electron microscopy（SEM）and transmission electronic microscopy（TEM）assay showed that GHaR could quickly destroy bacterial cell membranes,while GHa11R had a slight effect on membrane.GHa11R was speculated that it may enter bacterial membrance to exert antibacterial effects.The results of membrane potential experiments showed that both GHa and its derived peptides cause cell membrane depolarization.But GHaR acted in a concentration-dependent manner,while GHa11R acted at 12.5μM.S.mutans did not develop resistance to GHaR and GHa11R within 30 days.The combination of GHaR and GHa11R had an additive antimicrobial effect on S.mutans.The antibiofilm assay showed that the inhibition rate of GHaR and GHa11R at 3.1μM on biofilm formation was88%-95%,which was about 40%more than the inhibitory effect of GHa.And the eradicating rate of mature biofilms was 87%-95%at 25μM.Fluorescence microscope and confocal laser scanning microscope（CLSM）observed that GHaR and GHa11R effectively eradicated the mature biofilm of S.mutans,making the thick and dense biofilm become thinner and looser.Extracellular polysaccharides（EPS）are the main component of the biofilm matrix.GHaR and GHa11R had inhibitory effects on it.The results of hemolysis experiments showed that GHaR and GHa11R had good selectivity in the presence of S.mutans,and the cell selectivity index（CSI）were 7.0 and 14.9,respectively.GHaR was toxic to human oral epithelial cells（HOECs）with half-maximal inhibitory concentration(IC₅₀)of 23.6μM.GHa11R had no cytotoxicity to HOECs,and the IC₅₀>200μM.In conclusion,the antibacterial activity,antibacterial efficiency and anti-biofilm activity of GHaR and GHa11R were improved compared with GHa.In order to obtain more efficient and low-toxic AMPs,we selected GHaR as a template to design a series of derived peptides（GHaR6R,GHaR7R,GHaR8R,GHaR9R,GHaR9W）.Except for GHaR7R,the hemolytic toxicity of the other derived peptides was effectively reduced.GHaR9R had poor antibacterial activity.So GHaR6R,GHaR7R,GHaR8R,and GHaR9W were selected for research.The derived peptides（4×MIC）killed the bacteria within 15 min.In addition,the derived peptides also had the characteristics of high temperature resistance,acid and alkali resistance.They maintained stability in the presence of K⁺,Mg²⁺,under different storage conditions or in the presence of saliva.GHaR7R,GHaR8R,and GHaR9W took antimicrobial action after contacting with bacteria,leading to destroy the bacterial cell membrane,changing the morphology of the bacteria,and making the content flow out.While the effect of GHaR6R was relatively weak.S.mutans did not develop resistance to these derived peptides within 30 days.Both quantitative and qualitative biofilms experiments showed that GHaR6R,GHaR7R,GHaR8R,and GHaR9W had good anti-biofilm activity.These derived peptides were less toxic to HOECs at 1×MIC.In conclusion,GHaR6R,GHaR8R,and GHaR9W maintained good antimicrobial activity while reducing hemolysis.GHaR and GHa11R are novel antimicrobial candidates for S.mutans infection and prevention of dental caries.The design and synthesis of GHaR6R,GHaR8R and GHaR9W provide ideas for the further design of high efficiency and low toxicity of AMPs.