Inhibition Of Gram-positive Bacterial Biofilm By Antibacterial Peptides Temporin And Brevinin From Hylarana Guentheri Skin Of Hainan

Pathogenic bacteria have caused enormous losses to humans in the medical care of the world.According to statistics from the USA Centers for Disease Control,there are more than two millions infected patients caused by bacteria each year in the United States,including 23,000 patients caused by drug-resistant pathogens.Antibacterial peptides(AMPs)are the basic effector molecules from the innate immune system of body fluids.They are widely found in animals and are the first line of defense against pathogens.These bioactive peptides of 7 to 50 amino acids in length are also involved in the regulation of cellular processes,such as apoptosis and regulation of endocrine and neuroendocrine systems.AMP can be based on its biosynthetic mechanism(ribosomes and non-ribosomes),biological sources(bacteria,plants,animals,etc.),biological activity(antibacterial,antiviral,antifungal,etc.),physicochemical properties of antimicrobial peptides(hydrophobicity,amphipathy,cation,etc.)are classified.Three antibacterial peptides of GHc,GHd and GR23 were isolated and purified from the skin secretion of Hylarana guentheri.The GHc,GHd and GR23 used in the experiments of this research paper,which were obtained by chemical synthesis,and the related biological activities of them were studied and analyzed.Through the helical wheel and 3D structure prediction analysis,GHc and GHd possessd a-helical structures,hydrophilic amino acids and hydrophobic amino acids,which arranged on both sides with perfect amphiphilicity.The results of circular dichroism showed that GHc and GHd mainly dominated the random coil structure under physiological environment(0.9%NaCl),while a-helix in hydrophobic environment(30 mM SDS and 50%TFE).Plate punching showed that GHc and GHd had inhibitory activities against S.mutans,and the minimum inhibitory concentrations(MIC)were 12.6μM and 13.1 μM respectively.GHc and GHd could still maintain the inhibitory activity against S.mutans,which was treated by different physical and chemical factors(UV irradiation,NaCl concentration,heating temperature and pH).Thermal stability and structural stability are extremely important for the storage conditions and applications of antimicrobial peptides.The results of laser confocal microscopy and nucleic acid leak assay showed that GHc and GHd could change the permeability of S.mutans cell membrane,leading to the death of macromolecular substances in bacterial cells.Gel retardation experiments showed that GHc and GHd could bind to bacterial DNA in vitro by forming macromolecular complexes and reducing their mobility.S.mutans DNA synthesis decreased when GHc and GHd incubated with bacteria,which may be caused by AMPs acting on the DNA in the bacteria to interfere with its synthesis,or may be caused by changing the cell membrane to cause the leakage of macromolecular substances.The results of crystal violet staining and MTT assay for S.mutans attachment,which showed that the inhibition rates of S.mutans at 2MIC GHc(25.2 M)and GHd(26.2 M)were over 90%;The inhibition rate of S.mutans biofilm at 6 h was 61.6-67.2%;when the biofilm of S.mutans was grown for 24 h.the destruction rate of biofilm was reduced to about 20%.The results showed that GHc and GHd could inhibit the adhesion of S.mutans biofilm at the initial stage,and also had some damage to the biofilm formed at the early stage.However,with the prolongation of biofilm formation,the biofilm was formed and the rate of damage has dropped significantly.Both GHc and GHd inhibited the production of S.mulans extracellular polysaccharide(EPS),and the inhibition rate of GHc on S.mutans synthesis of water-soluble exopolysaccharide and water-insoluble exopolysaccharide at 12.6 M,the inhibition rates of S.mutans synthetic water-soluble exopolysaccharide and water-insoluble exopolysaccharide were 42.6%and 23.9%.respectively.The inhibition rates of S.mutans synthetic water-soluble exopolysaccharide and water-in soluble exopolysaccharide were 49.6%and 17.2%when GHd at 13.1 μM.Real-time PCR results showed that GHc and GHd could down-regulate the expression of S.mutans biosynthesis extracellular polysaccharide gene(glucosyltransferase,Gtfs).Both GHc and GHd were non-toxic to human red blood cells and oral epithelial cells,providing a good basis for further development and design.In this study,GR23 was synthesized by solid phase peptide synthesis.The prediction of the helical wheel and 3D structure of GR23 showed that it had perfect amphiphilicity,mainly in the a-helical structure.GR23 mainly dominated the random coil structure in a hydrophilic environment(PBS),while mainly dominated an a-helical structure in a hydrophobic environment.The plate punching and absorbance method showed that it had inhibitory activity against Staphylococcus aureus,and the minimum inhibitory concentration was 16.6 μM.GR23 maintained the inhibition of S.aureus after treated by different physical and chemical factors,showing thermal stability and structural stability.GR23 could change the permeability of S.aureus membranes,and caused the leakage of bacterial contents.Gel retardation experiments showed that GR23 could bind to S.aureus DNA in vitro.When GR23 was incubated with S.aureus for 1 h and 4 h,the total amount of DNA decreased,the results indicated that GR23 could interfere with bacterial DNA synthesis,and the decrease in DNA synthesis may also be related to the changes in cell membrane integrity.The anti-biofilm activity of GR23 was detected by crystal violet and MTT assay.The results showed that GR23 could inhibit the adhesion of S.aureus,biofilm growth and destroy the biofilm after 24 h of growth of S.aureus.Crystal violet and MTT assay showed that the inhibition rate of GR23 on S.aureus initial cell adhesion was above 90%when it was above 16.6 and the inhibition rates of S.aureus biofilm growth(6 h)were 68%and 41%.The inhibition rate of the biofilm after 24 h growth was reduced to 20%.GR23 also inhibited the growth of extracellular polysaccharides from the growth of S aureus,and the inhibition rates of water-soluble extracellular polysaccharides and water-insoluble extracellular polysaccharides were 71.8%and 52.2%,respectively.GR23(final concentration 200 μM)had no significant hemolytic activity on healthy human red blood cells.