Study On Bactericidal Activity Of Tne Polypeptide P-KKK Derived From OmpA Signal Peptide Of Escherichia Coli

Objective:To study and compare the bactericidal activity of the polypeptide P-KKK derived from the Escherichia coli outer membrane protein A(OmpA)signal peptide against standard bacterial strains and clinically isolated drug-resistant strains in order to provide important information for the development of new antibacterial agents.Methods:(1)Design of derived peptides:Based on the primary structure of theEscherichiacoliOmpAsignalpeptide(P-AQA)MKKTAIAIAVALAGFATVAQA(Methionine-Lysine-Lysine-Threonine-Alanine-Isoleucine-Alanine-Isoleucine-Alanine-Valine-Alanine-Leucine-Alanine-Glycin e-Phenylalanine-Alanine-Threonine-Valine-Alanine-Glutamine-Alanine)and functional characteristics,Using its primary structure as a template,only the 5amino acids at its C-terminus were replaced without changing the basic structure ofitsanteriorportion,andaderivedpeptide(P-KKK)was designed:MKKTAIAIAVALAGFARRKKK(Methionine-Lysine-Lysine-Threoni ne-Alanine-Isoleucine-Alanine-Isoleucine-Alanine-Valine-Alanine-Leucine-Ala nine-Glycine-Phenylalanine-Alanine-Arginine-Arginine-Lysine-Lysine-Lysine).(2)The first part of the experiment:Determination of bactericidal activity of the derived peptide P-KKK against standard strains:Using the agar plate counting method,different concentrations of the derived peptide P-KKK were mixed with several bacterial standard strains(Three kinds of G~+bacteria:Staphylococcus aureus,Enterococcus faecium,Bacillus subtilis;3 kinds of G~—bacteria:Escherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa)in the incubation system,and incubated in a constant-temperature water bath at37°C for 2 hours.After plating,the plate was incubated at 37°C for 18-24hours.The colony forming units(CFU)were then recorded on each agar plate and the bactericidal rate after the action of the derived polypeptide P-KKK was calculated.(3)The second part of the experiment:Determination of bactericidal activity of peptide P-KKK against resistant strains:Using the agar plate counting method,Different concentrations of the derived peptide P-KKK and several clinically-resolved drug-resistant strains(Two resistant strains of G~+bacteria:Staphylococcus aureus MRSA,high-level gentamicin-resistant HLGR Enterococcus faecium;two kinds of G~—resistant strains:extended-spectrum?-lactamase ESBL Escherichia coli,extended-spectrum?-lactamase ESBL Klebsiella pneumoniae)were mixed in the incubation system and incubated in a constant temperature water bath at 37°C for 2 hours.The agar plates were incubated at 37°C for 18 to 24 hours.The colony forming units(CFU)were then recorded on each agar plate and the bactericidal rate after the action of the derived polypeptide P-KKK was calculated.Results:(1)The derived peptide P-KKK has strong bactericidal activity against the three G~+strains,among which the bactericidal activity is the strongest against Bacillus subtilis,and there is no significant difference in the bactericidal activity against Staphylococcus aureus and Enterococcus faecium.At the concentration of 200 ug/mL,it can kill all of the G~+bacteria,and the sterilization rate at 40ug/mL is also 92.4%.The derived peptide P-KKK also had strong bactericidal activity against the three G~—bacterial standard strains.among which the bactericidal activity is the strongest against Klebsiella pneumoniae,and there is no significant difference in the bactericidal activity against Escherichia coli and Pseudomonas aeruginosa.When the concentration was 200ug/mL,it could kill 96.5%of G~—bacteria,and the sterilization rate was also in the range of 20%-96.4%at 40ug/mL.(2)The derived peptide P-KKK has strong bactericidal activity against two clinical isolates of G~+resistant strains.At a concentration of 40?g/mL,About 99.7%of MRSA Staphylococcus aureus and 99.9%of HLGR Enterococcus were killed.It also has strong bactericidal activity against two clinical isolates of G~—resistant strains.When the concentration of P-KKK peptide reached 200ug/mL,99.9%of ESBL Escherichia coli and ESBL Klebsiella pneumoniae could be killed.At a concentration of 40 ug/mL,the bactericidal activity against ESBL E.coli was17.3%,and the bactericidal activity against ESBL Klebsiella pneumoniae was61.3%.(3)There was no significant difference in the bactericidal activity of the derived peptide P-KKK against common bacterial standard strains and clinically isolated drug-resistant strains.However,the bactericidal activity of antibiotics against standard strains was about 100 times stronger than that of drug-resistant strains.Conclusions:(1)The E.coli OmpA signal peptide-derived peptide P-KKK has obvious bactericidal activity against the common G~+and G~—bacterial standard strains.The bactericidal activity of G~+bacteria is slightly stronger than that of G~—bacteria.The bactericidal activity of B.subtilis in the G+bacteria strains was the strongest,and the bactericidal activity of K.pneumoniae in the G~—bacteria strains was the strongest.(2)Bacillus subtilis was more sensitive to peptide-derived P-KKK molecules and M17 and C26 antibacterial peptide molecules studied earlier in the research group than other types of G+bacteria.This is related to the fact that Some structures ofBacillus subtilis are more Easier to be attacked by peptide molecules.(3)The E.coli OmpA signal peptide-derived peptide P-KKK also has strong bactericidal activity against clinically isolated drug-resistant strains,and showed no significant difference in bactericidal activity against standard strains.While the antibacterial activity of antibiotics against standard strains and clinically isolated resistant strains differed significantly.The antibacterial activity of antibiotics against standard strains was about 100 times stronger than that of resistant strains.It is suggested that the derived peptide P-KKK has a broad spectrum of bactericidal activity and a unique bactericidal mechanism.Further research on the structure and function of the derived peptide P-KKK will probably help develop a novel antibacterial drug that is sensitive to drug-resistant strains and is not susceptible to drug resistance.