Study On The Bactericidalactivity Of Human Phospholipase A₂ Amino-terminal Derived Peptide

Objective:To study the bactericidal activity of N-terminal derived peptides of human type I and type?phospholipase A₂?PLA₂?in vitro against Gram-positive?G⁺?bacteriaStaphylococcusaureus?S.aureus?and Gram-negative?G^-?bacteria Escherichia coli?E.coli?,and the relationship between the molecular structure of the PLA₂ N-terminal derived peptide and the bactericidal activity.Method:?1?Synthesis of derived peptides:?1?Based on the structure of human type I PLA₂ N-terminal peptide??-A12?,?-A1N?hydrophobic weakened?,I-Q4R?alkaline enhanced?,I-Q4D?alkaline weakened?,I-C11R?alkaline enhanced?and I-Q4R-C11R?alkaline enhanced?were synthesized by amino acid substitution.?2?Based on the structure of human type II PLA₂N-terminal peptide??-N15?,?-H6R?alkaline enhanced?,?-N1A-H6R-T12L?hydrophobic enhanced?,?-N4R-H6R?alkaline enhanced?,?-H6R-T12R?alkaline enhanced?,?-H6R-T12V?hydrophobic enhanced?and II-N4R-H6R-T13R?alkaline enhanced?were synthesized.?2?Experiment Part I:Determination of bactericidal activity of derived peptides:different concentrations of derived peptides were incubated with G⁺bacteria S.aureus and G^-bacteria E.coli in a constant temperature water bath at 37?for 2h,then plated with agar and placed at 37?incubating the incubator for 18-24 h,recording the number of colonies on each plate,and calculating the bactericidal rate.?3?Experiment Part II:Derived peptide II-H6R-T12V combined with antibiotics:the derived peptide II-H6R-T12V was co-administered with cefazolin to S.aureus,and the derived peptide II-H6R-T12V was co-administered with cefodizime to E.coli,and then the bactericidal rate was calculated by agar plate counting method.Results:?1?Human type I PLA₂ N-terminal peptide?I-A12?and derived peptide I-A1N,I-Q4R,I-Q4D,I-C11R and I-Q4R-C11R possessed bactericidal activity against S.aureus,the strongest of which was the peptide I-A12,the weakest was derived peptide I-Q4D;For E.coli,the most potent bactericidal activity was polypeptide I-A12,the weakest was derived peptide I-Q4D and I-C11R,which did not possess bactericidal activity against E.coli;?2?Human type?PLA₂ N-terminal peptide??-N15?and the derived peptide II-H6R,II-N1A-H6R-T12L,II-N4R-H6R,II-H6R-T12R,II-H6R-T12Vand II-N4R-H6R-T13R possessed bactericidal activity against S.aureus,the strongest of which was derived peptide II-H6R-T12V,the weakest was the peptide II-N15;For bactericidal activity to E.coli,the strongest was?-N1A-H6R-T12L,the weakest was the peptide?-N15;?3?To S.aureus,derived peptide?-H6R-T12V combined with cefazolin,the bactericidal rate stronger than the single drug;Derived peptide?-H6R-T12V and cefodizime co-acting in E.coli,bactericidal rate stonger than single drug action.Conclusions:?1?The amino acids at positions 4 and 11 of the PLA₂N-terminal peptide?I-A12?are extremely important for their bactericidal activity and are replaced by acidic amino acids or basic amino acids shall be significantly reduce the bactericidal activity;The hydrophobic amino acid at position 1 is replaced by a neutral amino acid,and the bactericidal activity is also reduced.?2?To increase the positive charge in the PLA₂ N-terminal peptide??-N15?enhance the bactericidal activity.When the 12th amino acid is replaced with the hydrophobic amino acid,the bactericidal activity is obviously enhanced;?3?Derived peptide?-H6R-T12V and?-lactam antibiotics have synergistic bactericidal effect;?4?The bactericidal activity of PLA₂ N-terminal peptides and its derived peptide is related to the positive charge and hydrophobicity carried by it,and may be related to the spatial structure of peptide molecule and the difference of bacterial cell membrane.