Studies On The Molecular Design And Antimicrobial Mechanism Of DNA Action Of Antimicrobial Peptide BuforinⅡ

The study of bactericidal mechanisms of antimicrobial peptides was always aiming at the membrane action mechanism by almost researchers during several decades. There were a few peptides which molecular action mechanism involved in crossing the cell membrane without inducing severe membrane collapse, then interacting with cytoplasmic target. A 21-aa peptide BuforinⅡ, which was isolated from the stomach tissue of an Asian toad, Bufo bufo gargarizans and showed much stronger antimicrobial activities against a broad spectrum of microorganisms compared with other cationic polypeptides, was proposed to kill bacteria by efficiently crossing cell membrane without inducing severe membrane permeabilization and strongly binding to DNA. Different from the membrane-action peptides, the DNA-action peptides owned themselves distinctive bactericidal mechanisms, which was considerably worth to research in depth.In this paper, a novel peptide, named BF2-X, was designed out based on the structure-activity analysis of an analogue of BuforinⅡ, named BF2-A. The BF2-X was a hybrid peptide containing the N-terminal residues 5 to 13 of BF2-A and three repeats of the C- terminal regularα-helical motif RLLR, and the residues 8 valine were replaced by leucine. The results of bioinformatics analysis had showed that two cationic peptides had no transmembrane region, and compared with BF2-A, the helicity, positive charge, hydrophobicity rate and C-terminal amphipathy of BF2-X had remarkably enhanced. Both peptides showed a random coil structure in an aqueous solution, while displaying a typicalα-helical structure in a membrane mimic condition. BF2-X exhibited higherα-helical contents than BF2-A in hydrophobic environment. BF2-X showed stronger antimicrobial activities against a broad spectrum of microorganisms than parent peptide BF2-A. Two analogues had no hemolytic activities and cytotoxicity, however showed endotoxin neutralization and thermal stability. BF2-A/X owned certain resistance for pepsin, trypsase and papain, but could not resist the enzymolysis of proteinase K.BF2-X could cause more calcein leakage from large unilamellar vesicles in a dose-dependent manner than BF2-A did. However, both peptides didn't induce membrane of liposomes collapse. BF2-A/X could increase the outer membrane permeabilization of G- bacteria. BF2-A didn't cause significant membrane permeabilization for influx of ONPG into cells, and hardly caused the leakage of intracellular macromolecules, probably BF2-A slightly disturbed cell membrane causing the K+ leakage during peptide crossing phospholipids bilayer, but the cytomembrane treated by BF2-A was still intact. On the contrary, BF2-X could cause rapid killing kinetics in a dose-dependent manner, and obviously increased the inner membrane permeability, even induced the slight leakage of macromolecules in the cytoplasm. Though the damage degree of BF2-X for cell membrane was deeper than that of BF2-A, the cytoplasmic membrane of BF2-X treated cells didn't collapse. BF2-X with higherα-helical content penetrated the cells more efficiently than BF2-A.The nucleic acid was the action target of two peptides after penetrating the E.coli cells. They didn't breakdown the genomic DNA, but strongly binding to DNA and RNA. The ability of BF2-X binding to DNA was tenfold strong than that of BF2-A. Interestingly, the ability of BF2-A binding to RNA was quadruple strong than that of BF2-X. The first step of both peptides binding to DNA was the absorption of basic amino acid on phosphate group depended on electrostatic attraction. Then the peptides inserted the groove of DNA duplex with hydrophobic interaction. The direct intercalation involving phenylalanine and nucleic acid bases participate in the peptides-DNA interaction. The higher binding affinity of BF2-X to DNA attributed to more powerful ability of intercalation into groove and base pair of DNA. Both peptides tended to specific binding to two domains of E.coli genomic DNA, and one of them probably was a fragment of consensus sequence located on the 23S rRNA gene.Both peptides binding to DNA/RNA primarily influenced the transcription function of gene, not the replication of DNA. Thus the preferential inhibition of RNA-synthesis appeared to be the primary target of BF2-A/X and the effect of the peptides on DNA-synthesis appeared to be secondary. And BF2-X caused a more marked inhibition of DNA/RNA-synthesis than BF2-A. Moreover, two analogues could preferentially inhibit the protein-synthesis by the translation level. Interestingly, BF2-A induced a more remarkable inhibition of protein synthesis than BF2-X. They also could influence the oxidation phosphorylation of cells, and then obviously inhibiting the respiration of E.coli, inducing a drop in ATP content. Overall, these data strongly suggest that the decrease in bacterial viability was causally related to the fall in respiration-linked proton motive force, with the attendant loss of macromolecular biosynthesis ability.On the conclusion, a novel peptide BF2-X was designed out based on BF2-A in present study, and their antimicrobial mechanisms were researched and compared. The powerful antimicrobial activities of both peptides were closely related to penetrate the cytoplasm and binding to DNA/RNA, but not to disrupt cell membrane. The increase of membrane permeabilization, crossing-cytomembrane efficiency, the ability of binding to DNA and inhibiting DNA/RNA-synthesis explained why BF2-X displayed more excellent antimicrobial activity and rapid killing kinetics than BF2-A. These results obtained in this study should help the design and research of therapeutically useful antimicrobial peptides based on DNA-action peptides.