The Studies On The Design Of Antibacterial Peptides Targeted With DNA And Antimicrobial Mechanism

Studies shown that there were some antimicrobial peptides which target nucleic acid to exert theirantibacterial activity by interfacing the transmission of genetic information of pathogenic micro-organismsand synthesis of biological macromolecules. However, the relationship among the antibacterial activities,nucleic acid interaction mechanisms and peptide structures of antimicrobial peptides was still unclear.Designing the nucleic acid target antimicrobial peptide and explaining their antimicrobial activity wereimportant. In this study, we made the cell-penetrating peptide ppTG20which possessed DNA bindingactivity as template, baseing on the net charge and hydrophobicity which play important roles inantibacterial activity of antimicrobial peptides and penetrate membrane activity of cell penetrating peptides,and designed series of DNA target antimicrobial peptides. We analysed their antibacterial activities andmechanism of penetrating cell membrane and targeting DNA, and further summaried the law among netcharge, hydrophobicity and the antimicrobial activity of peptide.We used sequence modification method to design analogues of parent peptide ppTG20(net charge Q=+5, hydrophobic H=-0.13) with similar hydrophobic but different net charge, P1(Q=+3, H=-0.12) andP2(Q=+7, H=-0.15), analogues of parent peptide with the same net charge+5but different hydrophobic,P3(Q=+5, H=-0.02), P4(Q=+5, H=-0.24), P5(Q=+5, H=-0.33), analogues of parent peptide withthe same net charge+7but different hydrophobic, P6(Q=+7and H=-0.26) and P7(Q=+7H=-0.35).We predicted the secondary structure and biological activity of peptide by software which shown thatderivative peptides and parent peptides were amphiphilic cationic alpha-helical peptides which possessantibacterial, penetrating activities and DNA binding activities. The increase of net charge of the parentpeptide was accompanied by a corresponding increase in number of DNA binding sites. Keeping constantnet charge but reducing hydrophobicity of the peptide made number of DNA binding sites increased.Comparison of the GM (mean of the minimum inhibitory concentrations) of parent peptide and itsderivative peptides against Gram-negative E. coli, S. typhimurium, S. dysentery and P. aeruginosa andGram-positive L. monocytogenes, S. mutans, S. pyogenes, S. aureus and B. cereus shown that the orders ofantibacterial activity of peptides was P5>P7>P2>P6>P1>P4>ppTG20>P3. Increasing the net charge of theparent peptide induced antibacterial activity of peptides increasing, but increasing hydrophobicity of parentpeptide caused the antibacterial activity reduced. In the hydrophobic range of-0.13to-0.35, antibacterialactivity of peptides with the same net charge could be improved by reducing hydrophobicity of peptides.The correlation analysis also showed that the GM of peptides was significantly and positively correlatedwith hydrophobicity of peptides. The parent peptide and its derivative peptides showed low hemolysis rate(below10%) in the active concentration range and cytotoxic to tumor cells, they showed a certain degreeresistance to pepsin and trypsin, but they were sensitive to proteinase K.We used microbial adhesion to solvents method, liposome dye leakage analysis, membrane fluorescentprobes and flow cytometry to analysis mechanism of peptides’s penetration to the cell membrane of E. coliand S. pyogenes. Firstly, parent peptide and its derivative peptides significantly reduced the cell surfacehydrophobic after binding to the bacterial surface and increased permeability of the bacterial cellmembrane with time. Then, peptides adjusted to an amphipathic alpha-helix structure for the effects of cellmembrane hydrophobic environmental. They inserted and destroyed integrity of the cell membrane damage.P5(19.57%) induced the highest rate of cell membrane damage of E. coli, and the other peptides caused therate of membrane damage less than10%. Except P3, the peptides could cause significant damage of the cellmembrane of S. pyogenes and showed higher injury rate compared with the cell membrane damage of E.coli. Except P3, peptides could penetrate the E. coli cell membrane with slightly damage to the bacterial. P5showed the highest penetrating efficiency. Its cell-penetrating efficiency of E. coli and S. pyogenes was94.32%and28.31%respectively. Correlation analysis showed that penetrating E. coli cells efficiency ofpeptides significant negatively correlated with its MIC values and hydrophobicity, and highly significant negatively correlated with the outer and inter membrane permeability and membrane damage. Penetrating S.pyogenes cells efficiency of peptides significant negatively correlated with hydrophobicity of peptide, andhighly significantly and positively correlated with membrane damage. Results showed that mechanism ofpeptides against E. coli was not membrane action. The activity was affected by the hydrophobicity of thepeptide and the cell membrane permeability and membrane damage. Action of peptides against S. pyogeneswas membrane but with penetrating activity.Localization of the peptide onto cells was determined by confocal laser-scanning microscopyanalysis.The result showed that parent peptide and its derivative peptides could pass through the cell membrane of E.coli and S. pyogenes and accumulated in the cytoplasm except P3. The gel retardation, UV-visible spectraand fluorescence spectra analysis showed that parent peptide and derivative peptides binded to DNA andintercalated into the hydrophobic region of E. coli and S. pyogenes genomic DNA double helix structure，then formed peptide-DNA complexes. The order of binding constants of peptides with E. coli genome DNAwas P4>P6>P5>P7>P2>P1>ppTG20>P3. The order of binding constants of peptides with S. pyogenesgenome DNA was P5>P4>P6>P7>P2>P1>ppTG20>P3. Increasing the net charge or decreasing thehydrophobicity of the parent peptide could improve the ability of peptide’s binding to DNA. FlowCytometric Analysis showed that P4, P5, P6and P7could significantly increase the number of E. coli cellsin phase S. Except P3, parent peptide and its derivative peptides could significantly increase the number ofS. pyogenes cells in the phase S. P5(59.58%) affected S. pyogenes normal cell cycle were significantlyhigher than other peptides-treated group. Correlation analysis showed that the effect on E. coli cell cycle ofpeptides was highly significantly and negatively correlated with the MIC values and hydrophobicity of thepeptide, significantly positively correlated with cell membrane penetrating efficiency of peptides andbinding constants of peptides with E. coli genome DNA. The effect on S. pyogenes cell cycle of peptideswas significantly and negatively correlated with the MIC values, and so do with the hydrophobicity ofpeptide, the peptide-membrane interactions and the binding constants of peptides with S. pyogenes genomeDNA.We used magnetic beads coupled peptide to enrich peptide DNA-binding fragments, with specificprimers. P5specially binded to dnaA and rnhA. The gene expression was significantly down-regulated by2.79fold and2.36fold, respectively. P7specially binded with rnhA, made the gene expression level wasdown-regulated by2.24times. At the same time, The gene expression levels of of ssb, dnaG, dnaB, ligB,dpoI and dpoIII beta participated in the E. coli DNA replication dropped down, under the effect of thepeptide. P5and P7induced E. coli DNA repair gene recA significantly up-regulated1.72fold and1.18foldrespectively, recN gene was significantly up-regulated1.19times and1.29times. With the binding ofspecific gene sequence, E. coli DNA damage and DNA replication was inhibited. In addition, P5and P7significantly reduced E. coli DNA and RNA synthesis, P7greaterly affected RNA synthesis than that ofDNA synthesis.In summary, antimicrobial peptides designed based on the penetrating peptide could penetrate E. colicell membrane and bind DNA, resulting in inducing DNA damage and inhibition of DNA replication. Theantibacterial activity of peptides was correlated with hydrophobicity of peptide, and effect the membranepermeability by peptides and DNA-binding ability of peptides. But their action against S. pyogenes was theresult of the membrane damage and intracellular DNA-binding which was correlated with hydrophobicityof peptide and the cell membrane induced by peptide.