The Mechanism Of Hybrid Peptides Cecropin A-magainins On MRSA By Inhibition Of DNA Synthesis

ObjectiveTo investigate the antimicrobial mechanism of DNA action and the effect of biomacromolecule in physiological metabolism of Methicillin-resistant Staphylococcus aureus(MRSA) treated with CecropinA-Magainin, These results obtained in this study should helpthe design and study the therapeutical effect of useful antimicrobial peptides based on DNA-actionpeptides.Methods1. The minimum inhibitory concentration(MIC) of cecropinA–magainin hybrid peptide against MRSA was determined using a microdilution technique in 96-well microtiter plates.2. Using Confocal laser scanning microscope to observe the accumulation of hybrid peptides in MRSA cells.3. Analysis of the binding capacity of hybrid peptides and MRSA genomic DNA by gel retardation method.4. Spectral changes of the interaction between he hybrid peptide and MRSA DNA by ultraviolet spectrometry.5. Using the fluorescence spectrum analysis of CecropinA- magainin hybrid peptide and MRSA genomic DNA; Analysis of spectral changes in the titration of hybrid systems of hybrid peptides and EB-DNA; Analysis of the spectral changes of the titration of EB and hybrid peptide-DNA complex system; Finally, we calculate the binding constant and key points of peptide and MRSA genomic DNA.6. Using a full wavelength microplate to detect the effect of CecropinA-Magainin on DNA, RNA and total protein synthesis abilityof MRSA.7. Analysis ofthe expression activity of?-galactosidase and alkaline phosphatase of MRSA treated with CecropinA-Magainin.8. Using O2 microelectrode(OX50) to measure the effect of CecropinA-Magainin on cellular respiration of MRSA.9. The effect of CecropinA-Magainin on the production of ATP in MRSA cells was detected by using the biological luminescence analyzer.Results 1.The MIC of CecropinA–magainin hybrid peptide at which they exerted potent bactericidal effects against MRSA were tested and were found to be 64mg/L.2. Using laser scanning confocal microscope to observe MRSA treated with Fitc-peptide at MIC concentration at different time. We found that a proportion of 32.2% of the bacteria appeared fluorescence after 10 min. After 30 min, the proportion of hybrid peptides into the bacterial cells was higher(71.4%), and the fluorescence intensity was stronger.3. We found that the hybrid peptide has a strong binding ability in binding with MRSA genomic DNA by gel retardation analysis.4. The hybrid peptidecaused hypochromic effect of MRSA genomic DNA detected by UV spectrum.5. The competitive binding of DNA and EB was existed in solution system detected by fluorescence spectroscopy; And we calculate the binding constant is KM=1.87×107 and key points is n=0.13.6. Compared with the control group, two different concentrations of hybrid peptide could cause cell DNA, RNA and total protein synthesissignificantly inhibited(P < 0.05);7. The hybrid peptides inhibited the expression of two important enzymes?-galactosidase and alkaline phosphatase in the MRSA cell, and the results were consistent with the ability to inhibit protein synthesis;8. Compared with the control group, the dissolved oxygen rate at 1×MIC hybrid peptide concentration had no significant difference in Statistics in the first 50 min. There was a significant difference until 60 min. The higher the concentration of heterozygous peptide used, the stronger the inhibitionrote of cell respiration was.9. Compared with the control group, different concentrations of CecropinA–magainin can decrease the ATP production of MRSA. Theeffect was positively related withCecropinA-Magainin concentration(P< 0.05).ConclusionsIn summary, we can speculate the mechanisms of cecropinA–magainin hybrid peptide on MRSA DNA targeting effect is that the hybrid peptide first enters the bacterial cell,and then combines with MRSA genomic DNA.The hybrid peptide is embedded in the base pair of DNA, which causes the change of DNA conformation. The peptides further affects DNA replication, transcriptional function, inhibites of intracellular RNA, total protein synthesis capacity.The expression of intracellular enzyme activity decreased, and Cell respiration was inhibited and Cell oxidative phosphorylation ability is reduced. Finally, hybrid peptide causes cell death.