Studies On The Damage Mechanism Of Cecropina–magainin On Methicillin-resistant Staphylococcus Aureus Membrane

ObjectiveTo study the damage mechanism of cecropin A–magainin on clinical isolated methicillin-resistant Staphylococcus aureus(MRSA) membrane. This study results provides a theoretical basis for the development of new effective peptide antibiotics.Methods1. The minimum inhibitory concentration(MIC) of cecropin A–magainin hybrid peptide against MRSA was determined using a microdilution technique in 96-well microtiter plates.2. Electrostatic and hydrophilic cell surface properties of MRSA were characterized by using the microelectrophoresis and microbial adhesion to solvents method, respectively.3. Choose phospholipid to synthesis liposome that simulation with anionic charge of bacterial cell membrane structure, study the characteristics of liposome by cecropin A–magainin hybrid peptide. Use fluorospectro photometer to analyze the intensity of fluorescein leakage of liposome after interacted with the hybrid peptides. Use FITC to tag the hybrid peptides to determine the liposomes spectra of the FITC-peptide and determine again after the blank liposomes. use fluorospectro photometer to analyze the intensity of fluorescein intensity of the FITC-peptide with fluorescence quenching agent.4. Use cecropin A–magainin hybrid peptide and antibiotic to experiment coordinated growth inhibition of MRSA and detect whether hybrid peptides can increase the permeability of MRSA outer membrane.5. Mainly through the beta-galactose glycosides test to reflect the permeability of cytoplasm membrane after treated with cecropin A–magainin hybrid peptide.6. Detect the leakage of K+ after interaction with cecropin A–magainin hybrid peptide 毕 by absorption spectrometer.7 Observe the effects of cecropin A–magainin hybrid peptide on bacterial cell membrane with transmission electron microscope.8 Analysis dye PI with positive bacterial count for bacterial cell membrane integrity by cecropin A–magainin hybrid peptide by flow cytometry instrument.Results1. The MIC of cecropin A–magainin hybrid peptide at which they exerted potent bactericidal effects against MRSA were tested and were found to be 64μg/ml.2. After the hybrid peptides treatment, cecropin A–magainin hybrid peptide can improve bacteria electronegativity changed from-0.95 to-0.50; the surface hydrophobicity of MRSA improve from 2.97% to 9.22%.3. The cecropin A–magainin hybrid peptide cause the fluorescein leakage from liposomes;liposomes influence fluorescence spectrum of the FITC-peptide change from 550.4nm to 536.2nm, and increased the intensity of fluorescence;liposomes can protect FITC-peptide from fluorescence quenching agent quenching;4. The cecropin A–magainin hybrid peptides work with rifampin, erythromycin and minocycline respectively have a role of synergy;5. The absorbance value increased with the extension of the cecropin A–magainin hybrid peptides;6. The atomic absorption spectrometer detect the extracellular concentration of potassium is elevated after treated with the cecropin A–magainin hybrid peptide.7. with the extension of the cecropin A–magainin hybrid peptides observed the ultrastructure image of the bacterial cell membrane destroyed, and even caused the content leakage to the out side of the cell, and even cause proposed nuclear disappear;8. The hybrid peptide caused the fluorescent iodide corganism(PI) into the cell, and the ratio of positive cells increased.ConclusionsThrough the tests we can find that the cecropin A–magainin hybrid peptides have certain osmosis to MRSA cell membrane, and eventually lead to the breakage of MRSA cell membrane.