Research Of Screening For Antimicrobial Peptides By Mimic Cell Membrane Based On Peptide-membrane Interaction

Antimicrobial peptides are a class of small-molecule peptides with good thermal stabilityand broad spectrum antimicrobial activity. They are important components of the host innateimmune defense system. As their mode of action is markedly different from that of thetraditional antibiotics, antimicrobial peptides have been under consideration as promisingcandidates for novel antibacterial alternatives that can resist pathogens developing resistance.They have broad development prospect in the pharmaceutical, food, feed and other fields.However, the traditional separation and purification protocols are time-consuming andlabor-intensive, and give rise to problems such as low separation efficiency and productivity,which greatly limit the large-scale production and application of antimicrobial peptides. Theestablishment of rapid, efficient and targeted screening methods to separate antimicrobialpeptides has become a focus of increased research. Studies showed that the initial actiontarget of most antimicrobial peptides was the cell membrane system; most antimicrobialpeptides had membrane affinity properties. Mimic cell membrane constructed in vitro hasbeen used as a model membrane to study the action mechanism of antimicrobial peptides.However, it has never been used for screening antimicrobial peptide as an affinity media.Based on the affinity between antimicrobial peptides and mimic cell membranes, threeinnovative methods have been developed to screen antimicrobial peptides by use of mimiccell membranes, combining with solid phase extraction, HPLC and equilibrium dialysistechnologies. This research has important implications for large-scale development andutilization of new antimicrobial peptides resources, and human nutrition and healthimprovement.Firstly, the phospholipid mimic cell membrane stationary phase was prepared by thecoating method. The adsorption isotherms was measured to calculate the saturation fixedamount, in order to determine the addition amount of phospholipids during the preparation ofthe stationary phase and ensure the experimental reproducibility. The result of the fluorescentleakage experiment confirmed that the phospholipids existed in the lipid bilayer form on thesurface of the silica matrix. A novel antimicrobial peptide was successfully isolated from thepepsin hydrolyzate of ovalbumin using the phospholipid mimic cell membranes stationaryphase extraction combining with HPLC technique. Its amino acid sequence was determined asRVASMASEKMKI using matrix-assisted laser desorption/ionization quadrupole time of flightmass spectrometry. It was named as Opep12. The result showed that it was feasible to isolateantimicrobial peptides by the phospholipid mimic cell membranes stationary phase extraction.A mimic cell membrane chromatography method was developed using the lipid extractedfrom Escherichia coli. The chromatographic stationary phase had good stability in a certaintemperature (≤25℃) and salt concentration (≤50mmol/L) ranges. Buffer type, pH and saltconcentration influenced the retention behavior of the model peptides; buffer types couldchange the elution order of the three models peptide. The phosphate buffer (10mmol/L, pH7.2, containing50mmol/L NaCl) was used as the mobile phase for the analysis ofantimicrobial peptides in the E. coli mimic cell membrane chromatography. A novelantimicrobial peptide was separated from the anchovy crude peptide using the E. coli mimiccell membrane chromatography. The amino acid sequence was identified as GLARCLAGTL, and the peptide was named as Apep10. The mimic cell membrane chromatographyconstructed from the bacterial lipid reflected the real interaction between the antimicrobialpeptides and the bacterial lipid membranes, and realized the continuous collection of theantimicrobial peptides.The mimic cell membrane was then constructed from the lipid extract of Staphylococcusaureus to develop an equilibrium dialysis system. An antimicrobial peptide was screened fromthe protein hydrolyzate of the anchovy cooking wastewater by this method. The amino acidsequence was identified as GLSRLFTALK, and the peptide was named as ACWpep10. Theapplication verified the feasibility of the equilibrium dialysis combining with the mimic cellmembrane constructed from the bacterial lipid in the screening of antimicrobial peptide, andrealized the targeted screening of the antimicrobial peptides against certain strains.The analysis of the physical and chemical properties of the three antimicrobial peptidesshowed that all these peptides were cationic peptides with hydrophobic properties. All thesethree peptides had antibacterial activities against the tested gram-negative bacteria andgram-positive bacteria, in which the gram-negative bacteria were more sensitive to Opep12and Apep10, and the gram-positive bacteria were more sensitive to ACWpep10. The peptidescould expose the hydrophobic region of the lipid bilayer, increase the cell membranepermeability and damage the membrane integrity of the susceptible bacteria, leading to theoverflow of the intracellular K+ions and the nucleic acid substances.The correlation between the interaction extents of seven model antimicrobial peptideswith the mimic cell membranes and their antibacterial activities were compared. The analysisresults showed that the peptide-membrane binding degree in the phospholipid mimicmembrane stationary phase extraction was not significantly correlated with (P>0.05) with theMIC values against E. coli and S. aureus, the capacity factor in the E. coli mimic cellmembrane chromatography was highly significantly and negatively correlated (P<0.01) withthe MIC values against E. coli, and the peptide-membrane binding degree in the S. aureusmimic cell membrane equilibrium dialysis was significantly and negatively correlated(P<0.05) with the MIC values against S. aureus. The interaction of the model peptides withthe mimic cell membranes derived from the real bacterial lipids were more boinic, and couldpredict their antimicrobial activities to a certain extent.The results of confocal microscopy-Raman spectroscopy confirmed that ACWpep10could increase the membrane mobility in the lipid chain. The lipid chain became moredisorder, and the lateral slack was more relaxed after the treatment of ACWpep10. Theinteraction between ACWpep10and the S. aureus mimic cell membrane was stronger thanthat with the E. coli mimic cell membrane and the phospholipid mimic cell membrane. Basedon the results of capillary electrophoresis analysis, the binding constants between ACWpep10and the three mimic membranes were in the order as follows: S. aureus mimic cellmembrane>E. coli mimic cell membrane>phospholipid mimic cell membranes. The lipidcomposition analysis showed that the phospholipids compositions of the three kinds ofmembrane were quite different. The different hydrophobic alkyl chains and polar head chargestates of the phospholipids caused the different interaction between the mimic membranes andthe antimicrobial peptides, and ultimately led to the differences of their identifications andaffinities to antimicrobial peptides. In conclusion, three novel antimicrobial peptide screening methods were developed byusing the mimic cell membranes constructed from different lipids in this dissertation, basedon the principles of antimicrobial peptides interacting with the mimic cell membranes and thedegree of continuity of the separation operations. And three new cationic antimicrobialpeptides were isolated and purified from different sources using these methods. Themembrane action modes of the antimicrobial peptides were analyzed, and the resultsconfirmed that the bacterial cell membrane was one of the important targets for theseantimicrobial peptides exerting their activities. The interactions between the three mimicmembranes and the model peptides were compared. The results may provide a theoreticalbasis for the efficient screening of antimicrobial peptides by mimic cell membranes.