title: Localized permeabilization of E. coli membranes by the antimicrobial peptide Cecropin A.
abstract: Fluorescence microscopy enables detailed observation of the effects of the antimicrobial peptide Cecropin A on the outer membrane (OM) and cytoplasmic membrane (CM) of single E. coli cells with subsecond time resolution. Fluorescence from periplasmic GFP decays and cell growth halts when the OM is permeabilized. Fluorescence from the DNA stain Sytox Green rises when the CM is permeabilized and the stain enters the cytoplasm. The initial membrane disruptions are localized and stable. Septating cells are attacked earlier than nonseptating cells, and curved membrane surfaces are attacked in preference to cylindrical surfaces. Below a threshold bulk Cecropin A concentration, permeabilization is not observed over 30 min. Above this threshold, we observe a lag time of several minutes between Cecropin A addition and OM permeabilization and ∼30 s between OM and CM permeabilization. The long lag times and the existence of a threshold concentration for permeabilization suggest a nucleation mechanism. However, the roughly linear dependence of mean lag time on bulk peptide concentration is not easily reconciled with a nucleation step involving simultaneous insertion of multiple peptides into the bilayer. Monte Carlo simulations suggest that within seconds, the OM permeability becomes comparable to that of a pore of 100 nm diameter or of numerous small pores distributed over a similarly large area.
authors.affiliation: Department of Chemistry and ‡Molecular Biophysics Program, University of Wisconsin-Madison , 1101 University Avenue, Madison, Wisconsin 53706, United States.
authors.forename: Nambirajan
authors.initials: N
authors.lastname: Rangarajan
authors.forename: Somenath
authors.initials: S
authors.lastname: Bakshi
authors.forename: James C
authors.initials: JC
authors.lastname: Weisshaar
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects*; D002463:Cell Membrane Permeability / Q000187:drug effects*; D048168:Escherichia coli K12 / Q000187:drug effects*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009010:Monte Carlo Method
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D049452:Green Fluorescent Proteins; C031161:cecropin A