title: Effects of the antimicrobial peptide temporin L on cell morphology, membrane permeability and viability of Escherichia coli.
abstract: Antimicrobial peptides are produced by all organisms in response to microbial invasion and are considered as promising candidates for future antibiotics. There is a wealth of evidence that many of them interact and increase the permeability of bacterial membranes as part of their killing mechanism. However, it is not clear whether this is the lethal step. To address this issue, we studied the interaction of the antimicrobial peptide temporin L with Escherichia coli by using fluorescence, confocal and electron microscopy. The peptide previously isolated from skin secretions of the frog Rana temporaria has the sequence FVQWFSKFLGRIL-NH2. With regard to fluorescence microscopy, we applied, for the first time, a triple-staining method based on the fluorochromes 5-cyano-2,3-ditolyl tetrazolium chloride, 4',6-diamidino-2-phenylindole and FITC. This technique enabled us to identify, in the same sample, both living and total cells, as well as bacteria with altered membrane permeability. These results reveal that temporin L increases the permeability of the bacterial inner membrane in a dose-dependent manner without destroying the cell's integrity. At low peptide concentrations, the inner membrane becomes permeable to small molecules but does not allow the killing of bacteria. However, at high peptide concentrations, larger molecules, but not DNA, leak out, which results in cell death. Very interestingly, in contrast with many antimicrobial peptides, temporin L does not lyse E. coli cells but rather forms ghost-like bacteria, as observed by scanning and transmission electron microscopy. Besides shedding light on the mode of action of temporin L and possibly that of other antimicrobial peptides, the present study demonstrates the advantage of using the triple-fluorescence approach combined with microscopical techniques to explore the mechanism of membrane-active peptides in general.
authors.affiliation: Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Istituto Pasteur-Fondazione Cenci Bolognetti, Azienda Ospedale S. Andrea, and CNR Istituto di Biologia e Patologia Molecolari, Università La Sapienza, I-00185 Roma, Italy. marialuisa.mangoni@uniroma1.it
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Mangoni
authors.forename: Niv
authors.initials: N
authors.lastname: Papo
authors.forename: Donatella
authors.initials: D
authors.lastname: Barra
authors.forename: Maurizio
authors.initials: M
authors.lastname: Simmaco
authors.forename: Argante
authors.initials: A
authors.lastname: Bozzi
authors.forename: Antonio
authors.initials: A
authors.lastname: Di Giulio
authors.forename: Andrea C
authors.initials: AC
authors.lastname: Rinaldi
mesh_terms: D023181:Antimicrobial Cationic Peptides; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000187:drug effects*; D004926:Escherichia coli / Q000166:cytology* / Q000187:drug effects* / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D008854:Microscopy, Electron / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D023181:Antimicrobial Cationic Peptides; D005456:Fluorescent Dyes; D010455:Peptides; C469676:temporin L, Rana temporaria