Effects of the antimicrobial peptide temporin L on cell morphology, membrane permeability and viability of Escherichia coli

Maria Luisa Mangoni, Niv Papo, Donatella Barra, Maurizio Simmaco, Argante Bozzi, Antonio Di Giulio, Andrea C. Rinaldi

Research output: Contribution to journalArticlepeer-review

125 Scopus citations

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 FVQWFSKFLG-RIL-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.

Original languageEnglish
Pages (from-to)859-865
Number of pages7
JournalBiochemical Journal
Volume380
Issue number3
DOIs
StatePublished - 15 Jun 2004
Externally publishedYes

Keywords

  • Amphibian skin
  • Antimicrobial peptide
  • Escherichia coli
  • Fluorescence microscopy
  • Membrane permeability
  • Temporin L.

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