Antimicrobial peptides are produced by all organisms in response to microbial invasion and are considered as promising candidates for future antibiotics. Many of them interact with the bacterial membrane and increase its permeability. However, it is not clear whether this is the lethal step for the killing mechanism. To address this issue, we studied the interaction of the antimicrobial peptide temporin L, isolated from Rana temporaria skin secretion, with Escherichia coli by using fluorescence, confocal, and electron microscopy. 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 fluorescein isothiocyanate. This technique enabled us to identify, in the same sample, both living and total cells, as well as bacteria with altered membrane permeability. We found 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 to 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. Overall, this study demonstrates the advantage of using the triple fluorescent approach combined with microscopical techniques to explore the mechanism of membrane-active peptides in general.
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