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.

A defensin from clam Venerupis philippinarum: Molecular characterization, localization, antibacterial activity, and mechanism of action

“…FITC is a low-molecular-mass (389.4 Da) green fluorescent probe, which is unable to traverse the cytoplasmic membrane of intact cells and provides an indication of membrane integrity (Mangoni et al, 2004). In the present study, FITC was used as a fluorescence probe to visualize the cytoplasmic membrane-perturbing activity of rVpDef on M. luteus.…”

Investigation into the mechanism of action of the antimicrobial peptides Os and Os-C derived from a tick defensin

“…The triple-stain enables the simultaneous viewing of total (stained by DAPI) and viable (stained by CTC) cells, as well as cells with altered membrane permeability (stained by FITC). E. coli and B. subtilis cells were exposed to 0.77 µM Os and 1.74 µM Os-C and a concentration 10 times lower for 10 minutes, stained with CTC which fluoresces red in viable cells, DAPI (blue) which stains all DNA and FITC (green) which only stains cells with permeabilized membranes [20]. Control cells of both E. coli and B. subtilis showed no permeabilization, and most of the cells fluoresced red, which indicated viability ( Fig.…”

“…The method described by Mangoni et al [20], using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), 4',6-diamidino-2-phenylindole (DAPI) and fluorescein 5(6)-isothiocyanate (FITC) was used. Stationary phase E. coli and B. subtilis cultures were adjusted to a cell density of 64x10 6 CFU/mL in 10 mM NaP buffer, pH 7.4, and exposed to peptide concentrations of 0.77 µM and 3.75 µM and concentrations 10x lower (0.077 µM and 0.37 µM) for 10 minutes at 37°C in a shaking incubator.…”

“…Hartmann and colleagues used electron microscopy to observe that the peptides gramicidin S and PGLa caused damage of the bacterial envelope, disruption of osmoregulation and affected bacterial DNA [12]. Mangoni et al described a fluorescence triple staining method with which permeability and viability of cells caused by peptides, could simultaneously be visually evaluated [20]. By fluorescently labeling buforin II analogs and observing the location with microscopy, Park and colleagues revealed that substitution or insertion of proline caused the peptide to become membrane-acting [24].…”

Keefektifan Pelapisan Benih terhadap Peningkatan Mutu Benih Padi Selama Penyimpanan

“…Mekanisme kerja antibakteri minyak atsiri adalah dengan merusak sel bakteri. Kerusakan sel diawali dengan rusaknya membran sel yang berlanjut dengan keluarnya material isi sel dan akhirnya sel mengalami kematian (Mangoni et al 2004, Rasooli et al 2006. Kim et al (1995) dan Bennis et al (2004) dan menurunkan daya berkecambah benih lebih cepat dibanding pestisida kimia yang berbentuk serbuk padat.…”