Lactoferricins impair the cytosolic membrane of Escherichia coli within a few seconds and accumulate inside the cell

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Abstract

We report the real-time response of E. coli to lactoferricin-derived antimicrobial peptides (AMPs) on length-scales bridging microscopic cell-sizes to nanoscopic lipid packing using millisecond time-resolved synchrotron small-angle X-ray scattering. Coupling a multi-scale scattering data analysis to biophysical assays for peptide partitioning revealed that the AMPs rapidly permeabilize the cytosolic membrane within less than three seconds-much faster than previously considered. Final intracellular AMP concentrations of ~ 80 to 100 mM suggest an efficient obstruction of physiologically important processes as primary cause for bacterial killing. On the other hand, damage of the cell envelope and leakage occurred also at sublethal peptide concentrations, thus emerging as a collateral effect of AMP activity that does not kill the bacteria. This implies that the impairment of the membrane barrier is a necessary but not sufficient condition for microbial killing by lactoferricins. The most efficient AMP studied exceeds others in both speed of permeabilizing membranes and lowest intracellular peptide concentration needed to inhibit bacterial growth.

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The current manuscript is a biophysical study, reporting data analysis of scattering data and peptide partitioning assays in vitro. All relevant data are included and plotted in the manuscript.In addition SANS raw data are accessible (DOI in Methods and Materials section).The modelling code for data analysis consists of a standard chi-squared minimization algorithm. The implemented analytical functions are described in the Methods and Materials section of the manuscript.

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Enrico Federico Semeraro

Institute of Molecular Biosciences, University of Graz, Graz, Austria

For correspondence
enrico.semeraro@uni-graz.at

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The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-6096-1108
Lisa Marx

Institute of Molecular Biosciences, University of Graz, Graz, Austria

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The authors declare that no competing interests exist.
Johannes Mandl

Institute of Molecular Biosciences, University of Graz, Graz, Austria

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The authors declare that no competing interests exist.
Ilse Letofsky-Papst

Institute of Electron Microscopy and Nanoanalysis, Graz University of Technology, Graz, Austria

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The authors declare that no competing interests exist.
Claudia Mayrhofer

Center for Electron Microscopy, Graz University of Technology, Graz, Austria

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Moritz PK Frewein

Institut Laue-Langevin, Grenoble, France

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The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-0329-5305
Haden L. Scott

Center for Environmental Biotechnology, University of Tennessee at Knoxville, Knoxville, United States

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The authors declare that no competing interests exist.
Sylvain Prévost

Institut Laue-Langevin, Grenoble, France

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The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-6008-1987
Helmut Bergler

Institute of Molecular Biosciences, University of Graz, Graz, Austria

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The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-7724-309X
Karl Lohner

Institute of Molecular Biosciences, University of Graz, Graz, Austria

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The authors declare that no competing interests exist.
Georg Pabst

Institute of Molecular Biosciences, University of Graz, Graz, Austria

For correspondence
georg.pabst@uni-graz.at

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The authors declare that no competing interests exist.

Funding

Austrian Science Fund (P 30921)

Karl Lohner

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.