Bridging the Antimicrobial Activity of Two Lactoferricin Derivatives in E. coli and Lipid-Only Membranes.

Lisa Marx,Moritz P Frewein,Johannes Mandl,Enrico F Semeraro,Nermina Malanovic,Johannes Kremser,Georg Pabst,Karl Lohner

doi:10.3389/fmedt.2021.625975

Lisa Marx, Moritz P Frewein + Show 6 more

Open Access

https://doi.org/10.3389/fmedt.2021.625975

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Abstract

We coupled the antimicrobial activity of two well-studied lactoferricin derivatives, LF11-215 and LF11-324, in Escherichia coli and different lipid-only mimics of its cytoplasmic membrane using a common thermodynamic framework for peptide partitioning. In particular, we combined an improved analysis of microdilution assays with ζ-potential measurements, which allowed us to discriminate between the maximum number of surface-adsorbed peptides and peptides fully partitioned into the bacteria. At the same time, we measured the partitioning of the peptides into vesicles composed of phosphatidylethanolamine (PE), phosphatidylgylcerol (PG), and cardiolipin (CL) mixtures using tryptophan fluorescence and determined their membrane activity using a dye leakage assay and small-angle X-ray scattering. We found that the vast majority of LF11-215 and LF11-324 readily enter inner bacterial compartments, whereas only 1−5% remain surface bound. We observed comparable membrane binding of both peptides in membrane mimics containing PE and different molar ratios of PG and CL. The peptides' activity caused a concentration-dependent dye leakage in all studied membrane mimics; however, it also led to the formation of large aggregates, part of which contained collapsed multibilayers with sandwiched peptides in the interstitial space between membranes. This effect was least pronounced in pure PG vesicles, requiring also the highest peptide concentration to induce membrane permeabilization. In PE-containing systems, we additionally observed an effective shielding of the fluorescent dyes from leakage even at highest peptide concentrations, suggesting a coupling of the peptide activity to vesicle fusion, being mediated by the intrinsic lipid curvatures of PE and CL. Our results thus show that LF11-215 and LF11-324 effectively target inner bacterial components, while the stored elastic stress makes membranes more vulnerable to peptide translocation.

Highlights

The history of research on antimicrobial peptides (AMPs) as promising agents to combat infectious diseases is long and rich in diverse aspects
Data were analyzed assuming that the AMP-induced delayed bacterial growth is entirely due to a lower number density of survived cells, which is supported by the observation that the growth rate in the exponential phase does not depend on peptide concentration (Supplementary Figure 1A), i.e., the growth of the viable fraction of cells is similar to bacterial growth in absence of AMPs
Efficacy and Partitioning We started our analysis of peptide activity in E. coli with measuring a range of inhibitory concentrations, including the minimum inhibitory concentration (MIC), by means of a slightly modified susceptibility assay, and for different bacterial concentrations

Summary

Introduction

The history of research on antimicrobial peptides (AMPs) as promising agents to combat infectious diseases is long and rich in diverse aspects. Poreformation (barrel-stave, toroidal/worm-hole) [8] or changes in membrane topology [9, 10] have been reported at elevated AMP concentrations, eventually leading to complete micellization [11] of the membrane, known as the carpet model [12]. It is increasingly becoming clear, that the observed effects strongly depend on the lipid composition of the used membrane mimics and the resulting collective membrane properties [13,14,15]. Do not exclude any AMP activity within the bacterial membranes [17]

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