The Antimicrobial Peptide 1018-K6 Interacts Distinctly with Eukaryotic and Bacterial Membranes, the Basis of Its Specificity and Bactericidal Activity

Abstract

Since penicillin was discovered, antibiotics have been critical in the fight against infections. However, antibiotic misuse has led to drug resistance, which now constitutes a serious health problem. In this context, antimicrobial peptides (AMPs) constitute a natural group of short proteins, varying in structure and length, that act against certain types of bacterial pathogens. The antimicrobial peptide 1018-K6 (VRLIVKVRIWRR- NH2) has significant bactericidal and antibiofilm activity against Listeria monocytogenes isolates, and against different strains and serotypes of Salmonella. Here, the mechanism of action of 1018-K6 was explored further to understand the peptide-membrane interactions relevant to its activity, and to define their determinants. We combined studies with model synthetic membranes (liposomes) and model biological membranes, assessing the absorption maximum and the quenching of 1018-K6 fluorescence in aqueous and lipid environments, the self-quenching of carboxyfluorescein, as well as performing lipid sedimentation assays. The data obtained reflect the differential interactions of the 1018-K6 peptide with eukaryotic and prokaryotic membranes, and the specific interactions and mechanisms of action in the three prokaryotic species studied: Salmonella Typhimurium^2GN, Escherichia coli^3GN, and Staphylococcus aureus^3GP. The AMP 1018-K6 is a candidate to prevent (food preservation) or treat (antibiotic use) infections caused by certain pathogenic bacteria, especially some that are resistant to current antibiotics.

Keywords: Salmonella spp.; Staphylococcus spp.; antimicrobial peptide; food preservation; membrane lipid therapy (melitherapy).

Figure 1

1018-K6 binding to multilamellar vesicles (MLVs) of model membranes (a,c,f) and Scatchard plots of the peptide-MLV interactions (b,d,g). The peptide binding affinity for MLVs was measured by spectroscopic fluorescence (a,b) and UV-Vis absorption (c,d), and by calculating the B_max and K_d values (e): ns, unsaturated binding curve). Binding curves (including the B_max and K_d values) and Scatchard analyses of the peptide-DOPE₇₈:POPG₁₈:CL₄ interaction with increasing peptide concentrations (f,g). All the measurements are the average of three experiments.

Figure 2

1018-K6 binding to multilamellar vesicles (MLVs) formed with lipids extracted from biological membranes (a,c) and Scatchard plots of the peptide-MLV interactions (b,d). The peptide binding affinity for the MLVs was measured using fluorescence (a,b) and UV-Vis absorption (c,d), and by calculating the B_max and K_d values (e). All the measurements are the average of three experiments.

Figure 3

Blue shift in Trp fluorescence of the 1018-K6 AMP in the presence or absence of MLVs (L, concentration up to 2 mM) (a) Δλ_max shift of AMP in model membranes of zwitterionic lipids (PC, pink), eukaryotic cells (PC₄₀:POPE₄₀:SM₁₅:PS₅, black), S. Typhimurium (DOPE₇₈:POPG₁₈:CL₄, green) and S. aureus (CL₄₂:POPG₅₈, orange). The data were analyzed with SPSS version 26 (IBM Analytics, Armonk, NY, USA) and the results were assessed with generalized linear mixed models (GLMMs), comparing the means with the Tukey test. All data are presented as the mean (M) ± standard error (SE): ^A, ^B values differ at p < 0.01; ^a, ^b values differ at p < 0.05. (b–e) Fluorescence emission spectra of 1018-K6 before and after the addition of MLVs simulating (b) zwitterionic membranes, (c) eukaryotic cells, (d) S. Typhimurium, and (e) S. aureus.

Figure 4

1018-K6 Trp fluorescence quenching by acrylamide in model membranes. (a) Stern-Volmer plots for acrylamide quenching of Trp fluorescence when 1018-K6 was maintained in aqueous buffer (1 μM, grey line) or lipidic environments (30 μM): model membranes of zwitterionic lipids (PC, pink line), eukaryotic cells (PC₄₀:POPE₄₀:SM₁₅:PS₅, black line), S. Typhimurium (DOPE₇₈:POPG₁₈:CL₄, green line), or S. aureus (CL₄₂:POPG₅₈, orange line). The results are expressed as the mean and standard error of three independent scans 30, 90 and 150 min after peptide:liposomes incubation. (b–f) Fluorescence spectra of the 1018-K6 AMP in (b) buffer, (c) zwitterionic lipid, (d) eukaryotic cells, (e) S. Typhimurium and (f) S. aureus before and after the addition of acrylamide (up to 0.1 M). Details of the peptide spectra in liposomes of CL₄₂:POPG₅₈, recording the extent of tryptophan quenching depending on the incubation time (f.1–f.3). (g) Non-scale schematic drawing.

Figure 5

Trp fluorescence quenching of 1018-K6 in biological membranes by acrylamide. (a) Stern–Volmer plots of Trp fluorescence acrylamide quenching for 1018-K6 in aqueous buffer (1 μM, grey line) or lipidic environments (30 μM): Salmonella spp., E. coli or S. aureus biological membranes. The results are expressed as the mean and standard error of three independent scans after a 30, 90 or 150 min peptide:liposome incubation. (b–d) Fluorescence spectra of AMP 1018-K6 partitioned into liposomes of (b) Salmonella spp., (c) E. coli, or (d) S. aureus before and after the addition of acrylamide (up to 0.1 M). Details of the peptide spectra in liposomes of S. aureus, recording the extent of tryptophan quenching at different incubation times (d.1,d.2).

Figure 6

1018-K6 Trp fluorescence quenching by nitromethane in model membranes. (a) Stern–Volmer plots for nitromethane quenching of Trp fluorescence from 1018-K6 in aqueous buffer (1 μM, grey line) and lipidic environments (30 μM): model membranes of zwitterionic lipids (PC, pink line), eukaryotic cell (PC₄₀:POPE₄₀:SM₁₅:PS₅, black line), S. Typhimurium (DOPE₇₈:POPG₁₈:CL₄, green line) or S. aureus (CL₄₂:POPG₅₈, orange line). The results are expressed as the mean and standard error of three independent scans after a 30, 90 or 150 min peptide:liposome incubation. (b–f) Fluorescence spectra of the 1018-K6 AMP in (b) buffer or (c) zwitterionic lipid, (d) eukaryotic cell, (e) S. Typhimurium and (f) S. aureus membranes before and after the addition of nitromethane (up to 0.1 M). (g) Non-scale schematic drawing.

Figure 7

1018-K6 Trp fluorescence quenching in biological membranes by nitromethane. (a) Stern–Volmer plots of Trp fluorescence quenching by nitromethane when 1018-K6 is maintained in aqueous buffer (1 μM, grey line) or lipidic environments (30 μM): biological membranes of Salmonella spp., E. coli or S. aureus. The results are expressed as the mean and standard error of three independent scans after a 30, 90 or 150 min peptide:liposome incubation. (b–d) Fluorescence spectra of AMP 1018-K6 partitioned into liposomes of (b) Salmonella spp., (c) E. coli, or (d) S. aureus before and after the addition of nitromethane (up to 0.1 M).

Figure 8

Permeabilization efficiency of AMP 1018-K6 (15 µM). (a) The kinetic release of carboxyfluorescein (CF) from LUVs derived from model zwitterionic (PC, pink line), eukaryotic cell (PC₄₀:POPE₄₀:SM₁₅:PS₅, black line), S. Typhimurium (DOPE₇₈:POPG₁₈:CL₄, green line) and S. aureus (CL₄₂:POPG₅₈, orange line) membranes, and from biological membranes (Salmonella spp. and S. aureus). All the data correspond to the mean ± standard error (SE) of three independent experiments. (b) Non-scale schematic drawing.

Figure 9

Kinetics of MLV aggregation in the presence of 1018-K6. (a) 6.25 µM; (b) 12.5 µM; (c) 20 µM; (d) 25 µM; (e) 50 µM. Model zwitterionic (pink), eukaryotic cell (black), S. Typhimurium (green), E. coli (blue) or S. aureus (orange) membranes were used at a concentration of 500 µM. The results are the mean of three independent experiments and the error bars represent the standard error (SE). In particular, effects of incubation time (peptide:liposomes) and of the lipid mixtures (different liposomes tested) on vesicle aggregation were statistically evaluated (f). The p-values were determined by two-way ANOVA analysis, performed using GraphPad Prism^® 8.0.1. ns means not significant; * p < 0.05; ** p < 0.01; **** p < 0.0001.