The activity of antimicrobial peptides stems from their interaction with bacterial membranes, which are disrupted according to a number of proposed mechanisms. Here, we investigate the interaction of a model antimicrobial peptide that contains a single arginine residue with vesicles containing model lipid membranes. The surfactant-like peptide Ala-Arg (AR) is studied in the form where both termini are capped (CONH-AR-NH, capAR) or uncapped (NH-AR-OH, AR). Lipid membranes are selected to correspond to model anionic membranes (POPE/POPG) resembling those in bacteria or model zwitterionic membranes (POPC/DOPC) similar to those found in mammalian cells. Viable antimicrobial agents should show activity against anionic membranes but not zwitterionic membranes. We find, using small-angle X-ray scattering (SAXS) and cryogenic-TEM (transmission electron microscopy) that, uniquely, capAR causes structuring of anionic membranes due to the incorporation of the peptide in the lipid bilayer with peptide β-sheet conformation revealed by circular dichroism spectroscopy (CD). There is a preferential interaction of the peptide with POPG (which is the only anionic lipid in the systems studied) due to electrostatic interactions and bidentate hydrogen bonding between arginine guanidinium and lipid phosphate groups. At a certain composition, this peptide leads to the remarkable tubulation of zwitterionic phosphatidylcholine (PC) vesicles, which is ascribed to the interaction of the peptide with the outer lipid membrane, which occurs without penetration into the membrane. In contrast, peptide AR has a minimal influence on the anionic lipid membranes (and no β-sheet peptide structure is observed) but causes thinning (lamellar decorrelation) of zwitterionic membranes. We also investigated the cytotoxicity (to fibroblasts) and antimicrobial activity of these two peptides against model Gram positive and Gram negative bacteria. A strong selective antimicrobial activity against Gram positive Listeria monocytogenes, which is an important food-borne pathogen, is observed for capAR. Peptide AR is active against all three studied bacteria. The activity of the peptides against bacteria and mammalian cells is related to the specific interactions uncovered through our SAXS, cryo-TEM, and CD measurements. Our results highlight the exquisite sensitivity to the charge distribution in these designed peptides and its effect on the interaction with lipid membranes bearing different charges, and ultimately on antimicrobial activity.

We study the self-assembly of arginine-capped bolaamphiphile peptide RAR (A: alanine, R: arginine) together with its binding to model membranes and its cytotoxicity and antimicrobial activity. Anionic 2-oleoyl-1-palmitoyl- sn-glycero-3-phospho-rac-(1-glycerol) sodium salt/2-oleoyl-1-palmitoyl- sn-glycero-3-phosphoethanolamine (POPG/POPE) vesicles and zwitterionic 1,2-dioleoyl- sn-glycero-3-phosphocholine/2-oleoyl-1-palmitoyl- sn-glycero-3-phosphocholine (POPC/DOPC) vesicles are used as model membranes to mimic bacterial and mammalian cell membranes, respectively. We show that RAR adopts a polyproline-II collagen-like conformation in water. Binding of RAR to POPG/POPE vesicles induces a strong correlation between the lipid bilayers, driven by RAR/POPG attractive electrostatic interaction together with a shift of the intramolecular POPE zwitterionic interaction toward an attractive electrostatic interaction with the RAR. Populations of RAR/POPG/POPE vesicles comprise different bilayer spacings, d and d, controlled by the conformation of the lipid chains corresponding to the L (gel-like) and L (liquid-crystal) phases, respectively. Cryo-TEM images reveal the presence of vesicles with no internal structure, compartmentalized thin-wall vesicles, or multilayer vesicles with uncorrelated layers and compartmentalization depending on the RAR/POPG/POPE composition. In contrast, the interaction of RAR with multilamellar POPC/DOPC vesicles leads to the decorrelation of the lipid bilayers. RAR was tolerated by skin fibroblast cells for a concentration up to 0.01 wt %, while 0.25 wt % RAR proved to be an efficient antibacterial agent against Gram-positive bacteria L. monocytogenes. Our results highlight the ability of RAR to distinguish between bacterial and mammalian cells and establish this peptide as a candidate to reduce the proliferation of L. monocytogenes bacteria.

Macroscopic capsules, with tunable properties based on hierarchical self-assembly on multiple lengthscales, are prepared from the co-operative self-assembly of polysaccharide and peptide amphiphiles. Different formulations can be used to create flexible membrane sacs in solution, soft capsules or rigid free-standing capsules. Samples are prepared by injecting a solution containing sodium alginate, with or without graphene oxide (GO), into a matrix consisting of a solution containing the peptide amphiphile PA C 16 -KKFF (K: lysine, F: phenylalanine), with or without CaCl 2 . Graphene oxide is added to the hybrid materials to modulate the mechanical properties of the capsules. Injection of sodium alginate solution into a pure PA matrix provides a flexible membrane sac in solution, while injection of NaAlg/GO solution into a PA matrix gives a soft capsule. Alternatively, a rigid free-standing capsule is made by injecting a NaAlg/GO solution into a PA + CaCl 2 matrix solution. A comprehensive insight into the hierarchical order within the capsules is provided through analysis of X-ray scattering data. A novel "Langmuir-Blodgett" mechanism is proposed to account for the formation of the sacs and capsules as the alginate solution is injected at the interface of the PA solution. The capsules show a unique antibacterial effect specific for the Gram positive bacterium Listeria monocytogenes, which is an important human pathogen. The hybrid nanostructured capsules thus have remarkable bioactivity and due to their tunable structural and functional properties are likely to have a diversity of other future applications.

Here it is demonstrated a novel approach in disinfection regimes where specific molecular acid resistance systems are inhibited aiming to eliminate microorganisms under acidic conditions. Despite the importance of the Glutamate Decarboxylase (GAD) system for survival of Listeria monocytogenes and other pathogens under acidic conditions, its potential inhibition by specific compounds that could lead to its elimination from foods or food preparation premises has not been studied. The effects of maleic acid on the acid resistance of L. monocytogenes were investigated and found that it has a higher antimicrobial activity under acidic conditions than other organic acids, while this could not be explained by its pKa or Ka values. The effects were found to be more pronounced on strains with higher GAD activity. Maleic acid affected the extracellular GABA levels while it did not affect the intracellular ones. Maleic acid had a major impact mainly on GadD2 activity as also shown in cell lysates. Furthermore, it was demonstrated that maleic acid is able to partly remove biofilms of L. monocytogenes. Maleic acid is able to inhibit the GAD of L. monocytogenes significantly enhancing its sensitivity to acidic conditions and together with its ability to remove biofilms, make a good candidate for disinfection regimes.