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A synergistic enhancement of activities has been described for the amphipathic cationic antimicrobial peptides magainin 2 and PGLa when tested in antimicrobial assays or in biophysical experiments using model membranes. In the presence of magainin 2, PGLa changes from an in-planar alignment parallel to the membrane surface to a more transmembrane orientation when investigated in membranes made from fully saturated PC or PC/PG, but not when one of the fatty acyl chains is unsaturated. Such lipid-mediated changes in the membrane topology of polypeptide domains could provide an interesting mechanism for the regulation of membrane proteins. Here we investigated the PGLa topology in a wide variety of membranes made of saturated or unsaturated PE, PC, and/or PG using 15 N solid-state NMR spectroscopy. In contrast to predictions made by previous models the data show that membrane curvature has only a minor effect on PGLa realignment. Furthermore, using 2 H solid-state NMR spectroscopy of deuterated phospholipid fatty acyl chains the order parameters of the lipids were investigated in the presence of PGLa, magainin, or equimolar peptide mixtures. Both peptides cause a pronounced decrease in the order parameters when oriented parallel to the membrane surface, an effect that reverts when PGLa flips into transmembrane alignments. Taken together, these data are suggestive that the magainin-induced disordering of fatty acyl chains provides an important driving force for PGLa realignment., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The emergence of bacterial resistance and hesitance in approving new drugs has bolstered research on membrane-active agents such as antimicrobial peptides and their synthetic derivatives as therapeutic alternatives against bacterial infections. Herein, we document the action of aryl-alkyl-lysines on liposomes mimicking bacterial membranes using solid-state nuclear magnetic resonance spectroscopy. A significant perturbation of the lipid thickness and order parameter of the lipid membrane was observed upon treatment with this class of compounds. Encouraged by these results, the ability of the most active compound (NCK-10) to interact with aggregates of lipopolysaccharides (LPSs) was studied. In vitro experiments showed that NCK-10 was able to prevent the LPS-induced stimulation of proinflammatory cytokines such as tumor necrosis factor-α and interleukin-6. The compound could also disrupt the biofilms of Pseudomonas aeruginosa in vitro and bring down the bacterial burden by more than 99% in a mice model of burn infections caused by the biofilms of P. aeruginosa ., Competing Interests: The authors declare no competing financial interest.

Antimicrobial peptides (AMPs) represent new alternatives to cope with the increasing number of multi-drug resistant microbial infections. Recently, a derivative of the frog-skin AMP esculentin-1a, Esc(1-21), was found to rapidly kill both the planktonic and biofilm forms of the Gram-negative bacterium Pseudomonas aeruginosa with a membrane-perturbing activity as a plausible mode of action. Lately, its diastereomer Esc(1-21)-1c containing two d-amino acids i.e. D Leu14 and D Ser17 revealed to be less cytotoxic, more stable to proteolytic degradation and more efficient in eradicating Pseudomonas biofilm. When tested in vitro against the free-living form of this pathogen, it displayed potent bactericidal activity, but this was weaker than that of the all-l peptide. To investigate the reason accounting for this difference, mechanistic studies were performed on Pseudomonas spheroplasts and anionic or zwitterionic membranes, mimicking the composition of microbial and mammalian membranes, respectively. Furthermore, structural studies by means of optical and nuclear magnetic resonance spectroscopies were carried out. Our results suggest that the different extent in the bactericidal activity between the two isomers is principally due to differences in their interaction with the bacterial cell wall components. Indeed, the lower ability in binding and perturbing anionic phospholipid bilayers for Esc(1-21)-1c contributes only in a small part to this difference, while the final effect of membrane thinning once the peptide is inserted into the membrane is identical to that provoked by Esc(1-21). In addition, the presence of two d-amino acids is sufficient to reduce the α-helical content of the peptide, in parallel with its lower cytotoxicity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The histidine-rich designer peptide LAH4-L1 exhibits antimicrobial and potent cell-penetrating activities for a wide variety of cargo including nucleic acids, polypeptides, adeno-associated viruses, and nanodots. The non-covalent complexes formed between the peptide and cargo enter the cell via an endosomal pathway where the pH changes from neutral to acidic. Here, we investigated the membrane interactions of the peptide with phospholipid bilayers and its membrane topology using static solid-state NMR spectroscopy. Oriented 15 N solid-state NMR indicates that in membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) 3:1 mol/mole and at neutral pH, the peptide adopts transmembrane topologies. Furthermore, 31 P and 2 H solid-state NMR spectra show that liquid crystalline 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and POPC/POPS 3:1 liposomes retain a bilayer macroscopic phase even at the highest peptide concentrations investigated, with an oblate orientational distribution of the phospholipids at a peptide/lipid ratio of 1:5. At pH 5, as it occurs in the endosome, the alignment of LAH4-L1 at a peptide/lipid ratio of 1:25 is predominantly parallel to POPC/POPS 3:1 bilayers (prolate deformation) when at the same time it induces a considerable decrease of the deuterium order parameter of POPC/ 2 H 31 -POPS 3:1. In addition, when studied in mechanically supported lipid membranes, a pronounced disordering of the phospholipid alignment is observed. In the presence of even higher peptide concentrations, lipid spectra are observed that suggest the formation of magnetically oriented or isotropic bicelles. This membrane-disruptive effect is enhanced for gel phase DMPC membranes. By protonation of the four histidines in acidic environments, the overall charge and hydrophobic moment of LAH4-L1 considerably change, and much of the peptide is released from the cargo. Thus, the amphipathic peptide sequences become available to disrupt the endosomal membrane and to assure highly efficient release from this organelle., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The antimicrobial peptide GL13K encompasses 13 amino acid residues and has been designed and optimized from the salivary protein BPIFA2 to exhibit potent bacteriocidal and anti-biofilm activity against Gram-negative and Gram-positive bacteria as well as anti-lipopolysaccharide activity in vitro and in vivo. Here, the peptide was analyzed in a variety of membrane environments by circular dichroism spectroscopy and by high-resolution multidimensional solution nuclear magnetic resonance (NMR) spectroscopy. Whereas in the absence of membranes a random coil conformation predominates, the peptide adopts a helical structure from residue 5 to 11 in the presence of dodecylphosphocholine micelles. In contrast, a predominantly β-sheet structure was observed in the presence of lipid bilayers carrying negatively charged phospholipids. Whereas 15 N solid-state NMR spectra are indicative of a partial alignment of the peptide 15 N- 1 H vector along the membrane surface, 2 H and 31 P solid-state NMR spectra indicate that in this configuration the peptide exhibits pronounced disordering activities on the phospholipid membrane, which is possibly related to antimicrobial action. GL13K, thus, undergoes a number of conformational transitions, including a random coil state in solution, a helical structure upon dilution at the surface of zwitterionic membranes, and β-sheet conformations at high peptide:lipid ratios.