Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to examine the ultrastructural changes in bacteria induced by antimicrobial peptides (AMPs). Both the β-stranded gramicidin S and the α-helical peptidyl-glycylleucine-carboxyamide (PGLa) are cationic amphiphilic AMPs known to interact with bacterial membranes. One representative Gram-negative strain, Escherichia coli ATCC 25922, and one representative Gram-positive strain, Staphylococcus aureus ATCC 25923, were exposed to the AMPs at sub-MICs and supra-MICs in salt-free medium. SEM revealed a shortening and swelling of the E. coli cells, and multiple blisters and bubbles formed on their surface. The S. aureus cells seemed to burst upon AMP exposure, showing open holes and deep craters in their envelope. TEM revealed the formation of intracellular membranous structures in both strains, which is attributed to a lateral expansion of the lipid membrane upon peptide insertion. Also, some morphological alterations in the DNA region were detected for S. aureus. After E. coli was incubated with AMPs in medium with low ionic strength, the cells appeared highly turgid compared to untreated controls. This observation suggests that the AMPs enhance osmosis through the inner membrane, before they eventually cause excessive leakage of the cellular contents. The adverse effect on the osmoregulatory capacity of the bacteria is attributed to the membrane-permeabilizing action of the amphiphilic peptides, even at low (sub-MIC) AMP concentrations. Altogether, the results demonstrate that both TEM and SEM, as well as appropriate sample preparation protocols, are needed to obtain detailed mechanistic insights into peptide function.

Damage of the Bacterial Cell Envelope by Antimicrobial Peptides Gramicidin S and PGLa as Revealed by Transmission and Scanning Electron Microscopy

We used solid-state NMR spectroscopy to investigate the oligomeric structure and insertion of protegrin-1 (PG-1), a β-hairpin antimicrobial peptide, in lipid bilayers that mimic either the bacterial inner membrane [palmitoyloleoylphosphatidyl ethanolamine and palmitoyloleoylphosphatidylglycerol (POPE/POPG) bilayers] or the red blood cell membrane [neutral palmitoyloleoylphosphatidylcholine (POPC)/cholesterol bilayers]. 1H spin diffusion from lipids to the peptide indicates that PG-1 contacts both the lipid acyl chains and the headgroups in the anionic membrane but resides far from the lipid chains in the POPC/cholesterol bilayer. 19F spin diffusion data indicates that 75% of the β-hairpins have homodimerized N strands and C strands in the anionic membrane. The resulting (NCCN)n multimer suggests a membrane-inserted β-barrel enclosing a water pore. The lipids surrounding the β-barrel have high orientational disorder and chain upturns, thus they may act as fillers for the pore. These results revise several features of the toroidal pore model, first proposed for magainin and subsequently applied to PG-1. In the POPC/cholesterol membrane, the N and C strands of PG-1 cluster into tetramers, suggesting the formation of β-sheets on the membrane surface. Thus, the membrane composition plays a decisive role in defining the assembly and insertion of PG-1. The different oligomeric structures of PG-1 help to explain its greater toxicity for bacteria than for eukaryotic cells.

https://www.pnas.org/content/pnas/103/44/16242.full.pdf

Membrane-dependent oligomeric structure and pore formation of a β-hairpin antimicrobial peptide in lipid bilayers from solid-state NMR

The replacement of para-substituted benzenes with saturated bi- and polycyclic bioisosteres – bicyclo[1.1.1]pentane, bicyclo[2.2.2]octane and cubane, – often increases the potency, selectivity and metabolic stability of bioactive compounds. The currently remaining challenge for chemists, however, is to rationally design, synthesize and validate the saturated bioisosteres for ortho- and meta-substituted benzenes.

Saturated bioisosteres of benzene: where to go next?

NMR is a versatile tool for studying interactions between antimicrobial peptides and lipid membranes. Different approaches using both liquid state and solid state NMR are outlined here, with an emphasis on solid state NMR methods, to study the structures of antimicrobial peptides in lipid bilayers as well as the effect of these peptides on model membranes. Different NMR techniques for observing both peptides and lipids are explained, including 2H, 13C, 15N, and 19F labels, or natural abundance 1H, 13C, or 31P. Previous studies in the field are extensively reviewed in easily accessible tables. © 2004 Wiley Periodicals, Inc. Concepts Magn Reson 23A: 89–120, 2004.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/cmr.a.20024

NMR methods for studying membrane‐active antimicrobial peptides

Concentration-Dependent Realignment of the Antimicrobial Peptide PGLa in Lipid Membranes Observed by Solid-State 19F-NMR

The non-natural amino acid 4-fluorophenylglycine (4F-Phg) was incorporated into several representative membrane-associated peptides for dual purpose. The (19)F-substituted ring is directly attached to the peptide backbone, so it not only provides a well-defined label for highly sensitive (19)F NMR studies but, in addition, the D and L enantiomers of the stiff side chain may serve as reporter groups on the transient peptide conformation during the biological function. Besides peptide synthesis, which is accompanied by racemisation of 4F-Phg, we also describe separation of the epimers by HPLC and removal of trifluoroacetic acid. As a first example, 18 different analogues of the fusogenic peptide "B18" were prepared and tested for induction of vesicle fusion; the results confirmed that hydrophobic sites tolerated 4F-Phg labelling. Similar fusion activities within each pair of epimers suggest that the peptide is less structured in the fusogenic transition state than in the helical ground state. In a second example, five doubly labelled analogues of the antimicrobial peptide gramicidin S were compared by using bacterial growth inhibition assays. This cyclic beta-sheet peptide could accommodate both L and D substituents on its hydrophobic face. As a third example, we tested six analogues of the antimicrobial peptide PGLa. The presence of d-4F-Phg reduced the biological activity of the peptide by interfering with its amphiphilic alpha-helical fold. Finally, to illustrate the numerous uses of l-4F-Phg in (19)F NMR spectroscopy, we characterised the interaction of labelled PGLa with uncharged and negatively charged membranes. Observing the signal of the free peptide in an aqueous suspension of unilamellar vesicles, we found a linear saturation behaviour that was dominated by electrostatic attraction of the cationic PGLa. Once the peptide is bound to the membrane, however, solid-state (19)F NMR spectroscopy of macroscopically oriented samples revealed that the charge density has virtually no further influence on the structure, alignment or mobility of the peptide.

Ute Möllmann

Papers

4‐Fluorophenylglycine as a Label for 19F NMR Structure Analysis of Membrane‐Associated Peptides