The effects of magainin 2-derived and rationally designed antimicrobial peptides on Mycoplasma pneumoniae.

Combating the spread of antimicrobial resistance (AMR) among bacteria requires a new class of antimicrobials, which desirably have a narrow spectrum because of their low propensity for the spread of AMR. Antimicrobial peptides (AMPs), which target the bacterial cell membrane, are promising seeds for novel antimicrobials because the cell membrane is essential for all cells. Previously, we reported the antimicrobial and haemolytic effects of a natural AMP, magainin 2 (Mag2), isolated from the skin of Xenopus laevis (the African clawed frog), four types of synthesised Mag2 derivatives, and three types of rationally designed AMPs on gram-positive and gram-negative bacteria. To identify novel antimicrobial seeds, we evaluated the effect of AMPs on Mycoplasma pneumoniae, which also exhibits AMR. We also evaluated the antimicrobial effects of an AMP, NK2A, which has been reported to have antimicrobial effects on Mycoplasma bovis, in addition to Mag2 and previously synthesised seven AMPs, on four strains of M. pneumoniae using colorimetric, biofilm, and killing assays. We found that three synthesised AMPs, namely 17base-Ac6c, 17base-Hybrid, and Block, had anti-M. pneumoniae (anti-Mp) effect at 8-30 µM, whereas others, including NK2A, did not have any such effect. For the further analysis, the membrane disruption activities of AMPs were measured by propidium iodide (PI) uptake assays, which suggested the direct interaction of AMPs to the cell membrane basically following the colorimetric, biofilm, and killing assay results. PI uptake assay, however, also showed the NK2A strong interaction to cell membrane, indicating unknown anti-Mp determinant factors related to the peptide sequences. Finally, we conclude that anti-Mp effect was not simply determined by the membrane disruption activities of AMPs, but also that the sequence of AMPs were important for killing of M. pneumoniae. These findings would be helpful for the development of AMPs for M. pneumoniae.

Traceless native chemical ligation of lipid-modified peptide surfactants by mixed micelle formation.

Biology utilizes multiple strategies, including sequestration in lipid vesicles, to raise the rate and specificity of chemical reactions through increases in effective molarity of reactants. We show that micelle-assisted reaction can facilitate native chemical ligations (NCLs) between a peptide-thioester - in which the thioester leaving group contains a lipid-like alkyl chain - and a Cys-peptide modified by a lipid-like moiety. Hydrophobic lipid modification of each peptide segment promotes the formation of mixed micelles, bringing the reacting peptides into close proximity and increasing the reaction rate. The approach enables the rapid synthesis of polypeptides using low concentrations of reactants without the need for thiol catalysts. After NCL, the lipid moiety is removed to yield an unmodified ligation product. This micelle-based methodology facilitates the generation of natural peptides, like Magainin 2, and the derivatization of the protein Ubiquitin. Formation of mixed micelles from lipid-modified reactants shows promise for accelerating chemical reactions in a traceless manner.

Qualitative and Quantitative Changes to Escherichia coli during Treatment with Magainin 2 Observed in Native Conditions by Atomic Force Microscopy.

The bacterial membrane has been suggested as a good target for future antibiotics, so it is important to understand how naturally occurring antibiotics like antimicrobial peptides (AMPs) disrupt those membranes. The interaction of the AMP magainin 2 (MAG2) with the bacterial cell membrane has been well characterized using supported lipid substrates, unilamellar vesicles, and spheroplasts created from bacterial cells. However, to fully understand how MAG2 kills bacteria, we must consider its effect on the outer membrane found in Gram-negative bacteria. Here, we use atomic force microscopy (AFM) to directly investigate MAG2 interaction with the outer membrane of Escherichia coli and characterize the biophysical consequences of MAG2 treatment under native conditions. While propidium iodide penetration indicates that MAG2 permeabilizes cells within seconds, a corresponding decrease in cellular turgor pressure is not observed until minutes after MAG2 application, suggesting that cellular homeostasis machinery may be responsible for helping the cell maintain turgor pressure despite a loss of membrane integrity. AFM imaging and force measurement modes applied in tandem reveal that the outer membrane becomes pitted, more flexible, and more adhesive after MAG2 treatment. MAG2 appears to have a highly disruptive effect on the outer membrane, extending the known mechanism of MAG2 to the Gram-negative outer membrane.

Synergy on Surfaces: Anti-Biofouling Interfaces Using Surface-Attached Antimicrobial Peptides PGLa and Magainin-2.

The synergistic effect of antimicrobial compounds is an important phenomenon that can increase the potency of treatment and might be useful against the formation of biofilms on surfaces. A strong inhibition of microbial viability on surfaces can potentially delay the development of biofilms on treated surfaces, thereby enhancing the performance of water-purification technologies and medical devices, for example, to prevent hospital-acquired infections. However, the synergistic effects of surface-immobilized antimicrobial peptides (AMPs) have not yet been reported. Here, we demonstrate the synergistic antimicrobial effects of the AMPs PGLa and magainin-2 on modified reverse-osmosis (RO) membranes. These AMPs are known to act synergistically in the free state, but their antimicrobial synergistic effects have not yet been reported in a surface-immobilized state. The AMPs were functionalized with alkyne linkers and covalently attached to RO membranes modified with azides, using a click chemistry reaction. The resulting RO membranes showed reduced contact angles, indicating increased wettability. X-ray photoelectron spectroscopy confirmed the presence of the two peptides on the membranes via changes in the amounts of carbon, oxygen, and sulfur, which led to an increased S/C ratio, probably because of the sulfur present in the methionine residue of the peptides. The synergistic activity was measured with the free peptides in solution and covalently bound on RO membrane surfaces by observing increased leakage of 5(6)-carboxyfluorescein from large unilamellar vesicles. The synergistic antimicrobial activity against Pseudomonas aeruginosa was observed using surface-activity assays, where the AMP-modified RO membranes showed an effective inhibition of P. aeruginosa biofilm growth, as compared with unmodified membranes. An enhanced activity of antimicrobials on surfaces might lead to potent antimicrobial surfaces, which could result in more fouling-resistant water-treatment membranes.

Assessing topology and surface orientation of an antimicrobial peptide magainin 2 using mechanically aligned bilayers and electron paramagnetic resonance spectroscopy.

Aligned CW-EPR membrane protein samples provide additional topology interactions that are absent from conventional randomly dispersed samples. These samples are aptly suited to studying antimicrobial peptides because of their dynamic peripheral topology. In this study, four consecutive substitutions of the model antimicrobial peptide magainin 2 were synthesized and labeled with the rigid TOAC spin label. The results revealed the helical tilts to be 66°â€¯±â€¯5°, 76°â€¯±â€¯5°, 70°â€¯±â€¯5°, and 72°â€¯±â€¯5° for the TOAC substitutions H7, S8, A9, and K10 respectively. These results are consistent with previously published literature. Using the EPR (electron paramagnetic resonance) mechanical alignment technique, these substitutions were used to critically assess the topology and surface orientation of the peptide with respect to the membrane. This methodology offers a rapid and simple approach to investigate the structural topology of antimicrobial peptides.

Evidence for phenylalanine zipper-mediated dimerization in the X-ray crystal structure of a magainin 2 analogue.

High-resolution structure elucidation has been challenging for the large group of host-defense peptides that form helices on or within membranes but do not manifest a strong folding propensity in aqueous solution. Here we report the crystal structure of an analogue of the widely studied host-defense peptide magainin 2. Magainin 2 (S8A, G13A, G18A) is a designed variant that displays enhanced antibacterial activity relative to the natural peptide. The crystal structure of magainin 2 (S8A, G13A, G18A), obtained for the racemic form, features a dimerization mode that has previously been proposed to play a role in the antibacterial activity of magainin 2 and related peptides.

Design and characterization of novel hybrid antimicrobial peptides based on cecropin A, LL-37 and magainin II.

Antimicrobial peptides (AMPs) are a naturally occurring component of the innate immune response of many organisms and can have activity against both Gram-negative and Gram-positive bacterial species. In order to optimize and improve the direct antimicrobial effect of AMPs against a broad spectrum of bacterial species, novel synthetic hybrids were rationally designed from cecropin A, LL-37 and magainin II. AMPs were selected based on their α-helical secondary structure and fragments of these were analyzed and combined in silico to determine which hybrid peptides would form the best amphipathic cationic α-helices. Four hybrid peptides were synthesized (CaLL, CaMA, LLaMA and MALL) and evaluated for direct antimicrobial activity against a range of bacterial species (Bacillus anthracis, Burkholderia cepacia, Francisella tularensis LVS and Yersinia pseudotuberculosis) alongside the original 'parent' AMPs. The hybrid peptides showed greater antimicrobial effects than the parent AMPs (in one case a parent is completely ineffective while a hybrid based on it removes all traces of bacteria by 3h), although they also demonstrated higher hemolytic properties. Modifications were then carried out to the most toxic hybrid AMP (CaLL) to further improve the therapeutic index. Modifications made to the hybrid lowered hemolytic activity and also lowered antimicrobial activity by various degrees. Overall, this work highlights the potential for rational design and synthesis of improved AMPs that have the capability to be used therapeutically for treatment of bacterial infections.

Enhanced membrane pore formation by multimeric/oligomeric antimicrobial peptides.

The pore-forming antibacterial peptide magainin 2 was made divalent, tetravalent, and octavalent via a copper(I)-mediated 1-3 dipolar cycloaddition reaction ("click" chemistry). This series of pore-forming compounds was tested in vitro for their ability to form pores in large unilamillar vesicles (LUVs). A large increase in the pore-forming capability was especially observed with the tetravalent and octavalent magainin compounds in the LUVs consisting of DOPC, and the octavalent magainin compound showed a marked increase with the DOPC/DOPG LUVs. Activity was observed in the low nanomolar range for these compounds.