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Most antimicrobial peptides (AMPs) damage the cell membrane of bacterial cells and induce rapid leakage of the internal cell contents, which is a main cause of their bactericidal activity. One of the AMPs, magainin 2 (Mag), forms nanopores in giant unilamellar vesicles (GUVs) comprising phosphatidylcholine (PC) and phosphatidylglycerol (PG), inducing leakage of fluorescent probes. In this study, to elucidate the Mag-induced pore formation in lipid bilayer region in E. coli cell membrane, we examined the interaction of Mag with single GUVs comprising E. coli polar lipids (E. coli-lipid-GUVs). First, we investigated the Mag-induced leakage of a fluorescent probe AF488 from single E. coli-lipid-GUVs, and found that Mag caused rupture of GUVs, inducing rapid AF488 leakage. The rate constant of Mag-induced GUV rupture increased with the Mag concentration. Using fluorescence microscopy with a time resolution of 5 ms, we revealed the GUV rupture process: first, a small micropore was observed in the GUV membrane, then the pore radius increased within 50 ms without changing the GUV diameter, the thickness of the membrane at the pore rim concomitantly increased, and eventually membrane aggregates were formed. Mag bound to only the outer monolayer of the GUV before GUV rupture, which increased the area of the GUV bilayer. We also examined the physical properties of E. coli-lipid-GUVs themselves. We found that the rate constant of the constant tension-induced rupture of E. coli-lipid-GUVs was higher than that of PG/PC-GUVs. Based on these results, we discussed the Mag-induced rupture of E. coli-lipid-GUVs and its mechanism.

Bacterial biofilms have long been recognized as a source of persistent infections and industrial contamination with their intransigence generally attributed to their protective layer of extracellular polymeric substances (EPS). EPS, consisting of secreted nucleic acids, proteins, and polysaccharides, make it difficult to fully eliminate biofilms by conventional chemical or physical means. Since most bacteria are capable of forming biofilms, understanding how biofilms respond to new antibiotic compounds and components of the immune system has important ramifications. Antimicrobial peptides (AMPs) are both potential novel antibiotic compounds and part of the immune response in many different organisms. Here, we use atomic force microscopy to investigate the biomechanical changes that occur in individual cells when a biofilm is exposed to the AMP magainin 2 (MAG2), which acts by permeabilizing bacterial membranes. While MAG2 is able to prevent biofilm initiation, cells in an established biofilm can withstand exposure to high concentrations of MAG2. Treated cells in the biofilm are classified into two distinct populations after treatment: one population of cells is indistinguishable from untreated cells, maintaining cellular turgor pressure and a smooth outer surface, and the second population of cells are softer than untreated cells and have a rough outer surface after treatment. Notably, the latter population is similar to planktonic cells treated with MAG2. The EPS likely reduces the local MAG2 concentration around the stiffer cells since once the EPS was enzymatically removed, all cells became softer and had rough outer surfaces. Thus, while MAG2 appears to have the same mechanism of action in biofilm cells as in planktonic ones, MAG2 cannot eradicate a biofilm unless coupled with the removal of the EPS.

The effect of membrane potential on plasma membrane damage generated by antimicrobial peptides (AMPs) is an important, yet poorly characterized, process. Here, we studied the effect of membrane potential (φm) on pore formation by magainin 2 (Mag) in single giant unilamellar vesicles (GUVs) composed of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes. Various membrane potentials in GUVs containing gramicidin A were generated as a result of K+ concentration gradients. First, we examined Mag-generated membrane permeation of the water-soluble fluorescent probe calcein in single DOPG/DOPC-GUVs in the presence of membrane potential. The results indicate that the rate constant (kp) of Mag-induced pore formation increased with increasing negative membrane potentials. Analysis of the rim intensity of single GUVs interacting with low concentrations of a fluorescent probe, carboxyfluorescein-labeled Mag (CF-Mag), using confocal laser scanning microscopy (CLSM) shows that the concentration of CF-Mag in the membrane greatly increased with negative membrane potentials. This indicates that the binding constant of CF-Mag to the membrane increased with more negative membrane potentials. To elucidate the location of Mag in a GUV with φm during Mag-induced pore formation, we examined the interaction of Mag and a low concentration of a CF-Mag mixture with single GUVs containing the water-soluble fluorescent probe AF647 using CLSM. The data indicate that CF-Mag locates in the external leaflet of single GUVs until just before pore formation. Based on these data, we conclude that the increase in the surface concentration of Mag is one of the primary causes of the increase in kp with negative membrane potential.

Bacterial biofilms have long been recognized as a source of persistent infections and industrial contamination with their intransigence generally attributed to their protective layer of extracellular polymeric substances (EPS). EPS, consisting of secreted nucleic acids, proteins, and polysaccharides, make it difficult to fully eliminate biofilms by conventional chemical or physical means. Since most bacteria are capable of forming biofilms, understanding how biofilms respond to new antibiotic compounds and components of the immune system has important ramifications. Antimicrobial peptides (AMPs) are both potential novel antibiotic compounds and part of the immune response in many different organisms. Here, we use atomic force microscopy to investigate the biomechanical changes that occur in individual cells when a biofilm is exposed to the AMP magainin 2 (MAG2), which acts by permeabilizing bacterial membranes. While MAG2 is able to prevent biofilm initiation, cells in an established biofilm can withstand exposure to high concentrations of MAG2. Treated cells in the biofilm are classified into two distinct populations after treatment: one population of cells is indistinguishable from untreated cells, maintaining cellular turgor pressure and a smooth outer surface, and the second population of cells are softer than untreated cells and have a rough outer surface after treatment. Notably, the latter population is similar to planktonic cells treated with MAG2. The EPS likely reduces the local MAG2 concentration around the stiffer cells since once the EPS was enzymatically removed, all cells became softer and had rough outer surfaces. Thus, while MAG2 appears to have the same mechanism of action in biofilm cells as in planktonic ones, MAG2 cannot eradicate a biofilm unless coupled with the removal of the EPS.

PEGylation is frequently used to improve the efficacy of protein and peptide drugs. Recently, we investigated its effects on the action mechanism of the cyclic beta-sheet antimicrobial peptide tachyplesin I isolated from Tachypleus tridentatus [Y. Imura, M. Nishida, Y. Ogawa, Y. Takakura, K. Matsuzaki, Action Mechanism of Tachyplesin I and Effects of PEGylation, Biochim. Biophys. Acta 1768 (2007) 1160-1169]. PEGylation did not change the basic mechanism behind the membrane-permeabilizing effect of the peptide on liposomes, however, it decreased the antimicrobial activity and cytotoxicity. To obtain further information on the effects of PEGylation on the activities of antimicrobial peptides, we designed another structurally different PEGylated antimicrobial peptide (PEG-F5W, E19Q-magainin 2-amide) based on the alpha-helical peptide magainin 2 isolated from the African clawed frog Xenopus laevis. The PEGylated peptide induced the leakage of calcein from egg yolk L-alpha-phosphatidylglycerol/egg yolk L-alpha-phosphatidylcholine large unilamellar vesicles, however, the activity was weaker than that of the control peptides. The PEGylated peptide induced lipid flip-flop coupled to the leakage and was translocated into the inner leaflet of the bilayer, indicating that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. The cytotoxicity of the non-PEGylated peptides was nullified by PEGylation. At the same time, the antimicrobial activity was weakened only by 4 fold. The effects of PEGylation on the activity of magainin were compared with those for tachyplesin.