Search

To clarify the damage of lipid bilayer region in bacterial cell membrane caused by antimicrobial peptides (AMPs) and antimicrobial compounds (AMCs), their interactions with giant unilamellar vesicles (GUVs) of various lipid compositions have been examined. The findings revealed two main causes for the leakage: nanopore formation in the membrane and burst of GUVs. Although GUV burst has been explained previously based on the carpet model, the supporting evidence is limited. In this review, to better clarify the mechanism of GUV burst by AMPs, AMCs, and other membrane-active peptides, we described current knowledge of the conditions, characteristics, and detailed processes of GUV burst and the changes in the shape of the GUVs during burst. We identified several physical factors that affect GUV burst, such as membrane tension, electrostatic interaction, structural changes of GUV membrane such as membrane folding, and oil in the membrane. We also clarified one of the physical mechanisms underlying the instability of lipid bilayers that are associated with leakage in the carpet model. Based on these results, we propose a mechanism underlying some types of GUV burst induced by these substances: the growth of a nanopore to a micropore, resulting in GUV burst., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Most antimicrobial peptides (AMPs) induce pore formation and a burst of lipid bilayers and plasma membranes. This causes severe leakage of the internal contents and cell death. The AMP PGLa forms nanopores in giant unilamellar vesicles (GUVs) comprising dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG). We here elucidated the effect of the line tension of a prepore rim on PGLa-induced nanopore formation by investigating the interaction of PGLa with single GUVs comprising dioleoylphosphatidylethanolamine (DOPE)/DOPG (6:4) in buffer using the single GUV method. We found that PGLa forms nanopores in the GUV membrane, which evolved into a local burst and burst of GUVs. The rate of pore formation in DOPE/DOPG-GUVs was smaller than that in DOPC/DOPG-GUVs. PGLa is located only in the outer leaflet of a GUV bilayer just before a fluorescent probe AF647 leakage from the inside, indicating that this asymmetric distribution induces nanopore formation. PGLa-induced local burst and burst of GUVs were observed at 10 ms-time resolution. After nanopore formation started, dense particles and small vesicles appeared in the GUVs, followed by a decrease in the GUV diameter. The GUV was finally converted into smaller GUV or lipid membrane aggregates. We discuss the mechanisms of PGLa-induced nanopore formation and its direct evolution to a GUV burst.

The osmotic pressure (Π) method has recently been developed to quantitatively examine the effect of membrane tension (σ) on pore formation in giant unilamellar vesicles (GUVs) induced by antimicrobial peptides (AMPs). Here, we used the Π method to reveal the effect of σ on the interaction of an AMP, PGLa, with lipid bilayers comprising dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (4/6). PGLa induced leakage of fluorescent probes from single GUVs under Π, indicating nanopore formation. Membrane tension did not transform a PGLa-induced nanopore into a micropore nor cause GUV burst up to 3.4 mN/m, which is in contrast with the effect of σ on another AMP, magainin 2-induced pore formation, where lower σ resulted in GUV burst. The fraction of leaking GUVs at a specific time increased with increasing σ, indicating that the rate of PGLa-induced pore formation increases with increasing σ. The rate of transfer of fluorescent probe-labeled PGLa across the lipid bilayer without pore formation also increased with increasing σ. PGLa-induced pore formation requires a symmetric distribution of peptides in both leaflets of the GUV bilayer, and thus we infer that the increase in the rate of PGLa transfer from the outer leaflet to the inner leaflet underlies the increase in the rate of pore formation with increasing σ. On the basis of these results, we discuss the difference between the effect of σ on nanopore formation in GUV membranes induced by PGLa and that by magainin 2., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Positive membrane tension in the stretched plasma membrane of cells and in the stretched lipid bilayer of vesicles has been well analyzed quantitatively, whereas there is limited quantitative information on negative membrane tension in compressed plasma membranes and lipid bilayers. Here, we examined negative membrane tension quantitatively. First, we developed a theory to describe negative membrane tension by analyzing the free energy of lipid bilayers to obtain a theoretical equation for negative membrane tension. This allowed us to obtain an equation describing the negative membrane tension (σosm) for giant unilamellar vesicles (GUVs) in hypertonic solutions due to negative osmotic pressure (Π). Then, we experimentally estimated the negative membrane tension for GUVs in hypertonic solutions by measuring the rate constant (kr) of rupture of the GUVs induced by the constant tension (σex) due to an external force as a function of σex. We found that larger σex values were required to induce the rupture of GUVs under negative Π compared with GUVs in isotonic solution and quantitatively determined the negative membrane tension induced by Π (σosm) by the difference between these σex values. At small negative Π, the experimental values of negative σosm agree with their theoretical values within experimental error, but as negative Π increases, the deviation increases. Negative tension increased the stability of GUVs because higher tensions were required for GUV rupture, and the rate constant of antimicrobial peptide magainin 2-induced pore formation decreased., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

Antimicrobial peptides (AMPs) inhibit the proliferation of or kill bacterial cells. To measure these activities, several methods have been used, which provide only the average value of many cells. Here, we report the development of a method to examine the antimicrobial and bactericidal activities of AMPs at the single-cell level (i.e., single-cell analysis) and apply this strategy to examine the interaction of an AMP, magainin 2 (Mag), with Escherichia coli cells. Using this method, we monitored the proliferation of single cells on agar in a microchamber and measured the distribution of the number of cells in each microcolony using optical microscopy. For method A, we incubated cells in the presence of various concentrations of AMPs for 3 h. The fraction of microcolonies containing only a single cell, P single , increased with the Mag concentration and reached 1 at a specific concentration, which corresponded to the MIC. For method B, after the interaction of a cell suspension with an AMP for a specific time, an aliquot was diluted to stop the interaction, and the proliferation of single cells then was monitored after a 3-h incubation; this method permits the definition of P single ( t ), the fraction of dead cells after the interaction. For the interaction of Mag with E. coli cells, P To elucidate the activity of antimicrobial peptides (AMPs) against bacterial cells, it is important to estimate the interaction time that is sufficient to induce cell death. We have developed a method to examine the antimicrobial and bactericidal activities of AMPs at the single-cell level (i.e., single-cell analysis). Using this method, we monitored the proliferation of single cells on agar in a microchamber and measured the distribution of the number of cells in each microcolony using optical microscopy. We found that during the interaction of magainin 2 (Mag) with E. coli cells, the fraction of dead cells, single ( t ) increased with the interaction time, reaching ~1 at 10 and 20 min for 25 and 13 μM Mag, respectively. Thus, these results indicate that a short interaction time between Mag and E. coli cells is sufficient to induce bacterial cell death. IMPORTANCE To elucidate the activity of antimicrobial peptides (AMPs) against bacterial cells, it is important to estimate the interaction time that is sufficient to induce cell death. We have developed a method to examine the antimicrobial and bactericidal activities of AMPs at the single-cell level (i.e., single-cell analysis). Using this method, we monitored the proliferation of single cells on agar in a microchamber and measured the distribution of the number of cells in each microcolony using optical microscopy. We found that during the interaction of magainin 2 (Mag) with E. coli cells, the fraction of dead cells, P single ( t ), increased with the interaction time, rapidly reaching 1 (e.g., 10 min for 25 μM Mag). This result indicates that Mag induces cell death after a short time of interaction.

Osmotic pressure ( Π ) induces membrane tension in cells and lipid vesicles, which may affect the activity of antimicrobial peptides (AMPs) by an unknown mechanism. We recently quantitated the membrane tension of giant unilamellar vesicles (GUVs) due to Π under physiological conditions. Here, we applied this method to examine the effect of Π on the interaction of the AMP magainin 2 (Mag) with single GUVs. Under low Π values, Mag induced the formation of nanometer-scale pores, through which water-soluble fluorescent probe AF488 permeates across the membrane. The rate constant for Mag-induced pore formation ( k p ) increased with increasing Π . It has been proposed that the membrane tension in the GUV inner leaflet ( σ in ) caused by Mag binding to the outer leaflet plays a vital role in Mag-induced pore formation. During the interactions between Mag and GUVs under Π , the σ in increases due to Π , thereby increasing k p . The relationship between the k p and the total σ in due to Π and Mag agreed with that without Π . In contrast, Mag induced rupture of a subset of GUVs under higher Π . Using fluorescence microscopy with a high-speed camera, the GUV rupture process was revealed. First, a small micrometer-scale pore was observed in individual GUVs. Then, the pore radius increased within ∼100 ms without changing the GUV diameter and concomitantly the thickness of the membrane at the pore rim increased, and finally the GUV transformed into a membrane aggregate. Based on these results, we discussed the effect of Π on Mag-induced damage of GUV membranes.

The hydrophilic polymer polyethylene glycol-grafted phospholipid has been used extensively in the study of artificial vesicles, nanomedicine, and antimicrobial peptides/proteins. In this research, the effects of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)-2000] (abbreviated PEG-DOPE) on the deformation and poration of giant unilamellar vesicles (GUVs)-induced by anionic magnetite nanoparticles (NPs) have been investigated. For this, the size of the NPs used was 18 nm, and their concentration in the physiological solution was 2.00 μg/mL. GUVs were prepared using the natural swelling method comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and PEG-DOPE. The mole% of PEG-DOPE in the membranes were 0, 2, and 5%. The degree of deformation of the GUVs was quantified by the parameter compactness (Com), which is 1.0 for the spherical-shaped GUVs. The value of Com increases with time during the interactions of NPs with GUVs for any concentration of PEG-DOPE, but the rate of increase is significantly influenced by the PEG-DOPE concentration in the membranes. The average compactness increases with the increase of PEG-DOPE%, and after 60 min of NPs interaction, the values of average compactness for 0, 2, and 5% PEG-DOPE were 1.19 ± 0.02, 1.26 ± 0.03 and 1.35 ± 0.05, respectively. The fraction of deformation (Frd) also increased with the increase of PEG-DOPE%, and at 60 min, the values of Frd for 0 and 5% PEG-DOPE were 0.47 ± 0.02 and 0.63 ± 0.02, respectively. The fraction of poration (Frp) increased with the increase of PEG-DOPE, and at 60 min, the values of Frp for 0 and 5% PEG-DOPE were 0.25 ± 0.02 and 0.48 ± 0.02, respectively. Hence, the presence of PEG-grafted phospholipid in the membranes greatly enhances the anionic magnetite NPs-induced deformation and poration of giant vesicles. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Magainin 2 (Mag) induced rupture of E. coli -lipid GUVs, inducing rapid leakage. • Rate constant of Mag-induced rupture of GUVs (k r) increased with Mag concentration. • First a small micropore was formed, and grew rapidly with time to rupture the GUV. • Binding of Mag to the outer monolayer increased the area of the GUV bilayer. • Mechanical property of E. coli -lipid GUVs related to pore formation was elucidated. [ABSTRACT FROM AUTHOR]

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 (φ m) 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 (k p) 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 φ m 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 k p with negative membrane potential. Unlabelled Image • Magainin 2 (Mag) caused leakage of calcein from GUVs under membrane potential, φ m. • Rate constant of Mag-induced pore formation (k p) increased as ∣ φ m ∣ increased. • Binding constant of CF-Mag to GUV membrane increased with an increase in ∣ φ m ∣. • Under φ m , Mag locates in outer leaflet of GUV until just before pore formation. • Increase in Mag surface concentration is one of main causes of the increase in k p. [ABSTRACT FROM AUTHOR]