High Concentrations of the Antimicrobial Peptide Magainin 2 Induce Distinct Biomechanical Changes in Escherichia coli

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 between antimicrobial peptides PGLa and Magainin 2 (MAG2) provides an efficient way to enhance their antimicrobial ability. However, the underlying molecular mechanism of such synergy, especially the individual roles of each peptide, remains poorly understood. We combined a giant unilamellar vesicle leakage assay, in situ interfacial photovoltage testing, and molecular dynamics to investigate membrane poration under the action of PGLa, MAG2, or a PGLa/MAG2 mixture. Our results clearly show the different membrane action modes of the three systems and demonstrate the importance of forming PGLa-MAG2 heterodimers in the membrane poration process. PGLa inserted into and extracted from a membrane rapidly and continually with minimal aggregation and produced only transient, small pores. In contrast, MAG2 peptides tended to aggregate together on the membrane surface or only shallowly embed in the membrane. Additionally, the PGLa and MAG2 residues were well integrated into the membrane via the formation of PGLa-MAG2 heterodimers. The membrane defect produced by the rapid insertion of PGLa was stabilized by MAG2, which further recruited other peptides for the formation of PGLa-MAG2 heterodimers and even heterodimer clusters. Growth in pore size then occurred in a step-by-step process involving the formation and assembly of heterodimer clusters within the membrane. Our results provide insight into the complicated synergy that occurs between PGLa and MAG2 during membrane poration and will assist in the design of new antimicrobial peptides.

Unlocking melanoma Suppression: Insights from Plasma-Induced potent miRNAs through PI3K-AKT-ZEB1 axis

Antimicrobial activity and mechanisms of a derived antimicrobial peptide TroNKL-27 from golden pompano (Trachinotus ovatus) NK-lysin

The "pre-assembled state" of magainin 2 lysine-linked dimer determines its enhanced antimicrobial activity.

Designed histidine-rich amphipathic cationic peptides, such as LAH4, have enhanced membrane disruption and antibiotic properties when the peptide adopts an alignment parallel to the membrane surface. Although this was previously achieved by lowering the pH, here we have designed a new generation of histidine-rich peptides that adopt a surface alignment at neutral pH. In vitro, this new generation of peptides are powerful antibiotics in terms of the concentrations required for antibiotic activity; the spectrum of target bacteria, fungi, and parasites; and the speed with which they kill. Further modifications to the peptides, including the addition of more hydrophobic residues at the N terminus, the inclusion of a helix-breaking proline residue or using D-amino acids as building blocks, modulated the biophysical properties of the peptides and led to substantial changes in toxicity to human and parasite cells but had only a minimal effect on the antibacterial and antifungal activity. Using a range of biophysical methods, in particular solid-state NMR, we show that the peptides are highly efficient at disrupting the anionic lipid component of model membranes. However, we also show that effective pore formation in such model membranes may be related to, but is not essential for, high antimicrobial activity by cationic amphipathic helical peptides. The information in this study comprises a new layer of detail in the understanding of the action of cationic helical antimicrobial peptides and shows that rational design is capable of producing potentially therapeutic membrane active peptides with properties tailored to their function.

Antimicrobial peptides (AMPs) play crucial role as mediators of the primary host defense against microbial invasion. They are considered a promising alternative to antibiotics for multidrug resistant bacterial strains. For complete understanding of the antimicrobial defense mechanism, a detailed knowledge of the dynamics of peptide-membrane interactions, including atomistic studies on AMPs geometry and both peptide and membrane structural changes during the whole process is a prerequisite. We aim at clarifying the conformation dynamics of small linear AMPs in solution as a first step of in silico protocol for establishing a correspondence between certain amino-acid sequence motifs, secondary-structure elements, conformational dynamics in solution and the intensity and mode of interaction with the bacterial membrane. To this end, we use molecular dynamics simulations augmented by well-tempered metadynamics to study the free-energy landscape of two AMPs with close primary structure and different antibacterial activity – the native magainin 2 (MG2) and an analog (MG2m, with substitutions F5Y and F16W) in aqueous solution. We observe that upon solvation, the initial α-helical structures change differently. The native form remains structured, with three shorter α-helical motifs, connected by random coils, while the synthetic analog tends predominantly to a disordered conformation. Our results indicate the importance of the side-chains at positions 5 and 16 for maintaining the solvated peptide conformation. They also provide a modeling background for recent experimental observations, relating the higher α-helical content in solution (peptide pre-folding) in the case of small linear AMPs to a lower antibacterial activity.

Antibiotic activity is limited by the physical construction of the Gram-negative cell envelope. Species of the Burkholderia cepacia complex (Bcc) are known as intrinsically multidrug-resistant opportunistic pathogens with low permeability cell envelopes. Here, we re-examined a previously performed chemical-genetic screen of barcoded transposon mutants in B. cenocepacia K56-2, focusing on cell envelope structural and functional processes. We identified structures mechanistically important for resistance to singular and multiple antibiotic classes. For example, susceptibility to novobiocin, avibactam, and the LpxC inhibitor, PF-04753299, was linked to the BpeAB-OprB efflux pump, suggesting these drugs are substrates for this pump in B. cenocepacia. Defects in peptidoglycan precursor synthesis specifically increased susceptibility to cycloserine and revealed a new putative amino acid racemase, while defects in divisome accessory proteins increased susceptibility to multiple β-lactams. Additionally, disruption of the periplasmic disulfide bond formation system caused pleiotropic defects on outer membrane integrity and β-lactamase activity. Our findings highlight the layering of resistance mechanisms in the structure and function of the cell envelope. Consequently, we point out processes that can be targeted for developing antibiotic potentiators.IMPORTANCEThe Gram-negative cell envelope is a double-layered physical barrier that protects cells from extracellular stressors, such as antibiotics. The Burkholderia cell envelope is known to contain additional modifications that reduce permeability. We investigated Burkholderia cell envelope factors contributing to antibiotic resistance from a genome-wide view by re-examining data from a transposon mutant library exposed to an antibiotic panel. We identified susceptible phenotypes for defects in structures and functions in the outer membrane, periplasm, and cytoplasm. Overall, we show that resistance linked to the cell envelope is multifaceted and provides new targets for the development of antibiotic potentiators.

Continuous monitoring of mitochondrial membrane potential in hepatocyte cell suspensions

A single cavitation microbubble can transiently disrupt the cellular membrane, providing a chemical-free, targeted drug delivery mechanism. Here, we investigate the spatiotemporal dynamics of membrane permeabilization in an Escherichia coli (E. coli) monolayer exposed to a single cavitation event. Using high-resolution fluorescence microscopy and propidium iodide (PI) uptake as a marker of membrane disruption, we tracked the response of 5565 individual cells around the center of the cavitation event over timescales from microseconds to minutes and spatial scales from 1 to 165 µm. PI uptake rates exhibited a strong spatial dependence, with cells closer to the cavitation center showing rapid and extensive permeabilization. A modified Goldman equation describing PI concentrations inside and outside the cells was used and related to the spatiotemporal measurements of fluorescence intensity. The model accurately captured the first-order PI uptake kinetics, which resulted in saturated fluorescence intensity profiles. Additionally, the model predicted an exponential decay of permeability post-cavitation, implicitly suggesting that pore-resealing dynamics were taking place. Membrane permeability decreased with distance as 1/r, with a characteristic decay time of approximately 3.4 min. Our model thus predicts cell damage induced by a single cavitation event in both space and time. In the present case, where the cavitation bubble reached maximum radius of 29.7 µm, we found that at a distance of 11 µm from the cavitation center, ~ 50% of cell membranes are damaged and permeable to PI, but a pore-resealing mechanism reduces this damage to ~ 1% after 10 min. Our results are consistent with existing sonoporation studies and offer novel insights for optimizing cavitation-assisted drug delivery and biofilm disruption strategies.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-26554-4.

Magainin-2 (MAG2) is a polycationic antimicrobial peptide isolated from the skin of the African clawed frog Xenopus laevis. It has a wide spectrum of antimicrobial activities against gram-positive and gram-negative bacteria, fungi, and induces osmotic lysis of protozoa. MAG2 also possesses antiviral and antitumoral properties. These activities make this peptide a promising candidate for therapeutic applications. Recombinant expression systems are necessary for the affordable production of large amounts of the biologically active peptide. In this work, MAG2 has been cloned to the N-terminal of a family III carbohydrate-binding module fused to the linker sequence (LK-CBM3) from Clostridium thermocellum; a formic acid recognition site was introduced between the two modules for chemical cleavage of the peptide. The recombinant protein MAG2-LK-CBM3 was expressed in Escherichia coli BL21 (DE3) and MAG2 was successfully cleaved and purified from the fusion partner LK-CBM3. Its functionality was confirmed by testing its activity against gram-negative bacteria.

In this study we elucidated the effects of berberine, a major alkaloid component contained in medicinal herbs, such as Phellodendri Cortex and Coptidis Rhizoma, on ischemic neuronal damage in mouse organotypic hippocampal slice cultures (OHSCs) caused by oxygen and glucose deprivation (OGD) and N-methyl-D-aspartate (NMDA) -type glutamate receptor stimulation. Hippocampal slices obtained from 7-d-old ICR mice were cultured for 10 d before the experiments. Ischemia-related damage was induced by OGD (5, 15, 45 min) or NMDA (10 microM) treatment, and was evaluated by measuring propidium iodide (PI) uptake. Levels of apoptotic marker proteins, B-cell lymphoma 2 (Bcl-2) and phosphorylated-Bcl-2 (p-Bcl-2), in the OHSCs were measured as indices of biochemical neuronal cell damage by Western blotting. Berberine (5, 25 microM) or the NMDA antagonist MK-801 (25 microM) was added to the medium 30 min before OGD or NMDA treatment. OGD time-dependently increased PI uptake of the OHSCs. Both berberine (5, 25 microM) and MK-801 (25 microM) significantly inhibited PI uptake at 24 h after 45-min OGD treatment and PI uptake in OHSCs exposed to NMDA for 24 h. OGD treatment also significantly increased the level of p-Bcl-2 but not that of Bcl-2 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in OHSCs. Berberine (5-25 microM) significantly suppressed the OGD-induced increase of p-Bcl-2 level in OHSCs when tissue was exposed to the alkaloid prior to OGD or simultaneously with OGD. These findings suggest that berberine has protective effects against ischemic damage in mouse OHSCs and that the effects are at least partly mediated by suppression of Bcl-2 phosphorylation.

Neuroprotective effects of erythropoietin against sevoflurane-induced neuronal apoptosis in primary rat cortical neurons involving the EPOR-Erk1/2-Nrf2/Bach1 signal pathway.

A prostacyclin analog beraprost sodium attenuates radiocontrast media-induced LLC-PK1 cells injury

The main goal of this study was to develop a straightforward and rapid microplate assay for measuring propidium iodide (PI) in C2C12 cells. The PI method has proven to be an efficient quantitative assay for analyzing cell viability through PI fluorescence analysis. Importantly, the protocol takes less than 30 min and the results are reproducible. C2C12 cells were exposed to an increasing concentration of palmitate for a period of 24 h to induce cell death, and the PI fluorescence increased in a concentration-dependent manner. Evaluation of mitochondrial function and the production of reactive oxygen species confirmed the deleterious effects of palmitate. Also, the microplate PI assay demonstrated high sensitivity, as indicated by the detection of modest fluctuations in cell viability in response to catalase overexpression in palmitate-treated cells. The microplate PI assay, therefore, offers an accurate method for use in in-vitro studies.