Model Bacterial Membranes Research Articles

Effect of Glycone Diversity on the Interaction of Triterpenoid Saponins and Lipid Bilayers.

Triterpenoid saponins are organic compounds widely available in the plant kingdom. These molecules have received extensive attention due to their antibacterial activity against both Gram-negative and Gram-positive bacteria. Recent studies identified the antibacterial activity of saponins closely relates to their interaction with bacterial membrane lipids; however, molecular details of this interaction remain unclear. Increased understanding of the mechanisms to disrupt bacterial lipid bilayers can help to mitigate development of antibiotic resistance. Here, we examined the effect of chemical structure and deprotonation states of saponin on its interaction with a bacterial membrane model using molecular dynamics simulations. We run multiple simulations with a ternary lipid mixture of POPE/POPG/DPPG (80/15/5 mol %) and different saponin molecules. While all saponin structures can permanently bind the membrane, their location and orientation inside the bilayer depend on the sugar chains attached to their backbone. Similarly, cluster formation and stability also depend on the chemical structure of the saponin molecule. Deprotonation site affects interactions with the bilayer by modulating hydrophilicity of the molecules. At the low concentrations simulated in this work, there is no statistically significant change in the membrane properties upon saponin(s) binding, but the molecules do preferentially partition to POPE lipid environment.

The Mechanism of Action of SAAP-148 Antimicrobial Peptide as Studied with NMR and Molecular Dynamics Simulations

SAAP-148 is an antimicrobial peptide derived from LL-37. It exhibits excellent activity against drug-resistant bacteria and biofilms while resisting degradation in physiological conditions. Despite its optimal pharmacological properties, its mechanism of action at the molecular level has not been explored. The structural properties of SAAP-148 and its interaction with phospholipid membranes mimicking mammalian and bacterial cells were studied using liquid and solid-state NMR spectroscopy as well as molecular dynamics simulations. SAAP-148 is partially structured in solution and stabilizes its helical conformation when interacting with DPC micelles. The orientation of the helix within the micelles was defined by paramagnetic relaxation enhancements and found similar to that obtained using solid-state NMR, where the tilt and pitch angles were determined based on 15N chemical shift in oriented models of bacterial membranes (POPE/POPG). Molecular dynamic simulations revealed that SAAP-148 approaches the bacterial membrane by forming salt bridges between lysine and arginine residues and lipid phosphate groups while interacting minimally with mammalian models containing POPC and cholesterol. SAAP-148 stabilizes its helical fold onto bacterial-like membranes, placing its helix axis almost perpendicular to the surface normal, thus probably acting by a carpet-like mechanism on the bacterial membrane rather than forming well-defined pores.

Interaction of antibacterial CM11 peptide with the gram-positive and gram-negative bacterial membrane models: a molecular dynamics simulations study

Interaction of antibacterial CM11 peptide with the gram-positive and gram-negative bacterial membrane models: a molecular dynamics simulations study

Biophysical investigations of antimicrobial peptide mimics for mechanistic studies.

With a relentless rise in the growth of antimicrobial resistance, much attention is being focused on Antimicrobial peptides (AMP) and the development of AMP mimicking agents to combat this nefarious issue. However lesser reports come up with adequate reasoning behind the driving force of the membrane perturbing nature. Here, we shall be discussing the two branches of AMP mimics, peptoids and small molecules. Further, we explore the mode of action behind the antimicrobial properties displayed by the library of peptoids and small molecule mimics. To address this big challenge of understanding how the membrane active compound behaves, each of the compounds were introduced to the liposomes, mimicking the bacterial model membranes, and their interactions was recorded using solid state nuclear magnetic resonance spectroscopy. We observe that each of the mimics interact with the liposome as the global shape of the vesicles gets deformed. Also we observed a definite interaction with the fatty acyl chain as changes in order parameters were observed with respect to that of pure lipids. To develop a thorough understanding of the pore forming abilities of these compounds, a fluorescent dye release studies was executed using both bacterial and mammalian model membranes. The calcein dye release test revealed that the small molecule mimics undergo a cooperative effect. For the peptoids investigated, the extent of release could be correlated with their antimicrobial activity as well as the toxicity data.

Metadynamics study of benzyltrimethylammonium binding free energy to EmrE protein in all atom molecular dynamics simulations.

EmrE is a membrane protein found in bacteria such as E. coli that transports positively charged, aromatic toxins outside the cell. It is a parallel dimer and belongs to the small multidrug resistance (SMR) family. Although it is very small, 110 amino acids per monomer, its structure was not resolved at high resolution until recently. The toxin transport is associated with an alternating access movement of the protein monomers. The toxin binds to the protein while the two GLU14 residues of the EmrE are deprotonated in the high pH environment, and is released on the other side when a GLU14 is protonated in low pH. We use metadynamics to calculate the free energy of the binding path of benzyltrimethylammonium to EmrE in a model bacterial membrane. Two geometric reaction coordinates were chosen for the metadynamics runs, distance between center of mass of toxin and the binding site, the angle of the toxin plane with the z-axis. Our results indicate that the free energy of benzyltrimethylammonium binding to EmrE through a water-only path is about −40 kJ/mol. We have also used funnel metadynamics to observe the effect of lipid interaction with the toxin to the binding free energy.

Coarse grained antimicrobial coacervated nanoparticle dynamics.

Colistin is a potent polycationic polymyxin antibiotic that sees wide use topically for multidrug resistant Gram-negative bacteria. Its ability to break down the membrane is a double edged sword due to the non-selective nature of its mode of action. Ingestible colistin is a last resort against severe infections, but recent experimental work has shown there promise when it is injected in nanoparticle form with other constituents. Compared with free colisitin, the nanoparticle showed either retained or enhanced efficacy and a lower hepatotoxicity in mice. This work explores the coarse-grained dynamics of this system and its mode of action with model bacterial membranes, as well as the structure of the amorphous nanoparticle coacervate.

Clustering and binding of oleanolic acid saponins with bacterial membranes.

Oleanolic acid saponins are the most common and important triterpenoid saponins in the plant kingdom. They have received extensive attention for their potential medicinal value. Recent studies showed that their biological activities are closely mediated by interaction with lipid bilayers. Previous studies characterized the effect of hydrophilicity on the interactions of saponin with a bacterial membrane model. The amphiphilic nature of these compounds easily drives the formation of large clusters. On the other hand, some saponins contain carboxyl groups, which can be deprotonated at physiological pH. Both aggregation and deprotonation affect saponin-bilayer interactions. To study these interactions, we used molecular dynamics (MD) simulations to probe the effect of chemical structure and concentration on the interaction of four model saponins with a lipid bilayer. Cluster size and hydrophilicity directly impact the degree of binding and insertion of saponin into the bilayer. Large clusters can bind the bilayer and remain intact at the membrane interface in the remaining of the simulation; whereas hydrophobic interactions with the membrane interior play a key role in the insertion and dissociation of small saponin clusters. In addition, we examined the effect of deprotonation of carboxyl groups at different positions in the saponin molecules, which significantly changes their hydrophilicity and interaction mechanism with the lipid bilayer. Taken together, these results increase our understanding of saponin-lipid interactions that are relevant in the study of lipid membrane disruption and rupture as it pertains to antibacterial treatments.

Rational design of magnetoliposomes for enhanced interaction with bacterial membrane models

There is a growing need for alternatives to target and treat bacterial infection. Thus, the present work aims to develop and optimize the production of PEGylated magnetoliposomes (MLPs@PEG), by encapsulating superparamagnetic iron oxide nanoparticles (SPIONs) within fusogenic liposomes. A Box–Behnken design was applied to modulate size distribution variables, using lipid concentration, SPIONs amount and ultrasonication time as independent variables. As a result of the optimization, it was possible to obtain MLPs@PEG with a mean size of 182 nm, with polydispersity index (PDI) of 0.19, and SPIONs encapsulation efficiency (%EE) around 76%. Cytocompatibility assays showed that no toxicity was observed in fibroblasts, for iron concentrations up to 400μg/ml. Also, for safe lipid and iron concentrations, no hemolytic effect was detected. The fusogenicity of the nanosystems was first evaluated through lipid mixing assays, based on Förster resonance energy transfer (FRET), using liposomal membrane models, mimicking bacterial cytoplasmic membrane and eukaryotic plasma membrane. It was shown that the hybrid nanosystems preferentially interact with the bacterial membrane model. Confocal microscopy and fluorescence lifetime measurements, using giant unilamellar vesicles (GUVs), validated these results. Overall, the developed hybrid nanosystem may represent an efficient drug delivery system with improved targetability for bacterial membrane.

Modeling the Effect of Hydrophobicity on the Passive Permeation of Solutes across a Bacterial Model Membrane.

Passive diffusion across biomembranes is an important mechanism of permeation for multiple drugs, including antibiotics. However, this process is frequently neglected while studying drug uptake and, in our view, warrants further investigation. Here, we apply molecular dynamics simulations to investigate the impact of changes in molecular hydrophobicity on the permeability of a series of inhibitors of the quorum sensing of Pseudomonas aeruginosa, previously discovered by us, across a membrane model. Overall, we show that permeation across this membrane model does not correlate with the molecule's hydrophobicity. We demonstrate that using a simple model for permeation, based on the difference between the maximum and minimum of the free energy profile, outperforms the inhomogeneous solubility-diffusion model, yielding a permeability ranking that better agrees with the experimental results, especially for hydrophobic permeants. The calculated differences in permeability could not explain differences in in bacterio activity. Nevertheless, substantial differences in molecular orientation along the permeation pathway correlate with the in bacterio activity, emphasizing the importance of analyzing, at an atomistic level, the permeation pathway of these solutes.

Solid and Liquid Surface-Supported Bacterial Membrane Mimetics as a Platform for the Functional and Structural Studies of Antimicrobials.

Increasing antibiotic resistance has provoked the urgent need to investigate the interactions of antimicrobials with bacterial membranes. The reasons for emerging antibiotic resistance and innovations in novel therapeutic approaches are highly relevant to the mechanistic interactions between antibiotics and membranes. Due to the dynamic nature, complex compositions, and small sizes of native bacterial membranes, bacterial membrane mimetics have been developed to allow for the in vitro examination of structures, properties, dynamics, and interactions. In this review, three types of model membranes are discussed: monolayers, supported lipid bilayers, and supported asymmetric bilayers; this review highlights their advantages and constraints. From monolayers to asymmetric bilayers, biomimetic bacterial membranes replicate various properties of real bacterial membranes. The typical synthetic methods for fabricating each model membrane are introduced. Depending on the properties of lipids and their biological relevance, various lipid compositions have been used to mimic bacterial membranes. For example, mixtures of phosphatidylethanolamines (PE), phosphatidylglycerols (PG), and cardiolipins (CL) at various molar ratios have been used, approaching actual lipid compositions of Gram-positive bacterial membranes and inner membranes of Gram-negative bacteria. Asymmetric lipid bilayers can be fabricated on solid supports to emulate Gram-negative bacterial outer membranes. To probe the properties of the model bacterial membranes and interactions with antimicrobials, three common characterization techniques, including quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR), and neutron reflectometry (NR) are detailed in this review article. Finally, we provide examples showing that the combination of bacterial membrane models and characterization techniques is capable of providing crucial information in the design of new antimicrobials that combat bacterial resistance.

Membrane fluidity, composition, and charge affect the activity and selectivity of the AMP ascaphin-8

Ascaphins are cationic antimicrobial peptides that have been shown to have potential in the treatment of infectious diseases caused by multidrug-resistant pathogens (MDR). However, to date, their principal molecular target and mechanism of action are unknown. Results from peptide prediction software and molecular dynamics simulations confirmed that ascaphin-8 is an alpha-helical peptide. For the first time, the peptide was described as membranotrophic using biophysical approaches including calcein liposome leakage, Laurdan general polarization, and dynamic light scattering. Ascaphin-8’s activity and selectivity were modulated by rearranging the spatial distribution of lysine (Var-K5), aspartic acid (Var-D4) residues, or substitution of phenylalanine with tyrosine (Var-Y). The parental peptide and its variants presented high affinity toward the bacterial membrane model (≤2 μM), but lost activity in sterol-enriched membranes (mammal and fungal models, with cholesterol and ergosterol, respectively). The peptide-induced pore size was estimated to be >20 nm in the bacterial model, with no difference among peptides. The same pattern was observed in membrane fluidity (general polarization) assays, where all peptides reduced membrane fluidity of the bacterial model but not in the models containing sterols. The peptides also showed high activity toward MDR bacteria. Moreover, peptide sensitivity of the artificial membrane models compared with pathogenic bacterial isolates were in good agreement.

Formation of a Fully Anionic Supported Lipid Bilayer to Model Bacterial Inner Membrane for QCM-D Studies.

Supported lipid bilayers (SLBs) on quartz crystals are employed as versatile model systems for studying cell membrane behavior with the use of the highly sensitive technique of quartz crystal microbalance with dissipation monitoring (QCM-D). Since the lipids constituting cell membranes vary from predominantly zwitterionic lipids in mammalian cells to predominantly anionic lipids in the inner membrane of Gram-positive bacteria, the ability to create SLBs of different lipid compositions is essential for representing different cell membranes. While methods to generate stable zwitterionic SLBs and zwitterionic-dominant mixed zwitterionic–anionic SLBs on quartz crystals have been well established, there are no reports of being able to form predominantly or fully anionic SLBs. We describe here a method for forming entirely anionic SLBs by treating the quartz crystal with cationic (3-aminopropyl) trimethoxysilane (APTMS). The formation of the anionic SLB was tracked using QCM-D by monitoring the adsorption of anionic lipid vesicles to a quartz surface and subsequent bilayer formation. Anionic egg L-α-phosphatidylglycerol (PG) vesicles adsorbed on the surface-treated quartz crystal, but did not undergo the vesicle-to-bilayer transition to create an SLB. However, when PG was mixed with 10–40 mole% 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) (LPG), the mixed vesicles led to the formation of stable SLBs. The dynamics of SLB formation monitored by QCM-D showed that while SLB formation by zwitterionic lipids followed a two-step process of vesicle adsorption followed by the breakdown of the adsorbed vesicles (which in turn is a result of multiple events) to create the SLB, the PG/LPG mixed vesicles ruptured immediately on contacting the quartz surface resulting in a one-step process of SLB formation. The QCM-D data also enabled the quantitative characterization of the SLB by allowing estimation of the lipid surface density as well as the thickness of the hydrophobic region of the SLB. These fully anionic SLBs are valuable model systems to conduct QCM-D studies of the interactions of extraneous substances such as antimicrobial peptides and nanoparticles with Gram-positive bacterial membranes.

An Antimicrobial Peptide-Mimetic Methacrylate Random Copolymer Induces Domain Formation in a Model Bacterial Membrane.

To address the emerging issue of drug-resistant bacteria, membrane-active synthetic polymers have been designed and developed to mimic host-defense antimicrobial peptides (AMPs) as antibiotic alternatives. In this study, we investigated the domain formation induced by synthetic polymer mimics of AMPs using model membranes to elucidate the biophysical principles that govern their membrane-active mechanisms. To that end, lipid vesicles mimicking Escherichia coli(E. coli) membrane were prepared using an 8:2 (molar ratio) mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), sodium salt (POPG). Our studies using differential scanning calorimetry (DSC) and fluorescence microscopy indicated that cationic amphiphilic methacrylate random copolymers induced the phase separation to form POPE- or POPG-rich domains. A rhodamine-labeled polymer also showed the binding to separated domains in the membrane. Based on these results, we propose the mechanism that the copolymers induce domain formation by clustering of anionic POPG lipids similar to natural AMPs. In addition, the time-course of polymer binding to the GUV membrane was sigmoidal, suggesting the positive feedback loop in the membrane binding. We also hypothesize that this cooperative binding of the polymer is driven by the domain formation. This study demonstrates the potential of the amphiphilic copolymers to modulate the lipid organization of cell membranes, which may provide a new strategy to design membrane-active antimicrobial agents.

Antimicrobial peptides derived from the SPLUNC1 protein perturb bacterial and eukaryotic lipid model membranes

SPLUNC1 (short palate lung and nasal epithelial clone 1) is a multifunctional host defense protein found in the human respiratory tract that possesses antimicrobial properties. Through modification of the 4th alpha helical motif of the protein, five novel antimicrobial peptides (AMPs) were designed with different lengths, charges, hydrophobicities (H) and hydrophobic moments (μH). The goal of this study was to compare the biological activities of these AMPs by means of testing against paired clinical isolates of the Gram-negative (G(-)) bacteria Klebsiella pneumoniae, obtained from twelve patients before and after colistin treatments (half susceptible and half resistant to colistin).

Mechanical rigidity of bacterial membrane models: effects of lipids and peptides probed by MD

Antimicrobial peptides (AMPs) are membrane-targeting short sequence peptides that have potential for the development of new antibiotics. AMPs are generally cationic, and target negatively charged bacterial membranes, resulting in their disruption. The multiple peptide resistance factor (MprF) protein reduces AMP binding to bacterial membranes via the conversion of anionic phosphatidylglycerol (PG) lipids into either zwitterionic Alanyl-phosphatidylglycerol (Ala-PG) or cationic Lysyl-phosphatidylglycerol (Lys-PG) lipids, reducing the negative surface charge present on the membrane.

Simulation study of domain formation in a model bacterial membrane.

Recent experimental studies revealed that functional membrane microdomains (FMMs) are formed in prokaryotic cells which are structurally and functionally similar to the lipid rafts formed in eukaryotic cells. In this study, we employ coarse-grained molecular dynamics simulations to investigate the mechanism of domain formation and its physiochemical properties in a model methicillin-resistant staphylococcus aureus (MRSA) cell membrane. We find that domains are formed through lateral segregation of staphyloxanthin (STX), a carotenoid which shields the bacteria from the host's immune because of its antioxidant nature. Simulation results suggest that membrane integrity increases with the size of the domain, which is assessed by computing bond order parameter of the lipid tails, membrane expansion modulus and water permeability across the membrane. Various membrane domain proteins such as flotillin-like protein floA and penicillin binding protein (PBP2a) preferentially bind with the STX and accumulate in the membrane domain which is consistent with the recent experimental results.

Hyaluronic Acid Derivative Molecular Weight-Dependent Synthesis and Antimicrobial Effect of Hybrid Silver Nanoparticles.

Silver nanoparticles (Ag NPs) appeared as promising antimicrobial candidates to face the development of antibiotic resistance. Although reported as toxic towards mammalian cells, their combination with biomolecules have shown reduced toxicity, while maintaining the antimicrobial function. Herein, hyaluronic acid (HA) with low (40 kDa), medium (200 and 600 kDa) and high (2 MDa) molecular weight (Mw) was modified with adipic acid dihydrazide (ADH) and used as reducing and capping agents to synthesise antimicrobial hybrid Ag NPs. The Mw of the polymer played a crucial role in the morphology, size and antibacterial activity of the Ag NPs. The 600 and 200 kDa HA-ADH-Ag NPs were able to reduce the Escherichia coli and Staphylococcus aureus concentration by more than 3 logs, while the 40 kDa NPs reached ~2 logs reduction. The 2 MDa HA-ADH failed to form homogenous NPs with strong bactericidal activity. A mechanistic study of the interaction with a model bacterial membrane using Langmuir isotherms confirmed the greater interaction between bacteria and higher Mw polymers and the effect of the NP’s morphology. The nanocomposites low toxicity to human skin cells was demonstrated in vitro, showing more than 90% cell viability after incubation with the NPs.

The studies on the membrane activity of triester of phosphatidylcholine in artificial membrane systems.

Due to the increasing number of infections together with the appearance of bacteria exhibiting multi-drug resistance, new antibiotics are being sought. In this context the interest of the cationic lipoids increases because of their amphiphilic structure and positive charge that can stimulates the antibacterial action of these compounds. Thus, in this work we have performed the studies on the effect of one selected triesters of phosphatidylcholine, namely 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (EDPPC), on the model lipid membranes. The investigations included the analysis of the impact of EDPPC on multicomponent monolayers and bilayers consisting of the lipids naturally occurring in bacterial membranes (phosphatidylethanolamines (PE), phosphatidylglycerols (PG) and cardiolipin (CL)), mixed in proportions reflecting the lipid composition of these biomembranes. In the study, the Langmuir monolayers (registered on water and PBS buffer) and liposomes as model bacterial biomembranes were applied. The obtained results demonstrate that the presence of cationic lipoid in PE/PG and PE/PG/CL systems significantly modifies their properties and molecular organization. The incorporation of EDPPC into model bacterial membranes primarily impact on the intermolecular interactions. It was shown that the strength of the interaction between the cationic lipid and the components of the model membranes depends both on the composition of the membrane as well as on the type of subphase. Furthermore, the investigated cationic lipoid leads to the decrease of the ordering of acyl chains and thus to the increase of fluidity of membranes. The obtained results allow one to propose that EDPPC may behave as antibiotic active at the level of membrane.

Cardiolipin prevents pore formation in phosphatidylglycerol bacterial membrane models.

Several antimicrobial peptides, including magainin and the human cathelicidin LL-37, act by forming pores in bacterial membranes. Bacteria such as Staphylococcus aureus modify their membrane's cardiolipin composition to resist such types of perturbations that compromise their membrane stability. Here, we used molecular dynamic simulations to quantify the role of cardiolipin on the formation of pores in simple bacterial-like membrane models composed of phosphatidylglycerol and cardiolipin mixtures. Cardiolipin modified the structure and ordering of the lipid bilayer, making it less susceptible to mechanical changes. Accordingly, the free-energy barrier for the formation of a transmembrane pore and its kinetic instability augmented by increasing the cardiolipin concentration. This is attributed to the unfavorable positioning of cardiolipin near the formed pore, due to its small polar head and bulky hydrophobic body. Overall, our study demonstrates how cardiolipin prevents membrane-pore formation and this constitutes a plausible mechanism used by bacteria to act against stress perturbations and, thereby, gain resistance to antimicrobial agents.

Chitosan effects on monolayers of zwitterionic, anionic and a natural lipid extract from E. coli at physiological pH

Langmuir monolayers are used to simulate the biological membrane environment, acting as a mimetic system of the outer or the inner membrane leaflet. Herein, we analyze the interaction of membrane models with a partially N-acetylated chitosan (Ch35%) possessing a quasi-ideal random pattern of acetylation, full water solubility up to pH ≈ 8.5 and unusually high weight average molecular weight. Lipid monolayers containing dipalmitoyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl ethalonamine (DPPE), dipalmitoyl phosphatidyl glycerol (DPPG) or E. coli total lipid extract were spread onto subphases buffered at pH 4.5 or 7.4. The incorporation of Ch35% chitosan caused monolayer expansion and a general trend of decreasing monolayer rigidity with Ch35% concentration. Due to its relatively high content of N-acetylglucosamine (GlcNAc) units, Ch35% interactions with negatively charged monolayers and with E. coli extract were weaker than those involving zwitterionic monolayers or lipid rafts. While the smaller interaction with negatively charged lipids was unexpected, this finding can be attributed to the degree of acetylation (35%) which imparts a small number of charged groups for Ch35% to interact. Chitosan properties are therefore determinant for interactions with model cell membranes, which explains the variability in chitosan bactericide activity in the literature. This is the first study on the effects from chitosans on realistic models of bacterial membranes under physiological pH.