Carpet Model Research Articles

The Mechanism of Membrane Disruption by Cytotoxic Amyloid Oligomers Formed by Prion Protein(106–126) Is Dependent on Bilayer Composition

The formation of fibrillar aggregates has long been associated with neurodegenerative disorders such as Alzheimer and Parkinson diseases. Although fibrils are still considered important to the pathology of these disorders, it is now widely understood that smaller amyloid oligomers are the toxic entities along the misfolding pathway. One characteristic shared by the majority of amyloid oligomers is the ability to disrupt membranes, a commonality proposed to be responsible for their toxicity, although the mechanisms linking this to cell death are poorly understood. Here, we describe the physical basis for the cytotoxicity of oligomers formed by the prion protein (PrP)-derived amyloid peptide PrP(106-126). We show that oligomers of this peptide kill several mammalian cells lines, as well as mouse cerebellar organotypic cultures, and we also show that they exhibit antimicrobial activity. Physical perturbation of model membranes mimicking bacterial or mammalian cells was investigated using atomic force microscopy, polarized total internal reflection fluorescence microscopy, and NMR spectroscopy. Disruption of anionic membranes proceeds through a carpet or detergent model as proposed for other antimicrobial peptides. By contrast, when added to zwitterionic membranes containing cholesterol-rich ordered domains, PrP(106-126) oligomers induce a loss of domain separation and decreased membrane disorder. Loss of raft-like domains may lead to activation of apoptotic pathways, resulting in cell death. This work sheds new light on the physical mechanisms of amyloid cytotoxicity and is the first to clearly show membrane type-specific modes of action for a cytotoxic peptide.

Sonochemically Processed Cationic Nanocapsules: Efficient Antimicrobials with Membrane Disturbing Capacity

Bacterial-mediated diseases are a major healthcare concern worldwide due to the rapid spread of antibiotic-resistant bacteria. One strategy to manage the bacterial infections while avoiding the emergence of resistant strains implies specific targeting and disruption of bacteria membranes. This work evaluates the potential of nanostructured biopolymer derivatives, nanocapsules (NCs), to disrupt the bacteria cell walls and effectively kill planktonic microorganisms. Two biopolymers, chitosan and cellulose, were chemically modified to synthesize derivatives with improved cationic character (thiolated chitosan and aminocellulose) prior to their processing into nanocapsules via a one-step sonochemical process. The interactions of NCs, displaying an average size of around 250 nm, with bacteria membrane were evaluated using two membrane models: Langmuir monolayers and liposome bilayers composed of a l-α-phosphatidylglycerol phospholipid extracted from Escherichia coli. NCs possessed improved membrane disturbing capacity in comparison to the nonprocessed biopolymer derivatives, by drastically increasing the monolayer fluidity and inducing more than 50% leakage of a dye inserted in the bilayered liposomes. In addition, membrane disturbance was directly proportional to the NCs cationic charge. Whereas evidence showed that thiolated chitosan and aminocellulose interacted with the bacteria membrane through a "carpet model", the NCs were found to induce larger surface defects and high local perturbance through a "detergent model". Importantly, the degree of disruption caused by the biopolymer derivatives and NCs correlated well with the antimicrobial capacity against Escherichia coli, selectively killing bacteria cells without imparting toxicity to human fibroblasts.

Bayesian Total Internal Reflection Fluorescence Correlation Spectroscopy Reveals hIAPP-Induced Plasma Membrane Domain Organization in Live Cells

Amyloid fibril deposition of human islet amyloid polypeptide (hIAPP) in pancreatic islet cells is implicated in the pathogenesis of type II diabetes. A growing number of studies suggest that small peptide aggregates are cytotoxic via their interaction with the plasma membrane, which leads to membrane permeabilization or disruption. A recent study using imaging total internal reflection-fluorescence correlation spectroscopy (ITIR-FCS) showed that monomeric hIAPP induced the formation of cellular plasma membrane microdomains containing dense lipids, in addition to the modulation of membrane fluidity. However, the spatial organization of microdomains and their temporal evolution were only partially characterized due to limitations in the conventional analysis and interpretation of imaging FCS datasets. Here, we apply a previously developed Bayesian analysis procedure to ITIR-FCS data to resolve hIAPP-induced microdomain spatial organization and temporal dynamics. Our analysis enables the visualization of the temporal evolution of multiple diffusing species in the spatially heterogeneous cell membrane, lending support to the carpet model for the association mode of hIAPP aggregates with the plasma membrane. The presented Bayesian analysis procedure provides an automated and general approach to unbiased model-based interpretation of imaging FCS data, with broad applicability to resolving the heterogeneous spatial-temporal organization of biological membrane systems.

Mechanistic Aspects of Peptide-Membrane Interactions Determined by Optical, Dielectric and Piezoelectric Techniques: An Overview

Antimicrobial peptides (AMPs) have been isolated from a wide variety of organisms that include microorganisms, plants, insects, frogs and mammals. As part of the innate immune system expressed in many tissues, AMPs are able to provide protection against invasion of foreign microorganisms and exhibit a broad spectrum of activity against bacteria, fungi and/or virus. Non-AMPs cell-penetrating peptides have been used as carriers for overcoming the membrane barrier and helping in the delivery of various molecules into the cell. Physicochemical peptide-lipid interactions studies can provide us with reliable molecular information about microbe defense response, including the elucidation of the prevailing mechanisms of its action, such as the barrel-stave, toroidal pore, carpet and detergent-like models. In this paper, we present an overview of the peptide-lipid mechanisms of interaction as well as discuss alternative techniques that could help to elucidate the peptides functionality. Quartz crystal microbalance (QCM), surface plasmon resonance (SPR) spectroscopy and electrochemical impedance spectroscopy (EIS) are useful techniques to investigate in details of the peptide-membrane interaction. The techniques here discussed could also offer specific and low-cost methods that can to shed some light over the different modes of action of AMPs, contributing to the development of drugs against infectious diseases.

Investigating the Mechanism of Action of Antimicrobial Piscidin in Bacterial and Mammalian Lipid Membrane Mimics using Oriented Circular Dichroism

Antimicrobial peptides (AMPs) are a class of amphiphilic, often cationic peptides, which are able to target and permeabilize the lipid membranes of pathogenic microbes. AMPs are often studied for their potential as novel pharmaceuticals against bacteria, viruses, and even some cancers. Many possible mechanisms for their action have been proposed, but it is believed that at low peptide:lipid ratios (P/L), peptides bind parallel to the membrane surface, known as the “S-state,” while at high P/L, peptides insert into the membrane and bind in the “I-state” perpendicular to the membrane surface. It is proposed that peptides in the I-state form pores, while peptides that remain in the S-state use the carpet model of membrane disruption.Here, OCD is used to investigate the orientation and threshold concentration of Piscidin 1 and Piscidin 3 (P3) in bacterial and mammalian-mimicking lipid systems and thereby determine the mechanism of action of P1 and P3 in these lipid systems. Both peptides are a 22-residue alpha-helical AMPs isolated from the mast cells of hybrid striped sea bass. P1 is both more antimicrobial and hemolytic than P3. We hypothesize that the two peptides behave differently in bacterial versus human cells due to the differences in membrane composition and that P1 initiates its activity at a lower threshold concentration. Mammalian and bacterial membrane mimics have been made using 4:1 PC/CHL (phosphocholine and cholesterol, respectively) and 3:1 PC/PG (phosphoglycerol). The bilayer orientations of piscidin have been investigated over a large range of P/L ratios using OCD. Membrane thinning was studied by x-ray. These studies provide insight into the mechanism of action of an important class of AMPs and may help provide design principles for new drug candidates.

Update of Peptides with Antibacterial Activity

For many years a battle has been going on between bacteria and humans, with bacteria trying to survive against the antibiotics used by humans. Bacteria are found to be dominant in this battle since they can develop resistance. In fact, in the last decade multi-, extended- and pan-drug resistant bacteria have been isolated. On the other hand, the number of new antibiotics approved by the FDA has dramatically decreased during the last 20 years. Therefore, there is a desperate need for developing new types of antibacterial agents, where antimicrobial peptides may play an important role. This review provides an update of the recently identified antimicrobial peptides. Three valid approaches for developing a future antibacterial agent, as are the mechanisms of action as well as the in vitro and in vivo assays have been described in depth. In comparison to the antibacterial agents available at present, the targets for most of the antimicrobial peptides are not well known. However several proposals having been introduced for many antimicrobial peptides of different mechanisms of action, there still lies some uncertainty about their utility. Hundreds of antimicrobial peptides have been tested in vitro against all types of bacteria, but in this review we will highlight only those which have been tested against the most important Grampositive and Gram-negative bacteria. The last step to get a potential antibiotic includes studies with an in vivo model. Therefore only antimicrobial peptides with good activity are tested that have been described in this review. Keywords: Antimicrobial peptides, mechanism of action, barrel model, toroidal model, aggregate model, carpet model, “in vitro” activity, “in vivo” activity, melittin, indolicidin, cecropin A, magainin II, pexiganan, buforin

The Density Functions of Pore-Size and Particle-Size Distribution Established by Using Fractal Theory and their Applications

Based on the Sierpinski carpet and Menger sponge models, the density functions of pore-size and particle-size distribution are put forward. Using the proposed density functions, expressions of soil porosity and grain mass are obtained. Using the expression of soil porosity, the porosity of soil sample is correctly predicted. The expression of grain mass is used to predict the mass of grains. The results show that the mass predictions of grains of size smaller than 0.002mm are very close to the measure results.

Lactoferrin-Derived Antimicrobial Peptide Induces a Micellar Cubic Phase in a Model Membrane System

The observation of a micellar cubic phase is reported for a mixture of an antimicrobial peptide from the Lactoferrin family, LFampin 265-284, and a model membrane system of dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (3:1), as derived from small-angle x-ray diffraction (SAXD) measurements. The system shows remarkable thermotropic polymorphism: the peptide disrupts the lipid bilayer, forming a cubic phase of the space group Pm3n (t < 28°C), and as the temperature increases it shows a complex phase behavior (not fully clarified by SAXD). The onset, volume fraction of each phase, and phase parameters are seen to vary with peptide/lipid ratio and temperature. The obtained SAXD data represent the first experimental evidence, to our knowledge, of a micellar cubic phase in the context of antimicrobial peptide/membrane interaction. We propose that the micellization of the membrane according to the carpet model, for long proposed as a possible mechanism of action, can go through the formation of a cubic micellar phase.

Interaction of the cationic peptide bactenecin with mixed phospholipid monolayers at the air–water interface

The initial mechanism by which antimicrobial peptides target microbes occurs via electrostatic interactions; however, the mechanism is not well understood. We investigate the interaction of the antimicrobial peptide bactenecin with a 50:50w:w% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG) phospholipid mixture at the air–water interface with different NaCl concentrations (0.01, 0.05, 0.1, 0.5M) in the subphase. A larger shift of DPPC:DMPG isotherms was obtained for 0.1M salt concentration at lower and higher pressures, demonstrating the influence of the negative charge of DMPG molecules and the screening of the electrostatic interaction by the salt concentration. Raman spectroscopy of monolayers demonstrated the presence of cysteine–cysteine bridges in bactenecin loops. The peptide adsorption in DPPC:DMPG monolayers observed by AFM images suggests a self-assembled aggregation process, starting with filament-like networks. Domains similar to carpets were formed and pore structures were obtained after a critical peptide concentration, according to the carpet model.

Tryptophan orientations in membrane-bound gramicidin and melittin—a comparative linear dichroism study on transmembrane and surface-bound peptides

In the search for methods to study structure and function of membrane-associated proteins and peptides flow linear dichroism, LD, spectroscopy has emerged as a promising technique. Using shear-aligned lipid vesicles, conformations and binding geometries of membrane-bound bio-macromolecules can be assessed. Here we investigate anchoring properties and specific orientations of tryptophan relative to the peptide backbone and to the membrane normal for the model peptides gramicidin and melittin. We have monitored the conformational change associated with the refolding of non-channel gramicidin into its channel form, and quantitatively determined the average orientations of its tryptophan transition moments, suggesting that these residues adopt a well-defined orientation at the membrane interface. An important conclusion regards the structural variation of gramicidin between these two distinct transmembrane forms. Whilst circular dichroism (CD) spectra, as has been reported before, vary strongly between the two forms suggesting their structures might be quite different, the LD results clearly evidence both the peptide backbone orientation and tryptophan side-chain positioning to be very similar. The latter are oriented in accord with what is expected from their role to anchor peptide termini to the membrane surface. The variations in CD could be due to, the in LD observed, minor shifts in mutual orientation and distance between neighbouring tryptophans sensitively determining their exciton interactions. Our data dispute that the non-channel form of membrane-bound gramicidin would be any of the intertwined forms often observed in crystal as the positioning of tryptophans along the peptide axis would not be compatible with the strong interfacial positioning observed here. The general role of tryptophans as interfacial anchors is further assessed for melittin whose conformation shows considerable angular spread, consistent with a carpet model of its mechanism for induced membrane leakage, and a predominantly surface-aligned membrane orientation governed by amphipathic interactions.

A carpet-based mechanism for direct antimicrobial peptide activity against vaccinia virus membranes

Antimicrobial peptides have activity against a wide variety of biological membranes and are an important component of innate immunity in vertebrate as well as invertebrate systems. The mechanisms of action of these peptides are incompletely understood and a number of competing but not necessarily mutually exclusive models exist. In this study we examined the virucidal activity of four peptides, the human cathelicidin derived LL37, Xenopus alanine-substituted Magainin-2 amide, uperin-3.1, and a cecropin-LL37 hybrid against vaccinia virus. The peptides were shown to be differentially virucidal but all were shown to attack the viral envelope, with LL37 being the most effective and uperin-3.1 the least. Density gradient analysis of the treated virions indicated the virus outer membrane was efficiently removed by peptide action and suggests a mechanism of direct virus inactivation that is consistent with the carpet model for peptide-mediated membrane disruption. Interestingly, the least effective peptide uperin-3.1 was equally effective as the others at inducing susceptibility to neutralizing antibody. This suggests that in addition to direct killing by a carpet-based mechanism, the peptides may simultaneously operate a different mechanism that exposes sequestered antigen without membrane removal.

Revealing the Lytic Mechanism of the Antimicrobial Peptide Gomesin by Observing Giant Unilamellar Vesicles

Gomesin (Gm) is a potent cationic antimicrobial peptide from a Brazilian spider. Here we use optical and fluorescence microscopy to study the interaction of Gm, its low active linear analogue, [Ser(2,6,11,15)]-Gm (GmL), and a fluorescent labeled analogue, Gm-Rh, with giant unilamellar vesicles (GUVs) composed of mixtures of the neutral lipid palmitoyloleoyl phosphatidylcholine (POPC) with the negatively charged lipid palmitoyloleoyl phosphatidylglycerol (POPG) or cholesterol, so as to mimic bacterial and mammalian cell membranes, respectively. We observed the effect of injecting a peptide solution with a micropipet close to GUVs. As a result of peptide-lipid interaction, GUVs burst suddenly. Stable pores, which result in leaky vesicles, were not observed. Fluorescence microscopy of Gm-Rh injected on GUVs confirmed the high peptide/lipid affinity. These facts lead us to suggest that Gm and GmL disrupt the membrane via the carpet model. In order to quantify the lytic activity of both peptides against different membrane composition, a solution of GUVs was diluted in increasing concentration of peptides and the fraction of burst GUVs was measured as a function of time. The lytic activity of both peptides was enhanced by the presence of POPG and decreased upon addition of cholesterol. GmL exhibited lower lytic activity as compared to Gm, but this difference vanished at high POPG molar fraction.

Two categories of fractal models of rock and soil expressing volume and size-distribution of pores and grains

Based on the Sierpinski carpet and Menger sponge models, two categories of fractal models of rock and soil which are composed of the volume fractal model of pores, the volume fractal model of grains, pore-size or particle-size distribution fractal models are established and their relations are clarified in this paper. Through comparison and analysis, it is found that previous models can be unified by the two categories of fractal models, so the unified fractal models are formed. Experimental results presented by Katz indicate that the first category of fractal models can be used to express the fractal behavior of sandstone. A scanning electron microscope (SEM) will be used to study the microstructure of soft clay and it will be testified that the fractal behavior of soft clay suits the second category of fractal models.

Mechanisms Of Antimicrobial Peptide Action Determined Using Chemical And Collisional Quenching Assays

Peptide-lipid interactions are pertinent to antimicrobial peptides (AMP) activity, stringency, and selectivity. The ability of AMPs to disrupt the target cell's lipid bilayer has been described using the nonspecific “carpet” model or using models that assume stable transbilayer pores (e.g. barrel stave pores). With the carpet model, there is a strong electrostatic and hydrophobic attraction between peptide and the lipid interfacial groups such that the peptides blanket the cell membrane. Carpet model peptides can then kill the organism by disrupting the lipid bilayer and causing loss of cellular contents. The stable pore model consists of fixed transmembrane structures that rely on an amphipathic amino acid sequence to form membrane-spanning pores. Included with these models, peptides can also induce flip-flop of bilayer lipids or form transient pores. We have developed chemical and collisional quenching assays that help determine the mode of lipid disruption associated with naturally occurring and synthetically designed AMPs. The assays require large unilamellar vesicles (LUVs) with fluorophore-attached lipid head groups in both inner and outer leaflets of the bilayer. Only peptides with stable pore-forming or detergent-like activity allow quenchers access to the inner leaflet. The combination of these quenching assays with leakage experiments and cryo-electron microscopy allows for a more complete description of the mechanism of membrane disruption by peptides.

Pore Forming Properties Of Antimicrobial Peptides In Different Natural Lipid Environment

The biophysical characteristics and the pore formation dynamics of synthetic, naturally occurring peptides forming membrane-spanning channels, were investigated by using cells having high membrane resistance recorded in whole-cell configuration. The peptides were applied to (and removed from) the recorded cells in ≤50 ms that were: isolated photoreceptor rod outer segments (OS) of low vertebrates, their red blood cells (RBC), and human RBC. The OS membrane resistance was ≥5 GΩ once the OS main endogenous conductance was blocked with light; the RBC had lower resistances but high enough to allow the detection of the exogenous current produced by the peptides up to single channel resolution. Surprisingly, the synthetic, naturally occurring alamethicin F50/5 and selected analogs produced current amplitudes once inserted in the OS at least twice the ones in RBC: therefore, the lack of hemolysis does not ensure the lack of side-effects of an antimicrobial peptide. Once in the RBC membrane, the alameticines produced pores whose voltage-dependency and single channel properties were similar to the ones recorded in OS, showing that the pore forms only as a barrel-stave, independently by the lipid environment. Other modes of pore formation were investigated with cecropin-melittin hybrid peptide (Acetyl-KWKLFKKIGAVLKVL-CONH2), that produced reversible membrane permeabilization at concentrations up to 10 μM. This reversibility at such high concentrations is not expected in the case of a carpet model of pore formation, where micellation should be severe. At difference with alamethicines, membrane permeabilization was voltage-independent, repetitive peptide application caused the progressive increase of the steady-state current amplitude, and no discernible single-channel events were detected at low peptide concentrations. Collectively, these results indicate that cecropin-melittin hybrid peptide permeabilize the membrane according to a toroidal model of pore formation (instead of barrel-stave or carpet one).

On the physiology and pathophysiology of antimicrobial peptides.

Antimicrobial peptides (AMP) are a heterogeneous group of molecules involved in the nonspecific immune responses of a variety of organisms ranging from prokaryotes to mammals, including humans. AMP have various physical and biological properties, yet the most common feature is their antimicrobial effect. The majority of AMP disrupt the integrity of microbial cells by 1 of 3 known mechanisms--the barrel-stave pore model, the thoroidal pore model, or the carpet model. Results of growing numbers of descriptive and experimental studies show that altered expression of AMP in various tissues is important in the pathogenesis of several gastrointestinal, respiratory, and other diseases. We discuss novel approaches and strategies to further improve the promising future of therapeutic applications of AMP. The spread of antibiotic resistance increases the importance of developing a clinical role for AMP.

New strategies for novel antibiotics: peptides targeting bacterial cell membranes

Membranes are targets of host defence or antimicrobial peptides, effector molecules of innate immunity that evolved in nature to contend with invaders as an active system of defence. The different physicochemical properties of the lipids found in biological membranes allow antimicrobial peptides to discriminate between bacterial and mammalian cell membranes. Such cationic amphipathic peptides will interact predominantly with negatively charged lipids exposed on the outer leaflet of bacterial cell membranes. The molecular mechanism(s) of membrane rupture mutually depends on the nature of the peptide and membrane lipid composition. Biophysical studies demonstrated a complex behavior in terms of membrane perturbation, which can range from pore formation to micellization (carpet model). Peptides aligned parallel to the membrane surface can induce a quasi-interdigitated structure in the gel phase, while depending on the hydrophobic matching of the lipid bilayer core and the peptide membrane thinning or thickening can be observed in the fluid phase. As a consequence, besides of peptide-lipid pores, formation of peptide-enriched membrane domains and promotion of cubic structures can be observed, which adversely affect membrane integrity and function. A strategy using the membrane damaging properties of these peptides will form the basis for the development of such peptides as potential novel antibiotic drugs.

Pore Formation Induced by an Antimicrobial Peptide: Electrostatic Effects

We investigate the mode of action of Cateslytin, an antimicrobial peptide, on zwitterionic biomembranes by performing numerical simulations and electrophysiological measurements on membrane vesicles. Using this natural β-sheet antimicrobial peptide secreted during stress as a model we show that a single peptide is able to form a stable membrane pore of 1nm diameter of 0.25 nS conductance found both from calculation and electrical measurements. The resulting structure does not resemble the barrel-stave or carpet models earlier predicted, but is very close to that found in the simulation of α-helical peptides. Based on the simulation of a mutated peptide and the effects of small external electric fields, we conclude that electrostatic forces play a crucial role in the process of pore formation.

Effects of arginine density on the membrane-bound structure of a cationic antimicrobial peptide from solid-state NMR

Solid-state NMR spectroscopy is used to determine the membrane-bound topological structure of a cationic β-hairpin antimicrobial peptide in which the number of Arg residues has been halved. The parent peptide, PG-1, was previously found to form transmembrane β-barrels in anionic membranes where the Arg residues complex with the lipid phosphate groups to cause toroidal pore defects in the membrane. In comparison, the charge-attenuated and less active mutant studied here forms β-sheets that lie on the surface of the zwitterionic membrane and only partially insert into the anionic membrane. The mutant also exhibits much looser contact with the lipid headgroups. These results indicate that transmembrane insertion and tight Arg-phosphate association are two important elements for strong antimicrobial activities of this class of peptides. Comparison with other β-hairpin antimicrobial peptides studied so far further suggests a relative potency scale for the various mechanisms of action for the β-sheet family of antimicrobial peptides. The transmembrane insertion-toroidal pore mechanism is the most potent in disrupting the lipid bilayer, followed by the large-amplitude in-plane motional mechanism. The carpet model, where peptides aggregate on the membrane surface to cause lateral expansion and eventual micellization of the membrane, is a weaker mechanism of action.

Bedforms and sediment transport processes in the ephemeral streams of Kobo basin, Northern Ethiopia

Bedforms and channel deposits architecture were studied in the ephemeral streams of the structural basin of Kobo in northern Ethiopia. The Froude numbers, calculated at bankfull flow for a few of the study reaches, are in agreement with the extensive occurrence of horizontal, planar lamination. Small bedforms, not previously reported in the literature, are found and described as produced mainly by scouring rather than accretion processes. Equations to predict particle entrainment proved that large, individual boulders standing scattered on the stream bed can be transported at bankfull flow for long distances. Direct inspection of the old alluvium and the modern channel deposits allow to identify a typical arrangement within a single bed consisting of divisions with specific characteristics. This model of division association is explained in terms of vertical distribution of shear stress and compared with hyperconcentrated flow and traction carpet models in contrast with recent theories of flat asymmetrical bed waves progradation at Froude numbers close to unity.