Interaction Of Antimicrobial Peptides Research Articles

Unlocking the specificity of antimicrobial peptide interactions for membrane-targeted therapies

Antimicrobial peptides (AMPs) are increasingly recognized as potent therapeutic agents, with their selective affinity for pathological membranes, low toxicity profile, and minimal resistance development making them particularly attractive in the pharmaceutical landscape. This study offers a comprehensive analysis of the interaction between specific AMPs, including magainin-2, pleurocidin, CM15, LL37, and clavanin, with lipid bilayer models of very different compositions that have been ordinarily used as biological membrane models of healthy mammal, cancerous, and bacterial cells. Employing unbiased molecular dynamics simulations and metadynamics techniques, we have deciphered the intricate mechanisms by which these peptides recognize pathogenic and pathologic lipid patterns and integrate into lipid assemblies. Our findings reveal that the transverse component of the peptide's hydrophobic dipole moment is critical for membrane interaction, decisively influencing the molecule's orientation and expected therapeutic efficacy. Our approach also provides insight on the kinetic and dynamic dependence on the peptide orientation in the axial and azimuthal angles when coming close to the membrane. The aim is to establish a robust framework for the rational design of peptide-based, membrane-targeted therapies, as well as effective quantitative descriptors that can facilitate the automated design of novel AMPs for these therapies using machine learning methods.

Interaction of antimicrobial peptides with model membranes: a perspective towards new antibiotics

Interaction of antimicrobial peptides with model membranes: a perspective towards new antibiotics

Characterization of antimicrobial peptide interactions with a realistically phase separated model of the E. coli outer membrane via large-scale simulations

Characterization of antimicrobial peptide interactions with a realistically phase separated model of the E. coli outer membrane via large-scale simulations

Antimicrobial peptide interactions with bacterial cell membranes

Antimicrobial peptides (AMPs) are potential alternatives for common antibiotics because of their greater activity and efficiency against a broad range of viruses, bacteria, fungi, and parasites. In this project, two antimicrobial peptides including magainin 2 and protegrin 1 with α-helix and β-sheet secondary structures were selected to investigate their interactions with different lipid bilayers such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), POPC/POPG (7:3), POPC/POPS (7:3), POPG/POPE(1:3), and POPG/POPE(3:1). The obtained structures of the AMPs illustrated that protegrin 1 cannot maintain its secondary structure in the solution phase in contrast to magainin 2. The head groups of the lipid units play a key role in the stability of the lipid bilayers. The head parts of the lipid membranes by increasing the internal H-bond contribute to membrane compactness. The POPG and POPS units inside the POPC/POPG and POPC/POPS membranes increase the order of the POPC units. The cationic residues of the AMPs form remarkable electrostatic interactions with the negatively charged membrane surfaces, which play a key role in the stabilization process of the peptide secondary structures. The Arg residues of protegrin 1 and the Gly1, Lys4, Lys10, Lys11, Lys14, and Glu19 of the magainin 2 have the most important roles in the complexation process. The values of Gibbs binding energies (ΔG) indicate that the complexation process between AMPs and different bacterial membranes is favorable from the thermodynamic viewpoint and AMPs could form stable complexes with the lipid bilayers. As a result of ΔG values, protegrin 1 forms a more stable complex with POPG/POPE(3:1), while the α-helix has more affinity to the POPG/POPE(1:3) bacterial membranes. Therefore, it can be considered that β-sheet and α-helix AMPs are more effective against gram-positive and gram-negative bacteria, respectively. The results of this study can provide useful details about the antimicrobial peptide interactions with the bacterial cell, which can be employed for designing new antimicrobial materials with greater efficiency. Communicated by Ramaswamy H. Sarma

Molecular dynamics simulation of the interactions of antimicrobial peptides with phospholipids of the inner membrane of gram-negative bacteria

Molecular dynamics simulation of the interactions of antimicrobial peptides with phospholipids of the inner membrane of gram-negative bacteria

Polyunsaturated lipids promote membrane phase separation and antimicrobial sensitivity.

Gram-negative bacteria such as Acinetobacter baumannii sequester host lipids from the site of infection for incorporation into lipid synthesis pathways, altering the membrane lipidome. Changes in membrane composition from the incorporation of host-derived polyunsaturated fatty acids (PUFAs) help restore sensitivity to antimicrobials in several species of Gram-negative bacteria. Using coarse-grained simulations based on lipidomic data of A. baumannii inner membrane collected under three different growth conditions, we show PUFA-incorporation alters membrane biophysical properties, increasing the phase separation between ordered and disordered lipid domains resulting in thinner, less ordered membranes. We show that the changes in A. baumannii membrane biophysical properties on the incorporation of PUFA-containing lipids alter the conformational cycling of RND multidrug efflux pumps and restore sensitivity to some antimicrobials. Finally, we examine the interaction of antimicrobial peptides (AMP) with the simulated A. baumannii membranes to identify the effect of lipid saturation and alterations in membrane properties has on AMP-induced membrane disruption.

A Molecular Dynamics Study of Antimicrobial Peptide Interactions with the Lipopolysaccharides of theOuterBacterial Membrane.

With rising bacterial resistance, antimicrobial peptides (AMPs) have been widely investigated as potential antibacterial molecules to replace conventional antibiotics. Our understanding of the molecular mechanisms for membrane disruption are largely based on AMP interactions with the inner phospholipid bilayers of both Gram-negative and Gram-positive bacteria. Mechanisms for AMP translocation across the outer membrane of Gram-negative bacteria composed of lipopolysaccharides and the asymmetric lipid bilayer are complicated by the secondary structure adopted by the peptide in the different membrane environments. We have employed atomistic molecular dynamics and umbrella-sampling simulations with an aggregate duration of [Formula: see text] 6 microseconds to obtain the free energy landscape of CM15 peptide translocating through the lipopolysaccharide region of Gram-negative bacteria, E. coli. The peptide has a favorable binding-free energy (-130 kJ mol[Formula: see text]) in the O-antigen region with a large barrier (150 kJ mol[Formula: see text]) at the interface between the anionic core saccharides and upper bilayer leaflet made up of lipid-A molecules. Restraint-free molecular dynamics simulations show that the random coil structure is favored over the helix in both the extracellular aqueous region and the cation-rich core-saccharide regions of the outer membrane. The peptide and membrane properties are analyzed at each of the 100 ns duration of the umbrella-sampling windows toillustrate changes in peptide length, orientation, and hydration. Our study provides insights into the free energy landscape for the insertion of the AMP CM15 in the outer membrane of Gram-negative bacteria, and we discuss the implications of our findings with the broader question of how AMPs overcome this barrier during antimicrobial activity.

In situ solid-state NMR study of antimicrobial peptide interactions with erythrocyte membranes

Antimicrobial peptides are promising therapeutic agents to mitigate the global rise of antibiotic resistance. They generally act by perturbing the bacterial cell membrane and are thus less likely to induce resistance. Because they are membrane-active molecules, it is critical to verify and understand their potential action toward eukaryotic cells to help design effective and safe drugs. In this work, we studied the interaction of two antimicrobial peptides, aurein 1.2 and caerin 1.1, with red blood cell (RBC) membranes using in situ 31P and 2H solid-state NMR (SS-NMR). We established a protocol to integrate up to 25% of deuterated fatty acids in the membranes of ghosts, which are obtained when hemoglobin is removed from RBCs. Fatty acid incorporation and the integrity of the lipid bilayer were confirmed by SS-NMR and fluorescence confocal microscopy. Leakage assays were performed to assess the lytic power of the antimicrobial peptides. The in situ perturbation of the ghost membranes by aurein 1.2 and caerin 1.1 revealed by 31P and 2H SS-NMR is consistent with membrane perturbation through a carpet mechanism for aurein 1.2, whereas caerin 1.1 acts on RBCs via pore formation. These results are compatible with fluorescence microscopy images of the ghosts. The peptides interact with eukaryotic membranes following similar mechanisms that take place in bacteria, highlighting the importance of hydrophobicity when determining such interactions. Our work bridges model membranes and in vitro studies and provides an analytical toolbox to assess drug toxicity toward eukaryotic cells.

Lipid-mediated synergistic interactions of antimicrobial peptides in membranes

Biophysical and structural studies have shown that peptides interact with the membranes in a highly dynamic manner. Both peptides and lipids change and mutually adapt their conformation, membrane penetration and detailed morphology locally and globally forming supramolecular assemblies of variable shape and dimension. Charged amphipathic sequences such as antimicrobial and cell penetrating peptides tend to stay intercalated at the membrane interface. Although the membranes are soft and can adapt, at increasing peptide density they cause pronounced disruptions of the phospholipid bilayer packing.

Investigating Membrane-Mediated Antimicrobial Peptide Interactions with Synchrotron Radiation Far-Infrared Spectroscopy.

Synchrotron radiation‐based Fourier transform infrared spectroscopy enables access to vibrational information from mid over far infrared to even terahertz domains. This information may prove critical for the elucidation of fundamental bio‐molecular phenomena including folding‐mediated innate host defence mechanisms. Antimicrobial peptides (AMPs) represent one of such phenomena. These are major effector molecules of the innate immune system, which favour attack on microbial membranes. AMPs recognise and bind to the membranes whereupon they assemble into pores or channels destabilising the membranes leading to cell death. However, specific molecular interactions responsible for antimicrobial activities have yet to be fully understood. Herein we probe such interactions by assessing molecular specific variations in the near‐THz 400–40 cm−1 range for defined helical AMP templates in reconstituted phospholipid membranes. In particular, we show that a temperature‐dependent spectroscopic analysis, supported by 2D correlative tools, provides direct evidence for the membrane‐induced and folding‐mediated activity of AMPs. The far‐FTIR study offers a direct and information‐rich probe of membrane‐related antimicrobial interactions.

Polydiacetylene vesicles acting as colorimetric sensor for the detection of plantaricin LD1

The interaction of antimicrobial peptides with membrane lipids plays a major role in numerous physiological processes. In this study, polydiacetylene (PDA) vesicles were synthesized using 10, 12-tricosadiynoic acid (TRCDA) and 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). These vesicles were applied as artificial membrane biosensor for the detection of plantaricin LD1 purified from Lactobacillus plantarum LD1. Plantaricin LD1 (200 μg/mL) was able to interact with PDA vesicles by changing the color from blue to red with colorimetric response 30.26 ± 0.59. Nisin (200 μg/mL), used as control, also changed the color of the vesicles with CR% 50.56 ± 0.98 validating the assay. The vesicles treated with nisin and plantaricin LD1 showed increased infrared absorbance at 1411.46 and 1000-1150 cm−1 indicated the interaction of bacteriocins with phospholipids and fatty acids, respectively suggesting membrane-acting nature of these bacteriocins. Further, microscopic observation of bacteriocin-treated vesicles showed several damages indicating the interaction of bacteriocins. These findings suggest that the PDA vesicles may be used as bio-mimetic sensor for the detection of bacteriocins produced by several probiotics in food and therapeutic applications.

In-silico study of peptide-protein interaction of antimicrobial peptides potentially targeting SARS and SARS-CoV-2 nucleocapsid protein.

This study is an attempt to find a suitable therapy using antimicrobial peptides (AMPs) by identifying peptide-protein interaction of AMPs and nucleocapsid protein of SARS and SARS-CoV- 2. The AMPs were shortlisted from the APD3 database (Antimicrobial peptide database) based on various physicochemical parameters. The binding efficacy of AMPs was measured using the lowest energy score of the docked complexes with 10 selected AMPs. For SARS-CoV, AP00180 showed the best pose with a binding affinity value of − 6.4 kcal/mol. Prominent hydrogen bonding interactions were observed between Lys85 (nucleocapsid receptor) and Arg13 (antimicrobial peptide ligand) having the least intermolecular distance of 1.759 Å. For SARS-CoV-2, AP00549 was docked with a binding affinity value of − 3.4 kcal/mol and Arg119 and Glu14 of receptor nucleocapsid protein and ligand AMP having the least intermolecular distance of 2.104 The dynamic simulation was performed at 50 ns to check the stability of the final docked complexes, one with each protein. The two best AMPs were AP00180 (Human Defensin-5) for SARS and AP00549 (Plectasin) for SARS-CoV-2. From positive results of dynamic simulation and previously known knowledge that some AMPs interact with the nucleocapsid of coronaviruses, these AMPs might be used as a potential therapeutic agent for the treatment regime of SARS-CoV-2 and SARS infection.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40203-021-00103-z.

Physicochemical Features and Peculiarities of Interaction of AMP with the Membrane.

Antimicrobial peptides (AMPs) are anti-infectives that have the potential to be used as a novel and untapped class of biotherapeutics. Modes of action of antimicrobial peptides include interaction with the cell envelope (cell wall, outer- and inner-membrane). A comprehensive understanding of the peculiarities of interaction of antimicrobial peptides with the cell envelope is necessary to perform a rational design of new biotherapeutics, against which working out resistance is hard for microbes. In order to enable de novo design with low cost and high throughput, in silico predictive models have to be invoked. To develop an efficient predictive model, a comprehensive understanding of the sequence-to-function relationship is required. This knowledge will allow us to encode amino acid sequences expressively and to adequately choose the accurate AMP classifier. A shared protective layer of microbial cells is the inner, plasmatic membrane. The interaction of AMP with a biological membrane (native and/or artificial) has been comprehensively studied. We provide a review of mechanisms and results of interactions of AMP with the cell membrane, relying on the survey of physicochemical, aggregative, and structural features of AMPs. The potency and mechanism of AMP action are presented in terms of amino acid compositions and distributions of the polar and apolar residues along the chain, that is, in terms of the physicochemical features of peptides such as hydrophobicity, hydrophilicity, and amphiphilicity. The survey of current data highlights topics that should be taken into account to come up with a comprehensive explanation of the mechanisms of action of AMP and to uncover the physicochemical faces of peptides, essential to perform their function. Many different approaches have been used to classify AMPs, including machine learning. The survey of knowledge on sequences, structures, and modes of actions of AMP allows concluding that only possessing comprehensive information on physicochemical features of AMPs enables us to develop accurate classifiers and create effective methods of prediction. Consequently, this knowledge is necessary for the development of design tools for peptide-based antibiotics.

Spectroscopic and Microscopic Approaches for Investigating the Dynamic Interactions of Anti-microbial Peptides With Membranes and Cells.

The emergence of microbes resistant to conventional antibiotics is a burgeoning threat to humanity with significant impacts on the health of people and on the health system itself. Antimicrobial peptides (AMPs) hold promise as potential future alternatives to conventional drugs because they form an integral part of the defense systems of other species in the animal, plant, and fungal kingdoms. To aid the design of the next generation of AMPs optimized for human use, we must first understand the mechanism of action of existing AMPs with their targets, ideally in the context of the complex landscape of the living (microbial) cell. Advances in lasers, optics, detectors, fluid dynamics and various probes has enabled the experimentalist to measure the kinetics of molecule–membrane, molecule–molecule, and molecule–cell interactions with increasing spatial and temporal resolution. The purpose of this review is to highlight studies into these dynamic interactions with a view to improving our understanding of AMP mechanisms.

Application of Biophysical Techniques to Investigate the Interaction of Antimicrobial Peptides With Bacterial Cells.

Gaining new understanding on the mechanism of action of antimicrobial peptides is the basis for the design of new and more efficient antibiotics. To this aim, it is important to detect modifications occurring to both the peptide and the bacterial cell upon interaction; this will help to understand the peptide structural requirement, if any, at the base of the interaction as well as the pathways triggered by peptides ending in cell death. A limited number of papers have described the interaction of peptides with bacterial cells, although most of the studies published so far have been focused on model membrane-peptides interactions. Investigations carried out with bacterial cells highlighted the limitations connected to the use of oversimplified model membranes and, more importantly, helped to identify molecular targets of antimicrobial peptides and changes occurring to the bacterial membrane. In this review, details on the mechanism of action of antimicrobial peptides, as determined by the application of spectroscopic techniques, as well as scattering, microscopy, and calorimetry techniques, to complex systems such as peptide/bacteria mixtures are discussed.

Simulations of octapeptin–outer membrane interactions reveal conformational flexibility is linked to antimicrobial potency

The octapeptins are lipopeptide antibiotics that are structurally similar to polymyxins yet retain activity against polymyxin-resistant Gram-negative pathogens, suggesting they might be used to treat recalcitrant infections. However, the basis of their unique activity is unclear because of the difficulty in generating high-resolution experimental data of the interaction of antimicrobial peptides with lipid membranes. To elucidate these structure-activity relationships, we employed all-atom molecular dynamics simulations with umbrella sampling to investigate the conformational and energetic landscape of octapeptins interacting with bacterial outer membrane (OM). Specifically, we examined the interaction of octapeptin C4 and FADDI-115, lacking a single hydroxyl group compared with octapeptin C4, with the lipid A-phosphoethanolamine modified OM of Acinetobacter baumannii Octapeptin C4 and FADDI-115 both penetrated into the OM hydrophobic center but experienced different conformational transitions from an unfolded to a folded state that was highly dependent on the structural flexibility of their respective N-terminal fatty acyl groups. The additional hydroxyl group present in the fatty acyl group of octapeptin C4 resulted in the molecule becoming trapped in a semifolded state, leading to a higher free energy barrier for OM penetration. The free energy barrier for the translocation through the OM hydrophobic layer was ∼72 kcal/mol for octapeptin C4 and 62 kcal/mol for FADDI-115. Our results help to explain the lower antimicrobial activity previously observed for octapeptin C4 compared with FADDI-115 and more broadly improve our understanding of the structure-function relationships of octapeptins. These findings may facilitate the discovery of next-generation octapeptins against polymyxin-resistant Gram-negative 'superbugs.'

Spectroscopic, zeta potential and molecular dynamics studies of the interaction of antimicrobial peptides with model bacterial membrane.

Peptide-membrane interactions play a key role in the mechanisms of activity of antimicrobial peptides. Here, methods of fluorescence spectroscopy, zeta potential, and molecular dynamics modeling were used to study the interaction of new antimicrobial peptide megin with model bacterial membrane. The Gibbs free energy of -6kcal/mol characterizes the interaction of the peptides with liposomes containing DOPE and POPG lipids. Fluorescence data, acrylamide quenching, and MD simulations show that megin peptides are mainly located at the lipid/water interface and are aligned parallel to the bilayer surface in a carpet like manner. Measurements of zeta potential demonstrate the decrease of the negative potential of liposomes in the presence of peptides. The influence of megin on the membrane properties is also confirmed by molecular dynamics simulations. Insertion of peptides into the membrane disturbs lipid ordering, decreases the order parameters of lipids, and facilitates penetration of water molecules through the membrane. According to our results, we proposed that the megin antimicrobial activity can be explained by the carpet model of peptide activity.

Charge-based interactions of antimicrobial peptides and general drugs with lipid bilayers

Membrane-active agents (MAAs), such as antimicrobial peptides (AMPs) and chemotherapy drugs (CDs), induce ion pores/channels inside lipid bilayer membrane, as confirmed by standard electrophysiology experiments. A novel experimental method is described which detects agents directly at the membrane as confirmed for MAAs, CDs and aptamers. MAAs exhibit characteristic ‘charge based’ interactions with lipids. Electrostatic (ES) and van der Waals (vdW) contributions to the interaction energies have been estimated using molecular dynamics (MD) simulations. These results are consistent with the screened Coulomb interaction predictions recently developed for lipid bilayer binding of integral AMP channels. Energy- and distance-dependence of MAA-lipid interactions from MD simulations are represented by universal probability functions. A generalized model of MAA-lipid interactions is developed based on the charge and geometrical profiles of the participating lipids and AMPs. The corresponding driving force correlates directly with the stability of MAA-lipid structures as observed in electrophysiology experiments. We conclude that MAAs and similar agents that target lipid membranes exhibit physiological effects mainly due to ES and vdW interactions determined by their charge profiles.

A Comparative Study on Interactions of Antimicrobial Peptides L- and D-phenylseptin with 1,2-dimyristoyl-sn-glycero-3-phosphocholine

L-phenylseptin (L-Phes) and D-phenylseptin (D-Phes) are amphibian antimicrobial peptides isolated from the skin secretion of Hypsiboas punctatus. In the N-termini, L-Phes and D-Phes contain three consecutive phenylalanine residues, l-Phe-l-Phe-l-Phe and l-Phe-d-Phe-l-Phe, respectively. They are known to exhibit antimicrobial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Xanthomonas axonopodis pv. Glycines. However, their mechanism of action and the role of the D-amino acid residue have not been elucidated yet. In this study, the interactions of both peptides with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were investigated by means of quartz crystal microbalance, circular dichroism, vibrational circular dichroism, 31P solid-state NMR, and molecular dynamics simulation. Both peptides have similar binding constants to the DMPC lipid bilayers, in the order of 106 M−1, and form an α-helix structure in the DMPC lipid bilayers. Both the peptides induce similar changes in the dynamics of DMPC lipids. Thus, in spite of the difference in the conformations caused by the chirality at the N-terminus, the peptides showed similar behavior in the membrane-bound state, experimentally and computationally.

Formation and Characterization of Supported Lipid Bilayers Composed of Phosphatidylethanolamine and Phosphatidylglycerol by Vesicle Fusion, a Simple but Relevant Model for Bacterial Membranes.

Supported lipid bilayers (SLBs) are simple and robust biomimics with controlled lipid composition that are widely used as models of both mammalian and bacterial membranes. However, the lipids typically used for SLB formation poorly resemble those of bacterial cell membranes due to the lack of available protocols to form SLBs using mixtures of lipids relevant for bacteria such as phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). Although a few reports have been published recently on the formation of SLBs from Escherichia coli lipid extracts, a detailed understanding of these systems is challenging due to the complexity of the lipid composition in such natural extracts. Here, we present for the first time a simple and reliable protocol optimized to form high-quality SLBs using mixtures of PE and PG at compositions relevant for Gram-negative membranes. We show using neutron reflection and quartz microbalance not only that Ca2+ ions and temperature are key parameters for successful bilayer deposition but also that mass transfer to the surface is a limiting factor. Continuous flow of the lipid suspension is thus crucial for obtaining full SLB coverage. We furthermore characterize the resulting bilayers and report structural parameters, for the first time for PE and PG mixtures, which are in good agreement with those reported earlier for pure POPE vesicles. With this protocol in place, more suitable and reproducible studies can be conducted to understand biomolecular processes occurring at cell membranes, for example, for testing specificities and to unravel the mechanism of interaction of antimicrobial peptides.