Antimicrobial Peptide CM15 Research Articles

Interaction between Antimicrobial Peptide CM15 and a Model Cell Membrane Affected by CM15 Terminal Amidation and the Membrane Phase State.

Antimicrobial peptides (AMPs) have been proposed as an effective class of antimicrobial agents against microorganisms. In this work, the interaction between an antimicrobial peptide, CM15, and a negatively charged phospholipid bilayer, DPPG, was studied via sum frequency generation (SFG) vibrational spectroscopy. Two structurally correlated characteristic variables were introduced to reveal the interaction mechanism/efficiency, i.e. C-terminal amidation and temperature variation (∼20 °C, room temperature, and ∼35 °C, close to human body temperature). Experimental results indicated that owing to the increased positive charge, C-terminal amidation resulted in rapid adsorption onto the bilayer surface and efficient disruption of the outer layer, exhibiting less ordered insertion orientation. The elevated temperature (from ∼20 °C to ∼35 °C) promoted the penetration of both the outer and inner leaflets by the peptides and finally led to the disruption of the whole bilayer owing to the enhanced fluidity of the bilayer. From the perspective of the interaction mechanism, this experimental study provides two practical cues to understand the disruption process of the negatively charged model biomembranes, which can lay the structural foundation for designing and developing high-efficiency antimicrobial peptides.

Manipulating Active Structure and Function of Cationic Antimicrobial Peptide CM15 with the Polysulfonated Drug Suramin: A Step Closer to in Vivo Complexity.

Antimicrobial peptides (AMPs) kill bacteria by targeting their membranes through various mechanisms involving peptide assembly, often coupled with disorder‐to‐order structural transition. However, for several AMPs, similar conformational changes in cases in which small organic compounds of both endogenous and exogenous origin have induced folded peptide conformations have recently been reported. Thus, the function of AMPs and of natural host defence peptides can be significantly affected by the local complex molecular environment in vivo; nonetheless, this area is hardly explored. To address the relevance of such interactions with regard to structure and function, we have tested the effects of the therapeutic drug suramin on the membrane activity and antibacterial efficiency of CM15, a potent hybrid AMP. The results provided insight into a dynamic system in which peptide interaction with lipid bilayers is interfered with by the competitive binding of CM15 to suramin, resulting in an equilibrium dependent on peptide‐to‐drug ratio and vesicle surface charge. In vitro bacterial tests showed that when CM15⋅suramin complex formation dominates over membrane binding, antimicrobial activity is abolished. On the basis of this case study, it is proposed that small‐molecule secondary structure regulators can modify AMP function and that this should be considered and could potentially be exploited in future development of AMP‐based antimicrobial agents.

The molecular mechanism of structural changes in the antimicrobial peptide CM15 upon complex formation with drug molecule suramin: a computational analysis.

Dynamic increase of resistant bacterial infectious diseases continuously requires development of novel compounds against them. The molecular level understanding of the mechanism and interactions of natural host-defense peptides or antimicrobial peptides (AMPs) is an important step towards rational design and development of compounds inspired by their function. A particular set of these peptides have disordered structure, the ordering of which may modify their antimicrobial properties. Recent experiments demonstrate that such conformational transitions of AMPs could be mediated by the presence of small organic compounds, such as approved drug molecules. However, the molecular mechanisms underlying these structural changes are unclear. In this study, we apply molecular docking and molecular dynamics-based approaches to rigorously analyze the interactions between the drug suramin and the AMP CM15, a synthetic unstructured hybrid peptide. We characterize the energetic properties of putative CM15-suramin complexes revealing particular impacts of CM15 residues as well as the parts of suramin on these interactions. We find that α-helical content of the peptide is increased in the presence of suramin, which is in agreement with the experimental data. Kinetics analysis from canonical molecular dynamics and metadynamics simulations suggest that the effect of suramin does not promote the formation of α-helix but rather results from its ability to stabilize the α-helical population in the conformational pool of the peptide. Potentially, understanding the physico-chemical basis underlying the interactions between drug molecules and disordered AMPs will prove useful in strategies for antimicrobial compound development. Further on, the given computational protocol for the analysis of such flexible systems provide a basis for future theoretical investigation of similar biomolecular complexes.

Photothermal Off-Resonance Tapping for Rapid and Gentle Atomic Force Imaging of Live Cells.

Imaging living cells by atomic force microscopy (AFM) promises not only high-resolution topographical data, but additionally, mechanical contrast, both of which are not obtainable with other microscopy techniques. Such imaging is however challenging, as cells need to be measured with low interaction forces to prevent either deformation or detachment from the surface. Off-resonance modes which periodically probe the surface have been shown to be advantageous, as they provide excellent force control combined with large amplitudes, which help reduce lateral force interactions. However, the low actuation frequency in traditional off-resonance techniques limits the imaging speed significantly. Using photothermal actuation, we probe the surface by directly actuating the cantilever. Due to the much smaller mass that needs to be actuated, the achievable measurement frequency is increased by two orders of magnitude. Additionally, photothermal off-resonance tapping (PORT) retains the precise force control of conventional off-resonance modes and is therefore well suited to gentle imaging. Here, we show how photothermal off-resonance tapping can be used to study live cells by AFM. As an example of imaging mammalian cells, the initial attachment, as well as long-term detachment, of human thrombocytes is presented. The membrane disrupting effect of the antimicrobial peptide CM-15 is shown on the cell wall of Escherichia coli. Finally, the dissolution of the cell wall of Bacillus subtilis by lysozyme is shown. Taken together, these evolutionarily disparate forms of life exemplify the usefulness of PORT for live cell imaging in a multitude of biological disciplines.

Microfluidic bacterial traps for simultaneous fluorescence and atomic force microscopy

The atomic force microscope has become an established research tool for imaging microorganisms with unprecedented resolution. However, its use in microbiology has been limited by the difficulty of proper bacterial immobilization. Here, we have developed a microfluidic device that solves the issue of bacterial immobilization for atomic force microscopy under physiological conditions. Our device is able to rapidly immobilize bacteria in well-defined positions and subsequently release the cells for quick sample exchange. The developed device also allows simultaneous fluorescence analysis to assess the bacterial viability during atomic force microscope imaging. We demonstrated the potential of our approach for the immobilization of rod-shaped Escherichia coli and Bacillus subtilis. Using our device, we observed buffer-dependent morphological changes of the bacterial envelope mediated by the antimicrobial peptide CM15. Our approach to bacterial immobilization makes sample preparation much simpler and more reliable, thereby accelerating atomic force microscopy studies at the single-cell level.

Drug and dye binding induced folding of the intrinsically disordered antimicrobial peptide CM15

Drug binding induces the disorder-to-order conformational transition of the natively unfolded antimicrobial peptide CM15.

Antimicrobial Peptide Simulations and the Influence of Force Field on the Free Energy for Pore Formation in Lipid Bilayers

Due to antimicrobial resistance, the development of new drugs to combat bacterial and fungal infections is an important area of research. Nature uses short, charged, and amphipathic peptides for antimicrobial defense, many of which disrupt the lipid membrane in addition to other possible targets inside the cell. Computer simulations have revealed atomistic details for the interactions of antimicrobial peptides and cell-penetrating peptides with lipid bilayers. Strong interactions between the polar interface and the charged peptides can induce bilayer deformations - including membrane rupture and peptide stabilization of a hydrophilic pore. Here, we performed microsecond-long simulations of the antimicrobial peptide CM15 in a POPC bilayer expecting to observe pore formation (based on previous molecular dynamics simulations). We show that caution is needed when interpreting results of equilibrium peptide-membrane simulations, given the length of time single trajectories can dwell in local energy minima for 100's of ns to microseconds. While we did record significant membrane perturbations from the CM15 peptide, pores were not observed. We explain this discrepancy by computing the free energy for pore formation with different force fields. Our results show a large difference in the free energy barrier (ca. 40 kJ/mol) against pore formation predicted by the different force fields that would result in orders of magnitude differences in the simulation time required to observe spontaneous pore formation. This explains why previous simulations using the Berger lipid parameters reported pores induced by charged peptides, while with CHARMM based models pores were not observed in our long time-scale simulations. We reconcile some of the differences in the distance dependent free energies by shifting the free energy profiles to account for thickness differences between force fields. The shifted curves show that all the models describe small defects in lipid bilayers in a consistent manner, suggesting a common physical basis.

Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15

Antibiotics target specific biochemical mechanisms in bacteria. In response to new drugs, pathogenic bacteria rapidly develop resistance. In contrast, antimicrobial peptides (AMPs) have retained broad spectrum antibacterial potency over millions of years. We present single-cell fluorescence assays that detect reactive oxygen species (ROS) in the Escherichia coli cytoplasm in real time. Within 30 s of permeabilization of the cytoplasmic membrane by the cationic AMP CM15 [combining residues 1-7 of cecropin A (from moth) with residues 2-9 of melittin (bee venom)], three fluorescence signals report oxidative stress in the cytoplasm, apparently involving O2 (-), H2O2, and •OH. Mechanistic studies indicate that active respiration is a prerequisite to the CM15-induced oxidative damage. In anaerobic conditions, signals from ROS are greatly diminished and the minimum inhibitory concentration increases 20-fold. Evidently the natural human AMP LL-37 also induces a burst of ROS. Oxidative stress may prove a significant bacteriostatic mechanism for a variety of cationic AMPs. If so, host organisms may use the local oxygen level to modulate AMP potency.

Cyanylated Cysteine used to Observe the Interaction between the CM15 Antimicrobial Peptide and Neutral Lipid Membranes

Antimicrobial peptides, the first immune response, are known to provide protection against pathogens; there are three proposed pore formation models by which antimicrobial peptides are believed to disrupt membranes. Previous work used the artificial amino acid cyanylated cysteine to confirm that the synthetic hybrid antimicrobial peptide CM15 porates anionic lipid vesicles by the barrel stave mechanism, which requires a single dominant orientation of the peptide on the lipid surface. Recent molecular dynamics results suggest that CM15 might adopt a number of different structures when binding to neutral lipid bilayers, including those made primarily of phosphatidylcholine (PC) lipids. Infrared spectrometry of CM15 variants provides evidence that the lipid bound conformation of CM15 to neutral bilayers varies significantly from that in contact with anionic bilayers. Additionally, circular dichroism experiments give further insight into the different structures of CM15 in contact with neutral lipid membranes.

Comparative molecular dynamics simulations of the antimicrobial peptide CM15 in model lipid bilayers

We report altogether 3-μs molecular dynamics (MD) simulations of the antimicrobial peptide CM15 to systematically investigate its interaction with two model lipid bilayers, pure POPC and mixed POPG:POPC (1:2). Starting with either an α-helical or a random-coil conformation, CM15 is found to insert into both bilayers. Peptide-lipid interaction is stronger with the anionic POPG:POPC than the zwitterionic POPC, which is largely attributed to the electrostatic attraction between CM15 and the negatively charged POPG. Simulations initiated with CM15 as a random coil allowed us to study peptide folding at the lipid-water interface. Interestingly, CM15 folding appears to be faster in POPC than POPG:POPC, which may be explained by a lower activation energy barrier of structural rearrangement in the former system. Our data also suggest that compared with the random-coil conformation, CM15 in a pre-folded α-helix has significantly reduced interactions with the lipids, indicating that peptide initial structures may bias the simulation results considerably on the 100-ns timescale. The implications of this result should be considered when preparing and interpreting future AMP simulations.

Effects of D-Lysine Substitutions on the Activity and Selectivity of Antimicrobial Peptide CM15.

Despite their potent antimicrobial activity, the usefulness of antimicrobial peptides (AMPs) as antibiotics has been limited by their toxicity to eukaryotic cells and a lack of stability in vivo. In the present study we examined the effects of introducing D-lysine residues into a 15-residue hybrid AMP containing residues 1-7 of cecropin A and residues 2-9 of melittin (designated CM15). Diastereomeric analogs of CM15 containing between two and five D-lysine substitutions were evaluated for their antimicrobial activity, lysis of human erythrocytes, toxicity to murine macrophages, ability to disrupt cell membranes, and protease stability. All of the analogs caused rapid permeabilization of the Staphylococcus aureus cell envelope, as indicated by uptake of SYTOX green. CM15 also permeabilized the plasma membrane of RAW264.7 macrophages, but this was substantially diminished for the D-lysine containing analogs. The introduction of D-lysine caused moderate decreases in antimicrobial activity for all analogs studied. However, D-Lys substitution produced a much more pronounced reduction in toxicity to eukaryotic cells, leading to marked improvements in antimicrobial efficacy for some analogs. Circular dichroism studies indicated a progressive loss of helical secondary structure upon introduction of D-lysine residues, and there was a good correspondence between helical content and eukaryotic cell cytotoxicity. Overall, these studies show that the biological activity of CM15 analogs containing D-lysine depends on both the number and position of D-Lys substitutions, and that such substitutions can dramatically lower toxicity to eukaryotic cells with only minimal decreases in antimicrobial activity.

Kinetics of antimicrobial peptide activity measured on individual bacterial cells using high-speed atomic force microscopy

Observations of real time changes in living cells have contributed much to the field of cellular biology. Eluding the field thus far is the ability to image whole, living cells with nanometre resolution on a time scale that is relevant to dynamic cellular processes1,2. Here we investigate the kinetics of individual bacterial cell death using a novel high-speed atomic force microscope (AFM) optimized for imaging live cells in real time. The increased time resolution (13 seconds per image) allows the characterization of the initial stages of the action of the antimicrobial peptide (AmP) CM15 on individual Escherichia coli cells with nanometre resolution. Our results suggest that the killing process is a combination of a time-variable incubation phase (which takes seconds to minutes to complete) and a more rapid execution phase.

Influence of Phosphocholine Alkyl Chain Length on Peptide−Micelle Interactions and Micellar Size and Shape

The interaction with biological membranes is of functional importance for many peptides and proteins. Structural studies on such membrane-bound biomacromolecules are often carried out in solutions containing small membrane-mimetic assemblies of detergent molecules. To investigate the influence of the hydrophobic chain length on the structure, diffusional and dynamical behavior of a peptide bound to micelles, we studied the binding of three peptides to n-phosphocholines with n ranging from 8 to 16. The peptides studied are the 15 residue antimicrobial peptide CM15, the 25-residue transmembrane helix 7 of yeast V-ATPase (TM7), and the 35-residue bacterial toxin LdrD. To keep the dimension of the peptide-membrane-mimetic assembly small, micelles are typically used when studying membrane-bound peptides and proteins, for example, by solution NMR spectroscopy. Since they are readily available in deuterated form most often sodium-dodecylsulfate (SDS) and dodecylphosphocholine (DPC) are used as the micelle-forming detergent. Using NMR, CD, and SAXS, we found that all phosphocholines studied form spherical micelles in the presence and absence of small bound peptides and the diameters of the micelles are basically unchanged upon peptide binding. The size of the peptide relative to the micelle determines to what extent the secondary structure can form. For small peptides (up to approximately 25 residues) the use of shorter chain phosphocholines is recommended for solution NMR studies due to the favorable spectral quality and since they are as well-structured as in DPC. In contrast, larger peptides are better structured in micelles formed by detergents with chain lengths longer than DPC.

Cyanylated Cysteine: A Covalently Attached Vibrational Probe of Protein-Lipid Contacts.

Cyanylated cysteine, or β-thiocyanatoalanine, is an artificial amino acid that can be introduced into peptides and proteins by post-translational chemical modification of solvent-exposed cysteine side chains, and thus it can be used in any protein with a suitable expression and mutagenesis system. In this study, cyanylated cysteine is introduced at selected sites in two model peptides that have been shown to bind to membrane interfaces: a membrane-binding sequence of the human myelin basic protein and the antimicrobial peptide CM15. Far-UV circular dichroism indicates that the secondary structures of the bound peptides are not influenced by introduction of the artificial side chain. Infrared spectra of both systems in buffer and exposed to dodecylphosphocholine micelles indicate that the CN stretching absorption band of cyanylated cysteine can clearly distinguish between membrane burial and solvent exposure of the artificial side chain. Since infrared spectroscopy can be applied in a wide variety of lipid systems, and since cyanylated cysteine can be introduced into proteins of arbitrary size via mutagenesis and post-translational modification, this new probe could see wide use in characterizing the protein−lipid interactions of membrane proteins.

Cyanylated Cysteine Used To Map Membrane Binding and Inter-Peptide Contacts in a Model Antimicrobial Peptide

Using single-cysteine mutants of the potent antimicrobial peptide CM15 as a model system for binding at the membrane surface, we are developing an infrared probe to characterize site-specific side chain solvent exposure and the ps-time scale dynamics of both membrane-peptide interactions and peptide-peptide contacts. The selective cyanylation of a mutated cysteine residue covalently attaches a nitrile vibrational probe at the chosen site. The frequency and lineshape of the CN stretching vibration are sensitive to both solvent exposure and peptide aggregation. These sensitivities are applied at multiple label sites to reveal information about the structural aspects of CM15's perturbation of E. coli lipid bilayers.

Positioning of Micelle-Bound Peptides by Paramagnetic Relaxation Enhancements

Many peptides, proteins, and drugs interact with biological membranes, and knowing the mode of binding is essential to understanding their biological functions. To obtain the complete orientation and immersion depth of such a compound, the membrane-mimetic system (micelle) is placed in an aqueous buffer containing the soluble and inert paramagnetic contrast agent Gd(DTPA-BMA). Paramagnetic relaxation enhancements (PREs) of a specific nucleus then depend only on its distance from the surface. The positioning of a structurally characterized compound can be obtained by least-squares fitting of experimental PREs to the micelle center position. This liquid-state NMR approach, which does not rely on isotopic labeling or chemical modification, has been applied to determine the location of the presumed transmembrane region 7 of yeast V-ATPase (TM7) and the membrane-bound antimicrobial peptide CM15 in micelles. TM7 binds in a trans-micelle orientation with the N-terminus being slightly closer to the surface than the C-terminus. CM15 is immersed unexpectedly deep into the micelle with the more hydrophilic side of the helix being closer to the surface than the hydrophobic one.

Membrane Insertion and Bilayer Perturbation by Antimicrobial Peptide CM15

Antimicrobial peptides (AMPs) are an important component of innate immunity and have generated considerable interest as a potential new class of antibiotic. The biological activity of AMPs is strongly influenced by peptide-membrane interactions; however, for many of these peptides the molecular details of how they disrupt and/or translocate across target membranes are not known. CM15 is a linear, synthetic hybrid AMP composed of the first seven residues of the cecropin A and residues 2–9 of the bee venom peptide mellitin. Previous studies have shown that upon membrane binding CM15 folds into an α-helix with its helical axis aligned parallel to the bilayer surface and have implicated the formation of 2.2–3.8 nm pores in its bactericidal activity. Here we report site-directed spin labeling electron paramagnetic resonance studies examining the behavior of CM15 analogs labeled with a methanethiosulfonate spin label (MTSL) and a brominated MTSL as a function of increasing peptide concentration and utilize phospholipid-analog spin labels to assess the effects of CM15 binding and accumulation on the physical properties of membrane lipids. We find that as the concentration of membrane-bound CM15 is increased the N-terminal domain of the peptide becomes more deeply immersed in the lipid bilayer. Only minimal changes are observed in the rotational dynamics of membrane lipids, and changes in lipid dynamics are confined primarily to near the membrane surface. However, the accumulation of membrane-bound CM15 dramatically increases accessibility of lipid-analog spin labels to the polar relaxation agent, nickel (II) ethylenediaminediacetate, suggesting an increased permeability of the membrane to polar solutes. These results are discussed in relation to the molecular mechanism of membrane disruption by CM15.

Mapping the Orientation of Helices in Micelle-Bound Peptides by Paramagnetic Relaxation Waves

Many antimicrobial peptides form alpha-helices when bound to a membrane. In addition, around 80% of residues in membrane-bound proteins are found in alpha-helical regions. The orientation and location of such helical peptides and proteins in the membrane are key factors determining their function and activity. Here we present a new solution state NMR method for obtaining the orientation of helical peptides in a membrane-mimetic environment (micelle-bound) without any chemical perturbation of the peptide-micelle system. By monitoring proton longitudinal relaxation rates upon addition of the freely water-soluble and inert paramagnetic probe Gd(DTPA-BMA) to an alpha-helical peptide, a wavelike pattern with a periodicity of 3.6 residues per turn is observed. The tilt and azimuth (rotation) angle of the helix determine the shape of this paramagnetic relaxation wave and can be obtained by least-square fitting of measured relaxation enhancements. Results are presented for the 15-residue antimicrobial peptide CM15 which forms an amphipathic helix almost parallel to the surface of the micelle. Thus, a few fast experiments enable the identification of helical regions and determination of the helix orientation within the micelle without the need for covalent modification, isotopic labeling, or sophisticated equipment. This approach opens a path toward the topology determination of alpha-helical membrane-proteins without the need for a complete NOE-based structure determination.

Osmoprotection of Bacterial Cells from Toxicity Caused by Antimicrobial Hybrid Peptide CM15

Antimicrobial peptides exist ubiquitously as a host defense system in a broad range of species, including insects, amphibians, and mammals. The binding of these peptides is followed by the disruption of cytoplasmic membranes, leading to bacterial cell death; however, the precise mechanism of membrane destruction has remained controversial. In this study, we have examined the mechanism of action for the antimicrobial peptide, CM15 (KWKLFKKIGAVLKVL), a chimeric peptide of cecropin and mellitin. We find that the cytotoxicity of CM15 against either E. coli or Pseudomonas aeruginosa can be mitigated by the addition of sugar or poly(ethylene glycol) osmolytes to the extracellular media. The dependence of osmoprotection on solute size suggests the formation of pores with an effective diameter of 2.2-3.8 nm. In contrast, no osmoprotection was observed for cell killing by the cationic detergent dodecyltrimethylammonium bromide. Osmolytes also protected cells against the cytotoxicity of CM15 expressed intracellularly as a C-terminal extension of the carrier protein ketosteroid isomerase (KSI). Osmoprotection against the intracellularly produced peptide was also dependent on osmolyte size, in a manner that was in agreement with that observed for extracellularly added synthetic CM15. These data indicate that the formation of discrete pores in the cytoplasmic membrane is a key factor in the mechanism of bacterial killing by CM15.