Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from + 4 to + 5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an α-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Interaction of a pseudosubstrate peptide of protein kinase C and its myristoylated form with lipid vesicles: Only the myristoylated form translocates into the lipid bilayer

Lipopeptides derived from protein kinase C (PKC) pseudosubstrates have the ability to cross the plasma membrane in cells and modulate the activity of PKC in the cytoplasm. Myristoylation or palmitoylation appears to promote translocation across membranes, as the non-acylated peptides are membrane impermeant. We have investigated, by fluorescence spectroscopy, how myristoylation modulates the interaction of the PKC pseudosubstrate peptide KSIYRRGARRWRKL with lipid vesicles and translocation across the lipid bilayer. Our results indicate that myristoylated peptides are intimately associated with lipid vesicles and are not peripherally bound. When visualized under a microscope, myristoylation does appear to facilitate translocation across the lipid bilayer in multilamellar lipid vesicles. Translocation does not involve large-scale destabilization of the bilayer structure. Myristoylation promotes translocation into the hydrophobic interior of the lipid bilayer even when the non-acylated peptide has only weak affinity for membranes and is also only peripherally associated with lipid vesicles.     

Two interdependent mechanisms of antimicrobial activity allow for efficient killing in nylon-3-based polymeric mimics of innate immunity peptides

Novel synthetic mimics of antimicrobial peptides have been developed to exhibit structural properties and antimicrobial activity similar to those of natural antimicrobial peptides (AMPs) of the innate immune system. These molecules have a number of potential advantages over conventional antibiotics, including reduced bacterial resistance, cost-effective preparation, and customizable designs. In this study, we investigate a family of nylon-3 polymer-based antimicrobials. By combining vesicle dye leakage, bacterial permeation, and bactericidal assays with small-angle X-ray scattering (SAXS), we find that these polymers are capable of two interdependent mechanisms of action: permeation of bacterial membranes and binding to intracellular targets such as DNA, with the latter necessarily dependent on the former. We systemically examine polymer-induced membrane deformation modes across a range of lipid compositions that mimic both bacteria and mammalian cell membranes. The results show that the polymers' ability to generate negative Gaussian curvature (NGC), a topological requirement for membrane permeation and cellular entry, in model Escherichia coli membranes correlates with their ability to permeate membranes without complete membrane disruption and kill E. coli cells. Our findings suggest that these polymers operate with a concentration-dependent mechanism of action: at low concentrations permeation and DNA binding occur without membrane disruption, while at high concentrations complete disruption of the membrane occurs. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Dual mechanism of bacterial lethality for a cationic sequence-random copolymer that mimics host-defense antimicrobial peptides

Flexible sequence-random polymers containing cationic and lipophilic subunits that act as functional mimics of host-defense peptides have recently been reported. We used bacteria and lipid vesicles to study one such polymer, having an average length of 21 residues, that is active against both Gram-positive and Gram-negative bacteria. At low concentrations, this polymer is able to permeabilize model anionic membranes that mimic the lipid composition of Escherichia coli, Staphylococcus aureus, or Bacillus subtilis but is ineffective against model zwitterionic membranes, which explains its low hemolytic activity. The polymer is capable of binding to negatively charged vesicles, inducing segregation of anionic lipids. The appearance of anionic lipid-rich domains results in formation of phase-boundary defects through which leakage can occur. We had earlier proposed such a mechanism of membrane disruption for another antimicrobial agent. Experiments with the mutant E. coli ML-35p indicate that permeabilization is biphasic: at low concentrations, the polymer permeabilizes the outer and inner membranes; at higher polymer concentrations, permeabilization of the outer membrane is progressively diminished, while the inner membrane remains unaffected. Experiments with wild-type E. coli K12 show that the polymer blocks passage of solutes into the intermembrane space at high concentrations. Cell membrane integrity in E. coli K12 and S. aureus exhibits biphasic dependence on polymer concentration. Isothermal titration calorimetry indicates that the polymer associates with the negatively charged lipopolysaccharide of Gram-negative bacteria and with the lipoteichoic acid of Gram-positive bacteria. We propose that this polymer has two mechanisms of antibacterial action, one predominating at low concentrations of polymer and the other predominating at high concentrations.     

Thermodynamics of RTA3 peptide binding to membranes and consequences for antimicrobial activity

RTA3 is an α-helical, amphipathic peptide with broad-spectrum activity against Gram-negative bacteria and low mammalian cell toxicity. RTA3 contains a cysteine residue, replacement of which with an alanine or serine (RTA3-C15S) virtually abolishes antimicrobial activity. Much of the activity of RTA3 can be recovered in RTA3-C15L, indicating that the C15 residue functions largely as a bulky hydrophobic side chain promoting target cell membrane interactions. The poorly active RTA3-C15S is a useful variant for assessing the mechanistic aspects of RTA3 activity. Binding and membrane perturbation in vesicles containing different proportions of negative surface charge are analyzed in terms of amino acid-specific free energy contributions to interfacial binding, which likely underlie variations in antimicrobial activity amongst RTA3 variants. Comparison with published free energy scales indicates that the reduced electrostatic contribution to binding to membranes having reduced negative surface charge can be compensated in RTA3 (but not RTA3-C15S) by a slightly deeper insertion of the C-terminus of the peptide to maximize hydrophobic contributions to binding. Analysis of inner membrane (IM)- and outer membrane (OM)-selective permeabilization of Escherichiacoli demonstrates a broad similarity between peptide effects on vesicles with low negative surface charge (20% negatively charged lipids), E.coli membrane perturbation, and antimicrobial activity, supporting a role for membrane perturbation in the killing mechanism of RTA3. The results demonstrate that large variations in antimicrobial activity on subtle changes in amino acid sequence in helical amphipathic peptides can be rationalized in terms of the thermodynamics of peptide binding to membranes, allowing a more systematic understanding of antimicrobial activity in these peptides.     

Interaction of an artificial antimicrobial peptide with lipid membranes

Antimicrobial peptides constitute an important part of the innate immune defense and are promising new candidates for antibiotics. Naturally occurring antimicrobial peptides often possess hemolytic activity and are not suitable as drugs. Therefore, a range of new synthetic antimicrobial peptides have been developed in recent years with promising properties. But their mechanism of action is in most cases not fully understood. One of these peptides, called V4, is a cyclized 19 amino acid peptide whose amino acid sequence has been modeled upon the hydrophobic/cationic binding pattern found in Factor C of the horseshoe crab (Carcinoscorpius rotundicauda). In this work we used a combination of biophysical techniques to elucidate the mechanism of action of V4. Langmuir-Blodgett trough, atomic force microscopy, Fluorescence Correlation Spectroscopy, Dual Polarization Interference, and confocal microscopy experiments show how the hydrophobic and cationic properties of V4 lead to a) selective binding of the peptide to anionic lipids (POPG) versus zwitterionic lipids (POPC), b) aggregation of vesicles, and above a certain concentration threshold to c) integration of the peptide into the bilayer and finally d) to the disruption of the bilayer structure. The understanding of the mechanism of action of this peptide in relation to the properties of its constituent amino acids is a first step in designing better peptides in the future.     

Bilayer interaction and localization of cell penetrating peptides with model membranes: A comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43-58)

Cell-penetrating peptides (CPPs) are able to translocate problematic therapeutic cargoes across cellular membranes. The exact mechanisms of translocation are still under investigation. However, evidence for endocytic uptake is increasing. We investigated the interactions of CPPs with phospholipid bilayers as first step of translocation. To this purpose, we employed four independent techniques, comprising (i) liposome buffer equilibrium dialysis, (ii) Trp fluorescence quenching, (iii) fluorescence polarization, and (iv) determination of ζ-potentials. Using unilamellar vesicles (LUVs) of different phospholipid composition, we compared weakly cationic human calcitonin (hCT)-derived peptides with the oligocationic CPPs pVEC and penetratin (pAntp). Apparent partition coefficients of hCT-derived peptides in neutral POPC LUVs were dependent on amino acid composition and secondary structure; partitioning in negatively charged POPC/POPG (80:20) LUVs was increased and mainly governed by electrostatic interactions. For hCT(9-32) and its derivatives, D values raised from about 100-200 in POPC to about 1000 to 1500 when negatively charged lipids were present. Localization profiles of CPPs obtained by Trp fluorescence quenching were dependent on the charge density of LUVs. In POPC/POPG, hCT-derived CPPs were located on the bilayer surface, whereas pVEC and pAntp resided deeper in the membrane. In POPG LUVs, an increase of fluorescence polarization was observed for pVEC and pAntp but not for hCT-derived peptides. Generally, we found strong peptide-phospholipid interactions, especially when negatively charged lipids were present.     

Coupling Molecular Dynamics Simulations with Experiments for the Rational Design of Indolicidin-Analogous Antimicrobial Peptides

Antimicrobial peptides (AMPs) have attracted much interest in recent years because of their potential use as new-generation antibiotics. Indolicidin (IL) is a 13-residue cationic AMP that is effective against a broad spectrum of bacteria, fungi, and even viruses. Unfortunately, its high hemolytic activity retards its clinical applications. In this study, we adopted molecular dynamics (MD) simulations as an aid toward the rational design of IL analogues exhibiting high antimicrobial activity but low hemolysis. We employed long-timescale, multi-trajectory all-atom MD simulations to investigate the interactions of the peptide IL with model membranes. The lipid bilayer formed by the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was chosen as the model erythrocyte membrane; lipid bilayers formed from a mixture of POPC and the negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol were chosen to model bacterial membranes. MD simulations with a total simulation time of up to 4 μs revealed the mechanisms of the processes of IL adsorption onto and insertion into the membranes. The packing order of these lipid bilayers presumably correlated to the membrane stability upon IL adsorption and insertion. We used the degree of local membrane thinning and the reduction in the order parameter of the acyl chains of the lipids to characterize the membrane stability. The order of the mixed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol/POPC lipid bilayer reduced significantly upon the adsorption of IL. On the other hand, although the order of the pure-POPC lipid bilayer was perturbed slightly during the adsorption stage, the value was reduced more dramatically upon the insertion of IL into the membrane's hydrophobic region. The results imply that enhancing IL adsorption on the microbial membrane may amplify its antimicrobial activity, while the degree of hemolysis may be reduced through inhibition of IL insertion into the hydrophobic region of the erythrocyte membrane. In addition, through simulations, we identified the amino acids that are most responsible for the adsorption onto or insertion into the two model membranes. Positive charges are critical to the peptide's adsorption, whereas the presence of hydrophobic Trp8 and Trp9 leads to its deeper insertion. Combining the hypothetical relationships between the membrane disordering and the antimicrobial and hemolytical activities with the simulated results, we designed three new IL-analogous peptides: IL-K7 (Pro7 → Lys), IL-F89 (Trp8 and Trp9 → Phe), and IL-K7F89 (Pro7 → Lys; Trp8 and Trp9 → Phe). The hemolytic activity of IL-F89 is considerably lower than that of IL, whereas the antimicrobial activity of IL-K7 is greatly enhanced. In particular, the de novo peptide IL-K7F89 exhibits higher antimicrobial activity against Escherichia coli; its hemolytic activity decreased to only 10% of that of IL. Our simulated and experimental results correlated well. This approach—coupling MD simulations with experimental design—is a useful strategy toward the rational design of AMPs for potential therapeutic use.     

Different modes in antibiotic action of tritrpticin analogs, cathelicidin-derived Trp-rich and Pro/Arg-rich peptides

The cathelicidin-derived antimicrobial tritrpticin could be classified as either Trp-rich or Pro/Arg-rich peptide. We recently found that the sequence modification of tritrpticin focused on Trp and Pro residues led to considerable change in structure and antimicrobial potency and selectivity, but their mechanisms of microbial killing action were still unclear. Here, to better understand the bactericidal mechanisms of tritrpticin and its two analogs, TPA and TWF, we studied their effect on the viability of Gram-positive S. aureus and Gram-negative E. coli in relation to their membrane depolarization. Although TWF more effectively inhibited growth of S. aureus and E. coli than TPA, only a 30 min exposure to TPA was sufficient to kill both bacteria and TWF required a lag period of about 3-6 h for bactericidal activity. Their different bactericidal kinetics was associated with membrane permeabilization, i.e., TWF showed negligible ability to depolarize the cytoplasmic membrane potential of target cell membrane, whereas we observed significant membrane depolarization for TPA. In addition, while TPA caused rapid and large dye leakage from negatively charged model vesicles, TWF showed very little membrane-disrupting activity. Interestingly, we have looked for a synergism among the three peptides against E. coli, supporting that they are working with different modes of action. Collectively, our results suggest that TPA disrupts the ion gradients across the membrane, causing depolarization and a loss of microbial viability. By contrast, TWF more likely translocates across the cytoplasmic membrane without depolarization and then acts against one or more intracellular targets. Tritrpticin exhibits intermediate properties and appears to act via membrane depolarization coupled to secondary intracellular targeting.     

Reversible sheet-turn conformational change of a cell-penetrating peptide in lipid bilayers studied by solid-state NMR

The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The ¹³C chemical shifts of ¹³C, ¹⁵N-labeled residues in the peptide indicate a reversible conformational change from β-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25-50% anionic lipids, and in both saturated DMPC/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C-H order parameters of 0.23-0.52 for C^α and C^β sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet-turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage.     

The effect of binding of spider-derived antimicrobial peptides, oxyopinins, on lipid membranes

Oxyopinins (Oxki1 and Oxki2) are antimicrobial peptides isolated from the crude venom of the wolf spider Oxyopes kitabensis. The effect of oxyopinins on lipid bilayers was investigated using high-sensitivity titration calorimetry and ³¹P solid-state NMR spectroscopy. High-sensitivity titration calorimetry experiments showed that the binding of oxyopinins was exothermic, and the binding enthalpies (ΔH) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) small unilamellar vesicles (SUVs) were − 18.1 kcal/mol and − 15.0 kcal/mol for Oxki1 and Oxki2, respectively, and peptide partition coefficient (K_p) was found to be 3.9 × 10³ M^− 1. ³¹P NMR spectra of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes in the presence of oxyopinins indicated that they induced a positive curvature in lipid bilayers. The induced positive curvature was stronger in the presence of Oxki2 than in the presence of Oxki1. ³¹P NMR spectra of phosphaditylcholine (PC) membranes in the presence of Oxki2 showed that Oxki2 produced micellization of membranes at low peptide concentrations, but unsaturated PC membranes or acidic phospholipids prevented micellization from occurring. Furthermore, ³¹P NMR spectra using membrane lipids from E. coli suggested that Oxki1 was more disruptive to bacterial membranes than Oxki2. These results strongly correlate to the known biological activity of the oxyopinins.     

Interaction of bundled Ser-rich amphiphilic peptides with phospholipid membranes.

To investigate properties of hydrophilic bundled peptides and their interactions with phospholipid membranes, bundled peptides named [Trp2]- and [Trp12]-4alpha-46S9, which are composed of four fragments of amphiphilic 24-mer peptide, were designed and synthesized. Tryptophan (Trp) was introduced at the 2nd position from the N-terminal or at the centre (12th) of the helix to monitor the peptide-lipid interaction. Circular dichroism measurements indicated that the peptides had low alpha-helicities in a buffer solution (pH 7.4) and also in the presence of dipalmitoyl-DL-3-phosphatidylcholine (DPPC) vesicles. In the presence of DPPC/dipalmitoyl-DL-3-phosphatidylglycerol (DPPG) (3:1) vesicles, the measurement could not be taken because of turbidity induced by vesicle aggregation. Both peptides had moderate perturbation activity for both the neutral and acidic vesicles at 25 degrees C. The perturbation patterns at 50 degrees C were much different from those at 25 degrees C and the maximum activity reached 100% at a low peptide concentration. The results of the measurement of membrane fusion activity of peptides showed a similar tendency to that found in the perturbation experiment. A quenching experiment indicated that the Trp2 and Trp12 residues in [Trp2]- and [Trp12]-4alpha-46S9 were scarcely embedded in neutral lipid membranes.     

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH₂) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. ²H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. ³¹P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from ³¹P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and ³¹P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.     

Membrane interactions and biological activity of antimicrobial peptides from Australian scorpion

UyCT peptides are antimicrobial peptides isolated from the venom of the Australian scorpion. The activity of the UyCT peptides against Gram positive and Gram negative bacteria and red blood cells was determined. The membrane interactions of these peptides were evaluated by dye release (DR) of the fluorophore calcein from liposomes and isothermal titration calorimetry (ITC); and their secondary structure was determined by circular dichroism (CD). Three different lipid systems were used to mimic red blood cells, Escherichia coli and Staphylococcus aureus membranes. UyCT peptides exhibited broad spectrum antimicrobial activity with low MIC for S. aureus and multi-drug resistant Gram negative strains. Peptide combinations showed some synergy enhancing their potency but not hemolytic activity. The UyCT peptides adopted a helical structure in lipid environments and DR results confirmed that the mechanism of action is by disrupting the membrane. ITC data indicated that UyCT peptides preferred prokaryotic rather than eukaryotic membranes. The overall results suggest that UyCT peptides could be pharmaceutical leads for the treatment of Gram negative multiresistant bacterial infections, especially against Acinetobacter baumanni, and candidates for peptidomimetics to enhance their potency and minimize hemolysis. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Cell selectivity correlates with membrane-specific interactions: A case study on the antimicrobial peptide G15 derived from granulysin

A 15-residue peptide dimer G15 derived from the cell lytic protein granulysin has been shown to exert potent activity against microbes, including E. coli, but not against human Jurkat cells [Z. Wang, E. Choice, A. Kaspar, D. Hanson, S. Okada, S.C. Lyu, A.M. Krensky, C. Clayberger, Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J. Immunol. 165 (2000) 1486-1490]. We investigated the target membrane selectivity of G15 using fluorescence, circular dichroism and ³¹P NMR methods. The ANS uptake assay shows that the extent of E. coli outer membrane disruption depends on G15 concentration. ³¹P NMR spectra obtained from E. coli total lipid bilayers incorporated with G15 show disruption of lipid bilayers. Fluorescence binding studies on the interaction of G15 with synthetic liposomes formed of E. coli lipids suggest a tight binding of the peptide at the membrane interface. The peptide also binds to negatively charged POPC/POPG (3:1) lipid vesicles but fails to insert deep into the membrane interior. These results are supported by the peptide-induced changes in the measured isotropic chemical shift and T1 values of POPG in 3:1 POPC:POPG multilamellar vesicles while neither a non-lamellar phase nor a fragmentation of bilayers was observed from NMR studies. The circular dichroism studies reveal that the peptide exists as a random coil in solution but folds into a less ordered conformation upon binding to POPC/POPG (3:1) vesicles. However, G15 does not bind to lipid vesicles made of POPC/POPG/Chl (9:1:1) mixture, mimicking tumor cell membrane. These results explain the susceptibility of E. coli and the resistance of human Jurkat cells to G15, and may have implications in designing membrane-selective therapeutic agents.     

Action mechanism of tachyplesin I and effects of PEGylation

PEGylation of protein and peptide drugs is frequently used to improve in vivo efficacy. We investigated the action mechanism of tachyplesin I, a membrane-acting cyclic antimicrobial peptide from Tachypleus tridentatus and the effects of PEGylation on the mechanism. The PEGylated peptide induced the leakage of calcein from egg yolk l-α-phosphatidylglycerol/egg yolk l-α-phosphatidylcholine large unilamellar vesicles similarly to the parent peptide. Both peptides induced lipid flip-flop coupled to leakage and was translocated into the inner leaflet of the bilayer, indicating that tachyplesin I forms a toroidal pore and that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. Despite their similar activities against model membranes, the peptides showed very different biological activities. The cytotoxicity of tachyplesin I was greatly reduced by PEGylation, although the antimicrobial activity was significantly weakened. We investigated the enhancement of the permeability of inner membranes induced by the peptides. Our results suggested that outer membranes and peptidoglycan layers play an inhibitory role in the permeation of the PEG moiety. Furthermore, a reduction in DNA binding by PEGylation may also contribute to the weak activity of the PEGylated peptide.     

The interactions of antimicrobial peptides derived from lysozyme with model membrane systems

Two peptides, RAWVAWR-NH₂ and IVSDGNGMNAWVAWR-NH₂, derived from human and chicken lysozyme, respectively, exhibit antimicrobial activity. A comparison between the L-RAWVAWR, D-RAWVAWR, and the longer peptide has been carried out in membrane mimetic conditions to better understand how their interaction with lipid and detergent systems relates to the reported higher activity for the all L-peptide. Using CD and 2D ¹H NMR spectroscopy, the structures were studied with DPC and SDS micelles. Fluorescence spectroscopy was used to study peptide interactions with POPC and POPG vesicles and DOPC, DOPE, and DOPG mixed vesicle systems. Membrane-peptide interactions were also probed by ITC and DSC. The ability of fluorescein-labeled RAWVAWR to rapidly enter both E. coli and Staphylococcus aureus was visualized using confocal microscopy. Reflecting the bactericidal activity, the long peptide interacted very weakly with the lipids. The RAWVAWR-NH₂ peptides preferred lipids with negatively charged headgroups and interacted predominantly in the solvent-lipid interface, causing significant perturbation of membrane mimetics containing PG headgroups. Peptide structures determined by ¹H NMR indicated a well-ordered coiled structure for the short peptides and the C-terminus of the longer peptide. Using each technique, the two enantiomers of RAWVAWR-NH₂ interacted in an identical fashion with the lipids, indicating that any difference in activity in vivo is limited to interactions not involving the membrane lipids.     

Heterologous expression and solution structure of defensin from lentil Lens culinaris

A new defensin Lc-def, isolated from germinated seeds of the lentil Lens culinaris, has molecular mass 5440.4 Da and consists of 47 amino acid residues. Lc-def and its ¹⁵N-labeled analog were overexpressed in Escherichia coli. Antimicrobial activity of the recombinant protein was examined, and its spatial structure, dynamics, and interaction with lipid vesicles were studied by NMR spectroscopy. It was shown that Lc-def is active against fungi, but does not inhibit the growth of Gram-positive and Gram-negative bacteria. The peptide is monomeric in aqueous solution and contains one α-helix and triple-stranded β-sheet, which form cysteine-stabilized αβ motif (CSαβ) previously found in other plant defensins. The sterically neighboring loop1 and loop3 protrude from the defensin core and demonstrate significant mobility on the μs–ms timescale. Lc-def does not bind to the zwitterionic lipid (POPC) vesicles but interacts with the partially anionic (POPC/DOPG, 7:3) membranes under low-salt conditions. The Lc-def antifungal activity might be mediated through electrostatic interaction with anionic lipid components of fungal membranes.     

Membrane association and activity of 15/16-membered peptide antibiotics: Zervamicin IIB, ampullosporin A and antiamoebin I

Permeabilization of the phospholipid membrane, induced by the antibiotic peptides zervamicin IIB (ZER), ampullosporin A (AMP) and antiamoebin I (ANT) was investigated in a vesicular model system. Membrane-perturbing properties of these 15/16 residue peptides were examined by measuring the K⁺ transport across phosphatidyl choline (PC) membrane and by dissipation of the transmembrane potential. The membrane activities are found to decrease in the order ZER > AMP >> ANT, which correlates with the sequence of their binding affinities. To follow the insertion of the N-terminal Trp residue of ZER and AMP, the environmental sensitivity of its fluorescence was explored as well as the fluorescence quenching by water-soluble (iodide) and membrane-bound (5- and 16-doxyl stearic acids) quenchers. In contrast to AMP, the binding affinity of ZER as well as the depth of its Trp penetration is strongly influenced by the thickness of the membrane (diC_16:1PC, diC_18:1PC, C_16:0/C_18:1PC, diC_20:1PC). In thin membranes, ZER shows a higher tendency to transmembrane alignment. In thick membranes, the in-plane surface association of these peptaibols results in a deeper insertion of the Trp residue of AMP which is in agreement with model calculations on the localization of both peptide molecules at the hydrophilic-hydrophobic interface. The observed differences between the membrane affinities/activities of the studied peptaibols are discussed in relation to their hydrophobic and amphipathic properties.     

Cathelicidin-derived Trp/Pro-rich antimicrobial peptides with lysine peptoid residue (Nlys): therapeutic index and plausible mode of action.

Recently, we designed a novel cell-selective antimicrobial peptide (TPk) with intracellular mode of action from Pro --> Nlys (Lys peptoid residue) substitution in a noncell-selective cathelicidin-derived Trp/Pro-rich antimicrobial peptide, tritrpticin-amide (TP; VRRFPWWWPFLRR-NH(2)) (Biochemistry 2006; 45: 13007-13017). In this study, to elucidate the effect of Pro --> Nlys substitution on therapeutic index and mode of action of other noncell-selective cathelicidin-derived Trp/Pro-rich antimicrobial peptides and develop novel short antimicrobial peptides with high cell selectivity/therapeutic index, we synthesized Nlys-substituted antimicrobial peptides, TPk, STPk and INk, in which all proline residues of TP, symmetric TP-analogue (STP; KKFPWWWPFKK-NH(2)) and indolicidin (IN; ILPWKWPWWPWRR-NH(2)) were replaced by Nlys, respectively. Compared to parent Pro-containing peptides (TP, STP and IN), Nlys substituted peptides (TPk, STPk and Ink) had 4- to 26-fold higher cell selectivity/therapeutic index. Parent Pro-containing peptides induced a significant depolarization of the cytoplasmic membrane of intact Staphylococcus aureus at their MIC, whereas Nlys-substituted antimicrobial peptides did not cause visible membrane depolarization at their MIC. These results suggest that the antibacterial action of Nlys-substituted peptides is probably not due to the disruption of bacterial cytoplasmic membranes but the inhibition of intracellular components. Taken together, our results showed that Pro --> Nlys substitution in other noncell-selective Trp/Pro-rich antimicrobial peptides such as STP and IN as well as TP can improve the cell selectivity/therapeutic index and change the mode of antibacterial action from membrane-disrupting to intracellular targeting. In conclusion, our findings suggested that Pro --> Nlys substitution in noncell-selective Trp/Pro-rich antimicrobial peptides is a promising method to develop cell-selective antimicrobial peptides with intracellular target mechanism.