Enhanced membrane pore formation by multimeric/oligomeric antimicrobial peptides

The pore-forming antibacterial peptide magainin 2 was made divalent, tetravalent, and octavalent via a copper(I)-mediated 1-3 dipolar cycloaddition reaction ("click" chemistry). This series of pore-forming compounds was tested in vitro for their ability to form pores in large unilamillar vesicles (LUVs). A large increase in the pore-forming capability was especially observed with the tetravalent and octavalent magainin compounds in the LUVs consisting of DOPC, and the octavalent magainin compound showed a marked increase with the DOPC/DOPG LUVs. Activity was observed in the low nanomolar range for these compounds.     

Enhanced membrane pore formation through high-affinity targeted antimicrobial peptides

Many cationic antimicrobial peptides (AMPs) target the unique lipid composition of the prokaryotic cell membrane. However, the micromolar activities common for these peptides are considered weak in comparison to nisin, which follows a targeted, pore-forming mode of action. Here we show that AMPs can be modified with a high-affinity targeting module, which enables membrane permeabilization at low concentration. Magainin 2 and a truncated peptide analog were conjugated to vancomycin using click chemistry, and could be directed towards specific membrane embedded receptors both in model membrane systems and whole cells. Compared with untargeted vesicles, a gain in permeabilization efficacy of two orders of magnitude was reached with large unilamellar vesicles that included lipid II, the target of vancomycin. The truncated vancomycin-peptide conjugate showed an increased activity against vancomycin resistant Enterococci, whereas the full-length conjugate was more active against a targeted eukaryotic cell model: lipid II containing erythrocytes. This study highlights that AMPs can be made more selective and more potent against biological membranes that contain structures that can be targeted.     

Energetics of pore formation induced by membrane active peptides

Antimicrobial peptides are known to form pores in cell membranes. We study this process in model bilayers of various lipid compositions. We use two of the best-studied peptides, alamethicin and melittin, to represent peptides making two types of pores, that is, barrel-stave pores and toroidal pores. In both cases, the key control variable is the concentration of the bound peptides in the lipid bilayers (expressed in the peptide-lipid molar ratio, P/L). The method of oriented circular dichroism (OCD) was used to monitor the peptide orientation in bilayers as a function of P/L. The same samples were scanned by X-ray diffraction to measure the bilayer thickness. In all cases, the bilayer thickness decreases linearly with P/L and then levels off after P/L exceeds a lipid-dependent critical value, (P/L)*. OCD spectra showed that the helical peptides are oriented parallel to the bilayers as long as P/L < (P/L)*, but as P/L increases over (P/L)*, an increasing fraction of peptides changed orientation to become perpendicular to the bilayer. We analyzed the data by assuming an internal membrane tension associated with the membrane thinning. The free energy containing this tension term leads to a relation explaining the P/L-dependence observed in the OCD and X-ray diffraction measurements. We extracted the experimental parameters from this thermodynamic relation. We believe that they are the quantities that characterize the peptide-lipid interactions related to the mechanism of pore formation. We discuss the meaning of these parameters and compare their values for different lipids and for the two different types of pores. These experimental parameters are useful for further molecular analysis and are excellent targets for molecular dynamic simulation studies.     

Many-body effect of antimicrobial peptides: on the correlation between lipid's spontaneous curvature and pore formation

Recently we have shown that the free energy for pore formation induced by antimicrobial peptides contains a term representing peptide-peptide interactions mediated by membrane thinning. This many-body effect gives rise to the cooperative concentration dependence of peptide activities. Here we performed oriented circular dichroism and x-ray diffraction experiments to study the lipid dependence of this many-body effect. In particular we studied the correlation between lipid's spontaneous curvature and peptide's threshold concentration for pore formation by adding phosphatidylethanolamine and lysophosphocholine to phosphocholine bilayers. Previously it was argued that this correlation exhibited by magainin and melittin supported the toroidal model for the pores. Here we found similar correlations exhibited by melittin and alamethicin. We found that the main effect of varying the spontaneous curvature of lipid is to change the degree of membrane thinning, which in turn influences the threshold concentration for pore formation. We discuss how to interpret the lipid dependence of membrane thinning.     

Lipid‐dependent pore formation by antimicrobial peptides arenicin‐2 and melittin demonstrated by their proton transfer activity

This work presents a comparative study of proton transfer activity (PTA) of two cationic (+6) antimicrobial peptides, β‐structural arenicin‐2 and α‐helical melittin. A new approach was proposed for the detection of passive proton transfer by using proteoliposomes containing bacteriorhodopsin, which creates a small light‐induced electrochemical proton gradient ?ΔpH. Addition of several nanomoles of the peptides lowers ?ΔpH that is proximately indicative of the pore formation. The quantitative analysis of sigmoidal dependences of ?pH on the peptides concentration was carried out using liposomes prepared from PC, PC/PE, PC/PE/PI and PC/PG. Substitution of PC‐containing liposomes with PE‐containing ones, having negative spontaneous curvature, reduced the PTA of α‐helical melittin and increased that of β‐structural arenicin‐2. This result indicates an essential difference in the pore formation by these peptides. Further increase of PTA in response to arenicin‐2 (in contrast to melittin) was observed in the liposomes prepared from PC/PE/PI. The data analysis leads to the conclusion that PTA is influenced by (i) efficiency of the pore assemblage, which depends on the structure of pore‐forming peptides, and the spontaneous curvature of lipids and (ii) the presence of mobile protons in the polar head groups of phospholipids. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Interactions of peptides with liposomes: pore formation and fusion

Leakage from liposomes induced by several peptides is reviewed and a pore model is described. According to this model peptide molecules become incorporated into the vesicle bilayer and aggregate reversibly or irreversibly within the surface. When a peptide aggregate reaches a critical size, peptide translocation can occur and a pore is formed. With the peptide GALA the pores are stable and persist for at least 10 minutes. The model predicts that for a given lipid/peptide ratio, the extent of leakage should decrease as the vesicle diameter decreases, and for a given amount of peptide bound per vesicle less leakage would be observed at higher temperatures due to the increase in reversibility of surface aggregates of the peptide. Effect of membrane composition on pore formation is reviewed. When cholesterol was included in the liposomes the efficiency of inducation of leakage by the peptide GALA was reduced due to reduced binding and increased reversibility of surface aggregation of the peptide. Phospholipids which contain less ordered acyl-chains and have a slightly wedge-like shape, can better accommodate peptide surface aggregates, and consequently insertion and translocation of the peptide may be less favored. Demonstrations of antagonism between pore formation and fusion are presented. The choice of factors which promote vesicle aggregation, e.g., larger peptides, increased vesicle and peptide concentration results in enhanced vesicle fusion at the expense of formation of intravesicular pores. FTIR studies with HIV-1 fusion peptides indicate that in systems where extensive vesicle fusion occurred the beta conformation of the peptides was predominant, whereas the alpha conformation was exhibited in cases where leakage was the main outcome. Antagonism between leakage and fusion was exhibited by 1-palmitoyl-2-oleoylphosphatidylglycerol vesicles, where the order of addition of peptide (HIV(arg)) or Ca(2+)dictated whether pore formation or vesicle fusion would occur. The current study emphasizes that the addition of Ca(2+), which promotes vesicle aggregation can also reduce peptide translocation in isolated vesicles.     

Surface aggregation and membrane penetration by peptides: relation to pore formation and fusion

The peptide GALA undergoes a conformational change to an amphipathic alpha -helix when the pH is reduced, inducing leakage of contents from vesicles. Leakage from neutral or negativelycharged vesicles at pH 5.0 was similar and could be adequately explained by a mathematical model which assumed that GALA becomes incorporated into the vesicle bilayer and irreversibly aggregates to form a pore consisting of M =10+/-2 peptides. Increasing cholesterol content in the membranes resulted in reduced leakage, and increased reversibility of surface aggregation of the peptide. Employing fluorescently labelled peptides confirmed that the degree of reversibility of surface aggregation of GALA was significantly larger in cholesterol containing liposomes. Orientation of the peptide GALA in bilayers was determined by a bodipy-avidin/ biotin binding assay. The peptide was labelled by biotin at the N- or Cterminus and bodipy-avidin molecules were added externally or were preencapsulated in the vesicles. The peptides are arranged in the pore perpendicularly to the membrane, such that 3/4 of the N-termini are on the internal side of the membrane. The pores are stable and persist for at least 10 min. When the peptides form an aggregate of size smaller than M, the orientation of the peptide is mostly parallel to the surface and the biotinylated peptide does not translocate. When a critical size of the aggregate is attained, a rearrangement of the peptide occurs, which amounts to rapid penetration and formation of a pore structure. Induction of fusion by peptides may be antagonistic to pore formation, the outcome being dependent on vesicle aggregation.     

Dissociation of antimicrobial and hemolytic activities in cyclic peptide diastereomers by systematic alterations in amphipathicity

We have investigated the role of amphipathicity in a homologous series of head-to-tail cyclic antimicrobial peptides in efforts to delineate features resulting in high antimicrobial activity coupled with low hemolytic activity (i.e. a high therapeutic index). The peptide GS14, cyclo(VKLKVd-YPLKVKLd-YP), designed on the basis of gramicidin S (GS), exists in a preformed highly amphipathic beta-sheet conformation and was used as the base compound for this study. Fourteen diastereomers of GS14 were synthesized; each contained a different single enantiomeric substitution within the framework of GS14. The beta-sheet structure of all GS14 diastereomers was disrupted as determined by CD and NMR spectroscopy under aqueous conditions; however, all diastereomers exhibited differential structure inducibility in hydrophobic environments. Because the diastereomers all have the same composition, sequence, and intrinsic hydrophobicity, the amphipathicity of the diastereomers could be ranked based upon retention time from reversed-phase high performance liquid chromatography. There was a clear correlation showing that high amphipathicity resulted in high hemolytic activity and low antimicrobial activity in the diastereomers. The latter may be the result of increased affinity of highly amphipathic peptides to outer membrane components of Gram-negative microorganisms. The diastereomers possessing the most favorable therapeutic indices possessed some of the lowest amphipathicities, although there was a threshold value below which antimicrobial activity decreased. The best diastereomer exhibited 130-fold less hemolytic activity compared with GS14, as well as greatly increased antimicrobial activities, resulting in improvement in therapeutic indices of between 1,000- and 10,000-fold for a number of microorganisms. The therapeutic indices of this peptide were between 16- and 32-fold greater than GS for Gram-negative microorganisms and represents a significant improvement in specificity over GS. Our findings show that a highly amphipathic nature is not desirable in the design of constrained cyclic antimicrobial peptides and that an optimum amphipathicity can be defined by systematic enantiomeric substitutions.     

Elementary processes of antimicrobial peptide PGLa-induced pore formation in lipid bilayers

Antimicrobial peptide PGLa induces the leakage of intracellular content, leading to its bactericidal activity. However, the elementary process of PGLa-induced leakage remains poorly understood. Here, we examined the interaction of PGLa with lipid bilayers using the single giant unilamellar vesicle (GUV) method. We found that PGLa induced membrane permeation of calcein from GUVs comprised of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG) and its rate increased with time to reach a steady value, indicating that PGLa induced pores in the bilayer. The binding of PGLa to the GUV membrane raised its fractional area change, δ. At high PGLa concentrations, the time course of δ showed a two-step increase; δ increased to a value, δ₁, which was constant for an extended period before increasing to another constant value, δ₂, that persisted until aspiration of the GUV. To reveal the distribution of PGLa, we investigated the interaction of a mixture of PGLa and carboxyfluorescein (CF) -labeled PGLa (CF-PGLa) with single GUVs. The change of the fluorescence intensity of the GUV rim, I, over time showed a two-step increase from a steady value, I₁, to another, I₂, concomitant with the entering of CF-PGLa into the lumen of the GUV prior to AF647 leakage. The simultaneous measurement of δ and I indicated that their time courses were virtually the same and the ratios (δ₂/δ₁ and I₂/I₁) were almost 2. These results indicated that CF-PGLa translocated across the bilayer before membrane permeation. Based on these results, the elementary processes of the PGLa-induced pore formation were discussed.     

The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy

Some mastoparan peptides extracted from social wasps display antimicrobial activity and some are hemolytic and cytotoxic. Although the cell specificity of these peptides is complex and poorly understood, it is believed that their net charges and their hydrophobicity contribute to modulate their biological activities. We report a study, using fluorescence and circular dichroism spectroscopies, evaluating the influence of these two parameters on the lytic activities of five mastoparans in zwitterionic and anionic phospholipid vesicles. Four of these peptides, extracted from the venom of the social wasp Polybia paulista, present both acidic and basic residues with net charges ranging from +1 to +3 which were compared to Mastoparan-X with three basic residues and net charge +4. Previous studies revealed that these peptides have moderate-to-strong antibacterial activity against Gram-positive and Gram-negative microorganisms and some of them are hemolytic. Their affinity and lytic activity in zwitterionic vesicles decrease with the net electrical charges and the dose response curves are more cooperative for the less charged peptides. Higher charged peptides display higher affinity and lytic activity in anionic vesicles. The present study shows that the acidic residues play an important role in modulating the peptides’ lytic and biological activities and influence differently when the peptide is hydrophobic or when the acidic residue is in a hydrophilic peptide.     

Influence of antimicrobial peptides on the formation of nonlamellar lipid mesophases

We have studied the influence of four antimicrobial peptides of different secondary and ternary structure - melittin (Mel), protegrin-1 (PG-1), peptidyl-glycylleucine-carboxyamide (PGLa), and gramicidin S (GS) - on the lamellar-to-nonlamellar transition of palmitoyloleoyl phosphatidylethanolamine (POPE) applying differential scanning calorimetry and small-angle X-ray diffraction. None of the peptides studied led to the formation of an inverted hexagonal phase observed for pure POPE at high temperatures. Instead either cubic or lamellar phases were stabilized to different degrees. GS was most effective in inducing a cubic phase, whereas Mel fully stabilized the lamellar phase. The behavior of POPE in the presence of PG-1 and PGLa was intermediate to GS and Mel. In addition to the known role of membrane elasticity we propose two mechanisms, which cause stabilization of the lamellar phase: electrostatic repulsion and lipid/peptide pore formation. Both mechanisms prevent transmembrane contact required to form either an inverted hexagonal phase or fusion pores, as precursors of the cubic phase.     

抗菌肽活性及天然抗菌肽的改造

抗菌肽具有抗菌谱广、热稳定性强、分子量小及免疫原性小等特点,其杀菌机制独特,病原菌不易产生耐药性,有望开发成新一代肽类抗生素。本文主要综述了影响抗菌肽生物活性的生化性质,即螺旋度、疏水性、两亲性、正电荷数等,并从结构的角度论述了其对抗菌肽抑菌活性的影响。部分抗菌肽具有空间结构不稳定、溶血活性等缺点,限制了其临床应用。因此,对天然抗菌肽的改造也成为目前抗菌肽的研究热点,本文还综述了天然抗菌肽的改造方法。     

Membrane selectivity by W-tagging of antimicrobial peptides

A pronounced membrane selectivity is demonstrated for short, hydrophilic, and highly charged antimicrobial peptides, end-tagged with aromatic amino acid stretches. The mechanisms underlying this were investigated by a method combination of fluorescence and CD spectroscopy, ellipsometry, and Langmuir balance measurements, as well as with functional assays on cell toxicity and antimicrobial effects. End-tagging with oligotryptophan promotes peptide-induced lysis of phospholipid liposomes, as well as membrane rupture and killing of bacteria and fungi. This antimicrobial potency is accompanied by limited toxicity for human epithelial cells and low hemolysis. The functional selectivity displayed correlates to a pronounced selectivity of such peptides for anionic lipid membranes, combined with a markedly reduced membrane activity in the presence of cholesterol. As exemplified for GRR10W4N (GRRPRPRPRPWWWW-NH(2)), potent liposome rupture occurs for anionic lipid systems (dioleoylphosphatidylethanolamine (DOPE)/dioleoylphosphatidylglycerol (DOPG) and Escherichia coli lipid extract) while that of zwitterionic dioleoylphosphatidylcholine (DOPC)/cholesterol is largely absent under the conditions investigated. This pronounced membrane selectivity is due to both a lower peptide binding to the zwitterionic membranes (z≈-8-10mV) than to the anionic ones (z≈-35-40mV), and a lower degree of membrane incorporation in the zwitterionic membranes, particularly in the presence of cholesterol. Replacing cholesterol with ergosterol, thus mimicking fungal membranes, results in an increased sensitivity for peptide-induced lysis, in analogy to the antifungal properties of such peptides. Finally, the generality of the high membrane selectivity for other peptides of this type is demonstrated.     

Midgut juice components affect pore formation by the Bacillus thuringiensis insecticidal toxin Cry9Ca

The pore-forming ability of the Bacillus thuringiensis toxin Cry9Ca, its two single-site mutants R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at R164 was evaluated under a variety of experimental conditions using an electrophysiological assay. All four toxin preparations depolarized the apical membrane of freshly isolated third-instar Manduca sexta midguts bathing in a solution containing 122 mM KCl at pH 10.5, but the 55-kDa fragment was considerably more active than Cry9Ca and its mutants. The activity of the latter toxins was greatly enhanced, however, when the experiments were conducted in the presence of fifth-instar M. sexta midgut juice. This effect was also observed after midgut juice proteins had been denatured by heating at 95 °C or after inorganic ions and small molecules had been removed from the midgut juice by extensive dialysis. A similar stimulation of toxin activity was also observed when the experiments were carried out in the presence of the lipids extracted from an equivalent volume of midgut juice. Depolarization of the cell membrane was also greatly enhanced, in the absence of midgut juice, by the addition of a cocktail of water-soluble protease inhibitors. These results indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut.     

Evidence for widespread distribution of piscidin antimicrobial peptides in teleost fish

Antimicrobial peptides (AMPs) are increasingly recognized as a critical component of the host's defense against infection. Several types of AMPs have been recently identified from mucosal tissues or immune cells of a number of teleosts. Among these are the piscidins, which are 22 residue, alpha-helical AMPs that were originally isolated from mast cells of hybrid striped bass Morone saxatilis male x Morone chrysops female. Using an antibody specific for the conserved N-terminal amino acid sequence of piscidin 1, we used immunohistochemistry to probe skin, gill, and gastrointestinal tract of 39 teleosts representing 7 different orders. Nine fish species were piscidin-positive, with all of these species being in the Perciformes, the largest and most evolutionarily advanced order of teleosts. Piscidin-positive cells were identified in species belonging to the families Moronidae, Serranidae, Sciaenidae, Siganidae and Belontidae. Immunopositive cells were usually most consistent with mast cells, although in some species, the granule appearance and tinctorial properties diverged somewhat from those of a typical piscine mast cell. In addition, rodlet cells were piscidin-positive in one member of the family Cichlidae; to our knowledge, it is the first time that a host-associated chemical biomarker has been identified in rodlet cells. Our data suggest that piscidins are present in many evolutionarily advanced teleosts. Piscidin-immunoreactive cells were most common at sites of pathogen entry, including the skin, gill and gastrointestinal tract. These results strongly suggest that piscidins are a widespread and important component of many fishes' defense against disease.     

Characterization of histatin 5 with respect to amphipathicity, hydrophobicity, and effects on cell and mitochondrial membrane integrity excludes a candidacidal mechanism of pore formation

Histatin 5 is a 24-residue peptide from human saliva with antifungal properties. We recently demonstrated that histatin 5 translocates across the yeast membrane and targets to the mitochondria, suggesting an unusual antifungal mechanism (Helmerhorst, E. J., Breeuwer, P., van't Hof, W., Walgreen-Weterings, E., Oomen, L. C. J. M., Veerman, E. C. I., Nieuw Amerongen, A. V., and Abee, T. (1999) J. Biol. Chem. 274, 7286-7291). The present study used specifically designed synthetic analogs of histatin 5 to elucidate the role of peptide amphipathicity, hydrophobicity, and the propensity to adopt alpha-helical structures in relation to membrane permeabilization and fungicidal activity. Studies included circular dichroism measurements, evaluation of the effects on the cytoplasmic transmembrane potential and on the respiration of isolated mitochondria, and analysis of the peptide hydrophobicity/amphipathicity relationship (Eisenberg, D. (1984) Annu. Rev. Biochem. 53, 595-623). The 14-residue synthetic peptides used were dh-5, comprising the functional domain of histatin 5, and dhvar1 and dhvar4, both designed to maximize amphipathic characteristics. The results obtained show that the amphipathic analogs exhibited a high fungicidal activity, a high propensity to form an alpha-helix, dissipated the cytoplasmic transmembrane potential, and uncoupled the respiration of isolated mitochondria, similar to the pore-forming peptide PGLa (Peptide with N-terminal Glycine and C-terminal Leucine-amide). In contrast, histatin 5 and dh-5 showed fewer or none of these features. The difference in these functional characteristics between histatin 5 and dh-5 on the one hand and dhvar1, dhvar4, and PGLa on the other hand correlated well with their predicted affinity for membranes based on hydrophobicity/amphipathicity analysis. These data indicate that the salivary protein histatin 5 exerts its antifungal function through a mechanism other than pore formation.     

Paneth cell antimicrobial peptides: topographical distribution and quantification in human gastrointestinal tissues

Antimicrobial peptides and proteins are key effectors of innate immunity, expressed both by circulating phagocytic cells and by epithelial cells of mucosal tissues. In the human small intestine, Paneth cells are secretory epithelial cells that express the antimicrobials human alpha-defensin-5 (HD5), HD6, lysozyme and secretory phospholipase A(2) (sPLA(2)), and recent studies have implicated reduced HD5 and HD6 expression levels in the pathogenesis of ileal Crohn's disease. However, expression levels of these molecules have not been determined routinely by techniques that readily permit quantitative comparisons of their distribution between tissues and samples. Using quantitative real-time PCR with external standards and Northern blot analysis, we compared expression levels of mRNA encoding these four Paneth cell antimicrobial peptides, as well as circulating human neutrophil defensins in several different gastrointestinal tissues and the bone marrow. HD5 and HD6 were the most abundant antimicrobials expressed in the small intestine. The concentration of HD5 mRNA is approximately 5 x 10(5) copies per 10ng RNA in the jejunum and ileum; HD6 mRNA levels were about six times lower than those of HD5. With the exception of low levels in the pancreas (10(3) copies/10 ng RNA), the expression of HD5 and HD6 in tissues other than small intestine was at or below detectable limits. The expression of sPLA2 and lysozyme mRNA was observed in the small intestine (approximately, 3 x 10(3) and 9 x 10(3) copies/10 ng RNA, respectively), but also in several other tissues. Lysozyme expression was high in the duodenum (10(5) copies/10 ng RNA), and the protein localized to both Brunner's glands in the lamina propria and Paneth cells. By comparison, the hematopoietic alpha-defensins HNP1-3 mRNA were detected at 6 x 10(5) copies per 10 ng RNA in the bone marrow. These quantitative RT-PCR data from healthy tissues represents the first quantitative topographical assessment of antimicrobial expression in the gastrointestinal tract and provides a means to directly compare expression levels between healthy tissues and disease specimens for multiple antimicrobial peptides.     

Plant defense and antimicrobial peptides

Plants are constantly exposed to a large array of pathogenic organisms and the survival in these conditions demands quick defense responses which include the synthesis of defense peptides and proteins with antimicrobial properties. The main groups of antimicrobial peptides found in plants are thionins, defensins and lipid transfer proteins. They constitute interesting candidates to engineer disease resistance in plants.