Molecular mechanisms of interaction of rabbit CAP18 with outer membranes of gram-negative bacteria.

The mechanism of interaction of the cationic antimicrobial protein (18 kDa), CAP18, with the outer membrane of Gram-negative bacteria was investigated applying transmission electron microscopy and voltage-clamp techniques on artificial planar bilayer membranes. Electron micrographs of bacterial cells exposed to CAP18 showed damage to the outer membrane of the sensitive Escherichia coli strains F515 and ATCC 11775, whereas the membrane of the resistant Proteus mirabilis strain R45 remained intact. Electrical measurements on various planar asymmetric bilayer membranes, one side consisting of a phospholipid mixture and the other of different phospholipids or of lipopolysaccharide (reconstitution model of the outer membrane), yielded information about the influence of CAP18 on membrane integrity. Addition of CAP18 to the side with the varying lipid composition led to lipid-specific adsorption of CAP18 and subsequent induction of current fluctuations due to the formation of transient membrane lesions at a lipid-specific clamp voltage. We propose that the applied clamp voltage leads to reorientation of CAP18 molecules adsorbed to the bilayer into an active transmembrane configuration, allowing the formation of lesions by multimeric clustering.     

Influence of proline residues on the antibacterial and synergistic activities of alpha-helical peptides.

To investigate the influence of proline residues on the activity of alpha-helical peptides, variants were synthesized with insertions of proline residues to create peptides without proline, or with one or two prolines. The influence of the proline-induced bends was assessed by circular dichroism in the presence of liposomes, and the ability of the peptides to kill microorganisms, to permeabilize the outer and cytoplasmic membranes of Escherichia coli, to bind to liposomes, to form channels in planar lipid bilayers, and to synergize with conventional antibiotics. Representative peptides adopted alpha-helical conformations in phosphatidylcholine/phosphatidylglycerol (POPC/POPG, 7:3) liposomes as well as in 60% trifluoroethanol solution, as revealed by circular dichroism (CD) spectroscopy. However, the percent of helicity decreased as the number of proline residues increased. Tryptophan fluorescence spectroscopy showed that all of these peptides inserted into the membranes of liposomes as indicated by a blue shift in the emission maximum and an increase in the fluorescence intensity of the single tryptophan at residue 2. Quenching experiments further prove that the tryptophan residue was no longer accessible to the aqueous quencher KI. The peptide that lacked proline exhibited the highest activity [minimal inhibitory concentrations (MICs) of 0.5-4 microg/mL] against all tested Gram-negative and Gram-positive bacteria, but was hemolytic at 8 microg/mL. The single-proline peptides exhibited intermediate antibacterial activity. Peptides with two proline residues were even less active with moderate MICs only against E. coli. With only one exception from each group, the peptides were nonhemolytic. The ability of the peptides to demonstrate synergy in combination with conventional antibiotics increased as the antibacterial effectiveness decreased. All peptides bound to bacterial lipopolysaccharide and permeabilized the outer membrane of E. coli to similar extents. However, their ability to permeabilize the cytoplasmic membrane of E. coli as assessed by the unmasking of cytoplasmic beta-galactosidase decreased substantially as the number of proline residues increased. Correspondingly, increasing the number of proline residues caused a decreased ability to form channels in planar lipid bilayers, and the hemolytic, proline-free peptide tended to cause rapid breakage of planar membranes. Thus, the number of bends created by insertion of proline residues is an important determinant of antimicrobial, hemolytic, and synergistic activity.     

The interaction of cationic antimicrobial peptides with vesicles containing synthetic glycolipids as models of the outer membrane of gram-negative bacteria.

Two simple lipid A analogues methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside (GL1) and methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside 4-O-phosphate (GL2) were synthesized and used for preparing mixed phosphocholine vesicles as models of the outer membrane of gram-negative bacteria. The interaction of these model membranes with magainin 2, a representative of the alpha-helical membrane active peptides, and apidaecin Ib and drosocin, two insect Pro-rich peptides which do not act at the level of the cellular membrane, were studied by CD and dye-releasing experiments. The CD spectra of apidaecin Ib and drosocin in the presence of GL1- or GL2-containing vesicles were consistent with largely unordered structures, whereas, according to the CD spectra, magainin 2 adopted an amphipathic alpha-helical conformation, particularly in the presence of negatively charged bilayers. The ability of the peptides to fold into amphipathic conformations was strictly correlated to their ability to bind and to permeabilize phospholipid as well as glycolipid membranes. Apidaecin Ib and drosocin, which are unable to adopt an amphipathic structure, showed negligible dye-leakage activity even in the presence of GL2-containing vesicles. It is reasonable to suppose that, as for the killing mechanism, the two classes of antimicrobial peptides follow different patterns to cross the bacterial outer membrane.     

Antibacterial activity and pore-forming properties of ceratotoxins: a mechanism of action based on the barrel stave model

Ceratotoxins are alpha-helical cationic peptides isolated from the medfly Ceratitis capitata. These amphipathic peptides were found to display strong antibacterial activity and weak hemolytic activity. When reconstituted into planar lipid bilayers, ceratotoxins developed highly asymmetric I/V curves under voltage ramps and formed, in single-channel experiments, well-defined voltage-dependent ion channels according to the barrel stave model. The antibacterial activity and pore-forming properties of these molecules were well correlated. Similar experiments performed with synthesized truncated fragments showed that the C-terminal domain of ceratotoxins is strongly implicated in the formation of helical bundles in the membrane whereas the largely cationic N-terminal region is likely to anchor ceratotoxins on the lipid surface.     

Antibacterial activity of linear peptides spanning the carboxy-terminal beta-sheet domain of arthropod defensins

The antibacterial activity of peptides without disulfide bridges, spanning the carboxy-terminal segment of arthropod defensins, has been investigated. Although all the peptides have net positive charges, they exhibited varying antibacterial potencies and spectra. Atomic force and fluorescence microscopic analyses indicate that the peptides exert their activity by permeabilizing the outer and inner membranes of Gram-negative bacteria such as Escherichia coli. It appears that the plasticity observed in the activity of mammalian defensins with respect to sequence, number of disulfide bridges or net positive charge, is also observed in insect defensins.     

Structure and mode of action of the antimicrobial peptide arenicin

The solution structure and the mode of action of arenicin isoform 1, an antimicrobial peptide with a unique 18-residue loop structure, from the lugworm Arenicola marina were elucidated here. Arenicin folds into a two-stranded antiparallel beta-sheet. It exhibits high antibacterial activity at 37 and 4 degrees C against Gram-negative bacteria, including polymyxin B-resistant Proteus mirabilis. Bacterial killing occurs within minutes and is accompanied by membrane permeabilization, membrane detachment and release of cytoplasm. Interaction of arenicin with reconstituted membranes that mimic the lipopolysaccharide-containing outer membrane or the phospholipid-containing plasma membrane of Gram-negative bacteria exhibited no pronounced lipid specificity. Arenicin-induced current fluctuations in planar lipid bilayers correspond to the formation of short-lived heterogeneously structured lesions. Our results strongly suggest that membrane interaction plays a pivotal role in the antibacterial activity of arenicin.     

Interaction with phospholipid bilayers, ion channel formation, and antimicrobial activity of basic amphipathic alpha-helical model peptides of various chain lengths.

Basic amphipathic alpha-helical peptides Ac-(Leu-Ala-Arg-Leu)3 or 4-NHCH3 (4(3) or 4(4)) and H-(Leu-Ala-Arg-Leu)3-(Leu-Arg-Ala-Leu)2 or 3-OH (4(5) or 4(6)) were synthesized and studied in terms of their interactions with phospholipid membranes, biological activity, and ion channel-forming ability. CD study of the peptides showed that they form alpha-helical structures in the presence of phospholipid liposomes and thus they have amphipathic distribution of the side chains along the axis of the helix. A leakage study of carboxyfluorescein encapsulated in phospholipid vesicles indicated that the peptides possess a highly potent ability to perturb the membrane structure. Membrane current measurements using the planar lipid bilayer technique revealed that the peptide 4(6), which was long enough to span the lipid bilayer in the alpha-helical structure, formed cation-selective ion channels at a concentration of 0.5 microM in a planar diphytanoylphosphatidylcholine bilayer. In contrast, other shorter peptides failed to form discrete and stable channels though they occasionally induced an increase in the membrane current with erratic conductance levels. The probability of detecting a conductance increase was in the order of 4(6) greater than 4(5) greater than 4(4) greater than 4(3), which corresponds to the order of the peptide chain lengths. Furthermore, 4(6) but not 4(5) showed an antimicrobial activity against both Gram-positive and -negative bacteria. The structure of ion channels formed by 4(6) and the relationship between the peptide chain length and biological activity of the synthetic peptides are discussed.     

The interaction of arginine- and tryptophan-rich cyclic hexapeptides with Escherichia coli membranes.

Cyclization of R- and W-rich hexapeptides has been found to enhance specifically the antimicrobial activity against Gram-negative Escherichia coli. To gain insight into the role of the bacterial outer membrane in mediating selectivity, we assayed the activity of cyclic hexapeptides derived from the parent sequence c-(RRWWRF) against several E. coli strains and Bacillus subtilis, L-form bacteria, and E. coli lipopolysaccharide (LPS) mutant strains, and we also investigated the peptide-induced permeabilization of the outer and inner membrane of E. coli. Wall-deficient L-form bacteria were distinctly less susceptible than the wild type strain. The patterns of peptide-induced permeabilization of the outer and inner E. coli membranes correlated well with the antimicrobial activity, confirming that membrane permeabilization is a detrimental effect of the peptides upon bacteria. Truncation of LPS had no influence on the activity of the cyclic parent peptide, but the highly active c-(RRWFWR), with three adjacent aromatic residues, required the complete LPS for maximal activity. Furthermore, differences in the activity of the parent peptide and its all-D sequence indicated stereospecific interactions with the LPS mutant strains. We suggest that, depending on the primary sequence of the peptides, either hydrophobic interactions with the fatty acid chains of lipid A, or electrostatic interactions disturbing the polar core region and interference with saccharide-saccharide interactions prevail in the barrier-disturbing effect upon the outer membrane and thereby provide peptide accessibility to the inner membrane. The results underline the importance of tryptophan and arginine residues and their relative location for a high antimicrobial effect, and the activity-modulating function of the outer membrane of E. coli. In addition to membrane permeabilization, the data provided evidence for the involvement of other mechanisms in growth inhibition and killing of bacteria.     

Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.     

Voltage gating in porin channels

Data from experiments in which porin channels are reconstituted into planar bilayer membranes are reviewed for their relevance to porin channel gating in vivo. Contradictory evidence concerning voltage gating indicates that the different results may stem from the variety of purification techniques employed. The likelihood of voltage gating as a property of E. coli porins in vivo is discussed in relation to the possible magnitude of the membrane potential across the outer membrane.     

Diversity of antimicrobial peptides and their mechanisms of action

Antimicrobial peptides encompass a wide variety of structural motifs. Many peptides have alpha-helical structures. The majority of these peptides are cationic and amphipathic but there are also hydrophobic alpha-helical peptides which possess antimicrobial activity. In addition, some beta-sheet peptides have antimicrobial activity and even antimicrobial alpha-helical peptides which have been modified to possess a beta-structure retain part of their antimicrobial activity. There are also antimicrobial peptides which are rich in a certain specific amino acid such as Trp or His. In addition, antimicrobial peptides exist with thio-ether rings, which are lipopeptides or which have macrocyclic Cys knots. In spite of the structural diversity, a common feature of the cationic antimicrobial peptides is that they all have an amphipathic structure which allows them to bind to the membrane interface. Indeed, most antimicrobial peptides interact with membranes and may be cytotoxic as a result of disturbance of the bacterial inner or outer membranes. Alternatively, a necessary but not sufficient property of these peptides may be to be able to pass through the membrane to reach a target inside the cell. The interaction of these peptides with biological membranes is not just a function of the peptide but is also modulated by the lipid components of the membrane. It is not likely that this diverse group of peptides has a single mechanism of action, but interaction of the peptides with membranes is an important requirement for most, if not all, antimicrobial peptides.     

Interaction of antibacterial peptides spanning the carboxy-terminal region of human beta-defensins 1-3 with phospholipids at the air-water interface and inner membrane of E. coli

Synthetic peptides Phd1-3 spanning the cationic carboxy-terminal region of human beta-defensins HBD-1-3 have been shown to have antibacterial activity. Gross morphological changes were seen in E. coli cells treated with these peptides. In this paper, we have studied the surface-active properties of peptides Phd1-3 and their interactions with different phospholipids using Langmuir-Blodgett monolayers. Compression isotherms and increase in pressure on insertion of peptides into lipid monolayers at different initial pressures indicate the affinity of these peptides for negatively charged lipids. Phd3 inserted less effectively into monolayers as compared to Phd1 and Phd2. The peptides differed in their ability to permeabilize the inner membrane of E. coli, with Phd3 being least effective. It is likely that the peptides kill Gram-negative bacteria by more than one mechanism. When hydrophobicity and net charge favor insertion into lipid membranes, then membrane permeabilization could be the primary event in the killing of bacteria. In cases where membrane insertion does not occur, interaction with phospholipid interface induces highly selective stress that leads to stasis and cell death, as proposed for polymyxin B and bactenecin.     

Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli.

Antimicrobial cationic peptides are prevalent throughout nature as part of the intrinsic defenses of most organisms, and have been proposed as a blueprint for the design of novel antimicrobial agents. They are known to interact with membranes, and it has been frequently proposed that this represents their antibacterial target. To see if this was a general mechanism of action, we studied the interaction, with model membranes and the cytoplasmic membrane of Escherichia coli, of 12 peptides representing all 4 structural classes of antimicrobial peptides. Planar lipid bilayer studies indicated that there was considerable variance in the interactions of the peptides with model phospholipid membranes, but generally both high concentrations of peptide and high transmembrane voltages (usually -180 mV) were required to observe conductance events (channels). The channels observed for most peptides varied widely in magnitude and duration. An assay was developed to measure the interaction with the Escherichia coli cytoplasmic membrane employing the membrane potential sensitive dye 3,5-dipropylthiacarbocyanine in the outer membrane barrier-defective E. coli strain DC2. It was demonstrated that individual peptides varied widely in their ability to depolarize the cytoplasmic membrane potential of E. coli, with certain peptides such as the loop peptide bactenecin and the alpha-helical peptide CP26 being unable to cause depolarization at the minimal inhibitory concentration (MIC), and others like gramicidin S causing maximal depolarization below the MIC. We discuss the mechanism of interaction with the cytoplasmic membrane in terms of the model of Matsuzaki et al. [(1998) Biochemistry 37, 15144-15153] and the possibility that the cytoplasmic membrane is not the target for some or even most cationic antimicrobial peptides.     

Wza: a new structural paradigm for outer membrane secretory proteins?

Gram-negative bacteria need to be able to transport a large variety of macromolecules across their outer membranes. In Escherichia coli, the passage of the group 1 capsular polysaccharide is mediated by an integral outer membrane protein, Wza. The crystal structure of Wza, determined recently, reveals a novel transmembrane alpha-helical barrel and a large central cavity within the core of the vase-shaped protein complex. The structure has similarities with that of the secretin protein, PilQ, which mediates the transition of type IV pili across the outer membrane. We propose that the large internal chamber, which can accommodate the secreted assembled macromolecule, is likely to be a common feature found in other outer membrane proteins involved in secretion processes.     

Cationic peptide-induced remodelling of model membranes: direct visualization by in situ atomic force microscopy

Our understanding of how antimicrobial and cell-penetrating peptides exert their action at cell membranes would benefit greatly from direct visualization of their modes of action and possible targets within the cell membrane. We previously described how the cationic antimicrobial peptide, indolicidin, interacted with mixed zwitterionic planar lipid bilayers as a function of both peptide concentration and lipid composition [Shaw, J.E. et al., 2006. J. Struct. Biol. 154 (1), 42-58]. In the present report, in situ atomic force microscopy was used to characterize the interactions between three families of cationic peptides: (1) tryptophan-rich antimicrobial peptides--indolicidin and two of its analogues, (2) an amphiphilic alpha-helical membranolytic peptide--melittin, and (3) an arginine-rich cell-penetrating peptide--Tat with phase-separated planar bilayers containing 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC)/1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) or DOPC/N-stearoyl-D-erythro-sphingosylphosphorylcholine (SM)/cholesterol. We found that these cationic peptides all induced remodelling of the model membranes in a concentration, and family-dependent manner. At low peptide concentration, these cationic peptides, despite their different biological roles, all appeared to reduce the interfacial line tension at the domain boundary between the liquid-ordered and liquid-disordered domains. Only at high peptide concentration was the membrane remodelling induced by these peptides morphologically distinct among the three families. While the transformation caused by indolicidin and its analogues were structurally similar, the concentration required to initiate the transformation was strongly dependent on the hydrophobicity of the peptide. Our use of lipid compositions with no net charge minimized the electrostatic interactions between the cationic peptides and the model supported bilayers. These results suggest that peptides within the same functional family have a common mechanism of action, and that membrane insertion of short cationic peptides at low peptide concentration may also alter membrane structure through a common mechanism regardless of the peptide's origin.     

The antibiotic activity of cationic linear amphipathic peptides: lessons from the action of leucine/lysine copolymers on bacteria of the class Mollicutes.

Peptides composed of leucyl and lysyl residues ('LK peptides') with different compositions and sequences were compared for their antibacterial activities using cell wall-less bacteria of the class Mollicutes (acholeplasmas, mycoplasmas and spiroplasmas) as targets. The antibacterial activity of the amphipathic alpha-helical peptides varied with their size, 15 residues being the optimal length, independent of the membrane hydrophobic core thickness and the amount of cholesterol. The 15-residue ideally amphipathic alpha helix with a +5 positive net charge (KLLKLLLKLLLKLLK) had the strongest antibacterial activity, similar to that of melittin. In contrast, scrambled peptides devoid of amphipathy and the less hydrophobic beta-sheeted peptides [(LK)nK], even those 15-residue long, were far less potent than the helical ones. Furthermore, the growth inhibitory activity of the peptides was correlated with their ability to abolish membrane potential. These data are fully consistent with a predominantly flat orientation of LK peptides at the lipid/water interface and strongly supports that these peptides and probably the linear polycationic amphipathic defence peptides act on bacterial membranes in four main steps according to the 'carpet' model: (a) interfacial partitioning with accumulation of monomers on the target membrane (limiting step); (b) peptide structural changes (conformation, aggregation, and orientation) induced by interactions with the lipid bilayer (as already shown with liposomes and erythrocytes); (c) plasma membrane permeabilization/depolarization via a detergent-like effect; and (d) rapid bacterial cell death if the extent of depolarization is maintained above a critical threshold.     

Inner field compensation as a tool for the characterization of asymmetric membranes and Peptide-membrane interactions

Symmetric and asymmetric planar lipid bilayers prepared according to the Montal-Mueller method are a powerful tool to characterize peptide-membrane interactions. Several electrical properties of lipid bilayers such as membrane current, membrane capacitance, and the inner membrane potential differences and their changes can be deduced. The time-resolved determination of peptide-induced changes in membrane capacitance and inner membrane potential difference are of high importance for the characterization of peptide-membrane interactions. Intercalation and accumulation of peptides lead to changes in membrane capacitance, and membrane interaction of charged peptides induces changes in the charge distribution within the membrane and with that to changes in the membrane potential profile. In this study, we establish time-resolved measurements of the capacitance minimization potential DeltaPsi on various asymmetric planar lipid bilayers using the inner field compensation method. The results are compared to the respective ones of inner membrane potential differences DeltaPhi determined from ion carrier transport measurements. Finally, the time courses of membrane capacitances and of DeltaPsi have been used to characterize the interaction of cathelicidins with reconstituted lipid matrices of various Gram-negative bacteria.     

The periplasmic maltose-binding protein modifies the channel-forming characteristics of maltoporin.

Maltoporin, a protein spanning Escherichia coli outer membranes, modifies electrical conductance of membranes due to its channel-forming properties. This observation was made by conductance measurements across planar bilayers which were derived from unextracted, isolated outer membrane vesicles using a porin-deficient E. coli strain. Alternatively, proteoliposomes reconstituted with detergent-solubilized homogeneous maltoporin and phospholipids were used. With either membrane preparation, channel conductance was observed, although no discrete conductance levels were detected. The presence of lipopolysaccharide, a bacterial glycolipid, was not required, nor did it affect channel activity. In the presence of the water-soluble periplasmic maltose-binding protein, conductance fluctuations occurred in discrete steps, demonstrating opening and closing events of channels. Multiple step sizes (1/3, 2/3 and 1 ns in 1 M KCl) in single channel traces suggest cooperative opening and closing of up to three channels. The action of maltose-binding protein is highly asymmetrical, and its affinity to maltoporin is very high (KD = 1.5 X 10(-7) M). Association of maltose-binding protein to maltoporin shifts, for a given polarity, the equilibrium between open and closed states in favour of closed states. This result matches earlier in vivo studies, and supports the physiological significance of the observations made.     

Refolding of an integral membrane protein. OmpA of Escherichia coli

OmpA is an integral membrane protein from the outer membrane of Escherichia coli. Purified, lipopolysaccharide-free OmpA was denatured by boiling in sodium dodecyl sulfate (SDS). Refolding was then induced by replacement of SDS with the nonionic detergent octylglucoside. The structure of both the denatured and refolded protein were investigated by SDS-gel electrophoresis, protease digestion, Raman and fluorescence spectroscopy. Refolded OmpA could be reconstituted into membranes of the synthetic lipid dimyristoylphosphatidylcholine. Thus, lipopolysaccharide is neither necessary for proper folding of OmpA nor for its insertion into lipid membranes. Based on this result, models for sorting of OmpA into the outer membrane of E. coli are discussed.     

Bacteriocin release proteins: mode of action, structure, and biotechnological application

Abstract: The mechanisms by which Gram-negative bacteria like Escherichia coli secrete bacteriocins into the culture medium is unique and quite different from the mechanism by which other proteins are translocated across the two bacterial membranes, namely through the known branches of the general secretory pathway. The release of bacteriocins requires the expression and activity of a so-called bacteriocin release protein and the presence of the detergent-resistant phospholipase A in the outer membrane. The bacteriocin release proteins are highly expressed small lipoproteins which are synthesized with a signal peptide that remains stable and which accumulates in the cytoplasmic membrane after cleavage. The combined action of these stable, accumulated signal peptides, the lipid-modified mature bacteriocin release proteins (BRPs) and phospholipase A cause the release of bacteriocins. The structure and mode of action of these BRPs as well as their application in the release of heterologous proteins by E. coli is described in this review.