Imaging the Lipid-Phase-Dependent Pore Formation of Equinatoxin II in Droplet Interface Bilayers

Using phase-separated droplet interface bilayers, we observe membrane binding and pore formation of a eukaryotic cytolysin, Equinatoxin II (EqtII). EqtII activity is known to depend on the presence of sphingomyelin in the target membrane and is enhanced by lipid phase separation. By imaging the ionic flux through individual pores in vitro, we observe that EqtII pores form predominantly within the liquid-disordered phase. We observe preferential binding of labeled EqtII at liquid-ordered/liquid-disordered domain boundaries before it accumulates in the liquid-disordered phase.     

Effect of lipid on the conformation of the N-terminal region of equinatoxin II: a synchrotron radiation circular dichroism spectroscopic study

Equinatoxin II (EqtII) is a protein toxin that lyses both red blood cells and artificial membranes. Lysis is dependent on the lipid composition, with small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC) and sphingomyelin (SM) (1:1 molar) being lysed more readily than those of phosphatidylcholine alone. Removing the N-terminus of EqtII prevents pore formation, but does not prevent membrane binding. A peptide corresponding to residues 1–32 of EqtII was found using NMR to adopt a helical structure in micelles. To further understand the structural changes that accompany membrane insertion, synchrotron radiation circular dichroism spectra of the N-terminal peptide in a range of model membranes have been analysed. The peptide structure was examined in water, dodecylphosphocholine (DPC) and DPC:SM (5:1) micelles, and SUVs composed of dioleoylphosphatidylcholine (DOPC) or DMPC, together with SM and cholesterol (Chol). The peptide adopted different conformations in different lipids. Although the presence of SM did not affect the conformation in micelles, inclusion of SM in the bilayer-forming lipid increased the helicity of the peptide. This effect was abolished when Chol was added in DOPC but not in DMPC, which may relate to liquid ordered versus disordered phase properties of the lipid. SM may act as a promoter of membrane organisation necessary for membrane lysis by EqtII.     

The effects of lipids on the structure of the eukaryotic cytolysin equinatoxin II: A synchrotron radiation circular dichroism spectroscopic study

Synchrotron radiation circular dichroism (SRCD) spectroscopy studies of the eukaryotic pore-forming protein equinatoxin II (EqtII) were carried out in solution and in the presence of micelles or small unilamellar vesicles (SUV) of different lipid composition. The SRCD structural data was correlated with calcein leakage from SUV and with partitioning of EqtII to liposomes, and micelles, according to haemolysis assays. The structure of EqtII in water and dodecylphosphocholine micelles as determined by SRCD was similar to the values calculated from crystal and solution structures of the protein, and no changes were observed with the addition of sphingomyelin (SM). SM is required to trigger pore formation in biological and model membranes, but our results suggest that SM alone is not sufficient to trigger dissociation of the N-terminal helix and further structural rearrangements required to produce a pore. Significant changes in conformation of EqtII were detected with unsaturated phospholipid (DOPC) vesicles when SM was added, but not with saturated phospholipids (DMPC), which suggests that not only is membrane curvature important, but also the fluidity of the bilayer. The SRCD data indicated that the EqtII structure in the presence of DOPC:SM SUV represents the ‘bound’ state and the ‘free’ state is represented by spectra for DOPC or DOPC:Chol vesicles, which correlates with the high lytic activity for SUV of DOPC:SM. The SRCD results provide insight into the lipid requirements for structural rearrangements associated with EqtII toxicity and lysis.     

Solid-state NMR study of membrane interactions of the pore-forming cytolysin, equinatoxin II

Equinatoxin II (EqtII) is a pore-forming protein from Actinia equina that lyses red blood cell and model membranes. Lysis is dependent on the presence of sphingomyelin (SM) and is greatest for vesicles composed of equimolar SM and phosphatidylcholine (PC). Since SM and cholesterol (Chol) interact strongly, forming domains or “rafts” in PC membranes, ³¹P and ²H solid-state NMR were used to investigate changes in the lipid order and bilayer morphology of multilamellar vesicles comprised of different ratios of dimyristoylphosphatidylcholine (DMPC), SM and Chol following addition of EqtII. The toxin affects the phase transition temperature of the lipid acyl chains, causes formation of small vesicle type structures with increasing temperature, and changes the T₂ relaxation time of the phospholipid headgroup, with a tendency to order the liquid disordered phases and disorder the more ordered lipid phases. The solid-state NMR results indicate that Chol stabilizes the DMPC bilayer in the presence of EqtII but leads to greater disruption when SM is in the bilayer. This supports the proposal that EqtII is more lytic when both SM and Chol are present as a consequence of the formation of domain boundaries between liquid ordered and disordered phases in lipid bilayers leading to membrane disruption.     

The effects of lipids on the structure of the eukaryotic cytolysin equinatoxin II: a synchrotron radiation circular dichroism spectroscopic study

Synchrotron radiation circular dichroism (SRCD) spectroscopy studies of the eukaryotic pore-forming protein equinatoxin II (EqtII) were carried out in solution and in the presence of micelles or small unilamellar vesicles (SUV) of different lipid composition. The SRCD structural data was correlated with calcein leakage from SUV and with partitioning of EqtII to liposomes, and micelles, according to haemolysis assays. The structure of EqtII in water and dodecylphosphocholine micelles as determined by SRCD was similar to the values calculated from crystal and solution structures of the protein, and no changes were observed with the addition of sphingomyelin (SM). SM is required to trigger pore formation in biological and model membranes, but our results suggest that SM alone is not sufficient to trigger dissociation of the N-terminal helix and further structural rearrangements required to produce a pore. Significant changes in conformation of EqtII were detected with unsaturated phospholipid (DOPC) vesicles when SM was added, but not with saturated phospholipids (DMPC), which suggests that not only is membrane curvature important, but also the fluidity of the bilayer. The SRCD data indicated that the EqtII structure in the presence of DOPC:SM SUV represents the 'bound' state and the 'free' state is represented by spectra for DOPC or DOPC:Chol vesicles, which correlates with the high lytic activity for SUV of DOPC:SM. The SRCD results provide insight into the lipid requirements for structural rearrangements associated with EqtII toxicity and lysis.     

The Influence of Hydrogen Bonding on Sphingomyelin/Colipid Interactions in Bilayer Membranes

The phospholipid acyl chain composition and order, the hydrogen bonding, and properties of the phospholipid headgroup all influence cholesterol/phospholipid interactions in hydrated bilayers. In this study, we examined the influence of hydrogen bonding on sphingomyelin (SM) colipid interactions in fluid uni- and multilamellar vesicles. We have compared the properties of oleoyl or palmitoyl SM with comparable dihydro-SMs, because the hydrogen bonding properties of SM and dihydro-SM differ. The association of cholestatrienol, a fluorescent cholesterol analog, with oleoyl sphingomyelin (OSM) was significantly stronger than its association with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in bilayers with equal acyl chain order. The association of cholestatrienol with dihydro-OSM, which lacks a trans double bond in the sphingoid base, was even stronger than the association with OSM, suggesting an important role for hydrogen bonding in stabilizing sterol/SM interactions. Furthermore, with saturated SM in the presence of 15 mol % cholesterol, cholesterol association with fluid dihydro-palmitoyl SM bilayers was stronger than seen with palmitoyl SM under similar conditions. The different hydrogen bonding properties in OSM and dihydro-OSM bilayers also influenced the segregation of palmitoyl ceramide and dipalmitoylglycerol into an ordered phase. The ordered, palmitoyl ceramide-rich phase started to form above 2 mol % in the dihydro-OSM bilayers but only above 6 mol % in the OSM bilayers. The lateral segregation of dipalmitoylglycerol was also much more pronounced in dihydro-OSM bilayers than in OSM bilayers. The results show that hydrogen bonding is important for sterol/SM and ceramide/SM interactions, as well as for the lateral segregation of a diglyceride. A possible molecular explanation for the different hydrogen bonding in SM and dihydro-SM bilayers is presented and discussed.     

Imaging the Lipid-Phase-Dependent Pore Formation of Equinatoxin II in Droplet Interface Bilayers

Using phase-separated droplet interface bilayers, we observe membrane binding and pore formation of a eukaryotic cytolysin, Equinatoxin II (EqtII). EqtII activity is known to depend on the presence of sphingomyelin in the target membrane and is enhanced by lipid phase separation. By imaging the ionic flux through individual pores in vitro, we observe that EqtII pores form predominantly within the liquid-disordered phase. We observe preferential binding of labeled EqtII at liquid-ordered/liquid-disordered domain boundaries before it accumulates in the liquid-disordered phase.     

Structural Evidence for Inter-Residue Hydrogen Bonding Observed for Cellobiose in Aqueous Solution

The structure of the disaccharide cellulose subunit cellobiose (4-O-β-D-glucopyranosyl-D-glucose) in solution has been determined via neutron diffraction with isotopic substitution (NDIS), computer modeling and nuclear magnetic resonance (NMR) spectroscopic studies. This study shows direct evidence for an intramolecular hydrogen bond between the reducing ring HO3 hydroxyl group and the non-reducing ring oxygen (O5′) that has been previously predicted by computation and NMR analysis. Moreover, this work shows that hydrogen bonding to the non-reducing ring O5′ oxygen is shared between water and the HO3 hydroxyl group with an average of 50% occupancy by each hydrogen-bond donor. The glycosidic torsion angles φ_H and ψ_H from the neutron diffraction-based model show a fairly tight distribution of angles around approximately 22^° and −40^°, respectively, in solution, consistent with the NMR measurements. Similarly, the hydroxymethyl torsional angles for both reducing and non-reducing rings are broadly consistent with the NMR measurements in this study, as well as with those from previous measurements for cellobiose in solution.     

Effects of the eukaryotic pore-forming cytolysin Equinatoxin II on lipid membranes and the role of sphingomyelin

Equinatoxin II (EqtII), a protein toxin from the sea anemone Actinia equina, readily creates pores in sphingomyelin-containing lipid membranes. The perturbation by EqtII of model lipid membranes composed of dimyristoylphosphatidycholine and sphingomyelin (10 mol %) was investigated using wideline phosphorus-31 and deuterium NMR. The preferential interaction between EqtII (0.1 and 0.4 mol %) and the individual bilayer lipids was studied by (31)P magic angle spinning NMR, and toxin-induced changes in bilayer morphology were examined by freeze-fracture electron microscopy. Both NMR and EM showed the formation of an additional lipid phase in sphingomyelin-containing mixed lipid multilamellar suspensions with 0.4 mol % EqtII. The new toxin-induced phase consisted of small unilamellar vesicles 20-40 nm in diameter. Deuterium NMR showed that the new lipid phase contains both dimyristoylphosphatidycholine and sphingomyelin. Solid-state (31)P NMR showed an increase in spin-lattice and a decrease in spin-spin relaxation times in mixed-lipid model membranes in the presence of EqtII, consistent with an increase in the intensity of low frequency motions. The (2)H and (31)P spectral intensity distributions confirmed a change in lipid mobility and showed the creation of an isotropic lipid phase, which was identified as the small vesicle structures visible by electron microscopy in the EqtII-lipid suspensions. The toxin appears to enhance slow motions in the membrane lipids and destabilize the membrane. This effect was greatly enhanced in sphingomyelin-containing mixed lipid membranes compared with pure phosphatidylcholine bilayers, suggesting a preferential interaction between the toxin and bilayer sphingomyelin.     

Molecular dynamics simulation of adrenocorticotropin (1-10) peptide in a solvated dodecylphosphocholine micelle

Adrenocorticotropin (ACTH) (1-10), an adrenocorticotropin hormone fragment, has been studied by molecular dynamics (MD) simulation in an NPT ensemble in an explicit dodecylphosphocholine (DPC) micelle. Two starting configurations of the peptide/micelle system, corresponding to the insertion and surface-binding modes, were used. A common equilibrated configuration, in which the peptide lies parallel to the micellar surface, was reached from both simulations. In the initial part of the simulations, distance restraints derived from NMR nuclear Overhauser enhancements were incorporated before the peptide reached an equilibrium configuration with respect to the micelle. Analyses of the trajectories from the subsequent free (unrestrained) MD simulation showed that ACTH (1-10) does not conform strictly to a helical structure. The loss of the helical structure is due to decreased intramolecular hydrogen bonding accompanied by an increase of hydrogen bonding between the amide protons of the peptide and the micellar head groups. However, the extent of the latter interaction is less pronounced than in the negatively charged SDS micelle. The final structure enhances the amphipathic nature of the peptide, facilitating better interactions at the water-hydrophobic interface. The primary hydrophobic interactions with the micelle came from the side chains of Met4, Phe7, and Trp9. All peptide bonds were either hydrated or were involved in intramolecular hydrogen bonding. The interactions with the DPC micelle, the conformation of the bound peptide, and the dynamics of the peptide, as revealed by the time correlation functions of the N-H bonds, were compared with those of the ACTH (1-10)/SDS system studied previously by MD simulations.     

Confinement-Dependent Friction in Peptide Bundles

Friction within globular proteins or between adhering macromolecules crucially determines the kinetics of protein folding, the formation, and the relaxation of self-assembled molecular systems. One fundamental question is how these friction effects depend on the local environment and in particular on the presence of water. In this model study, we use fully atomistic MD simulations with explicit water to obtain friction forces as a single polyglycine peptide chain is pulled out of a bundle of k adhering parallel polyglycine peptide chains. The whole system is periodically replicated along the peptide axes, so a stationary state at prescribed mean sliding velocity V is achieved. The aggregation number is varied between k = 2 (two peptide chains adhering to each other with plenty of water present at the adhesion sites) and k = 7 (one peptide chain pulled out from a close-packed cylindrical array of six neighboring peptide chains with no water inside the bundle). The friction coefficient per hydrogen bond, extrapolated to the viscous limit of vanishing pulling velocity V → 0, exhibits an increase by five orders of magnitude when going from k = 2 to k = 7. This dramatic confinement-induced friction enhancement we argue to be due to a combination of water depletion and increased hydrogen-bond cooperativity.     

Location and orientation relative to the micelle surface for glucagon in mixed micelles with dodecylphosphocholine EPR and NMR studies

The spin labels, 5-doxylstearate, 12-doxylstearate, 16-doxylstearate and 1-oxyl-2,2,6,6-tetramethyl-4-dodecylphospiperidine, have been incorporated into dodecylphospocholine micelles and mixed dodecylphosphocholine/ glucagon micelles. The EPR spectral parameters for the different spin labels and the ¹H- and ¹³C-NMR relaxation rates for nuclei of the detergent molecules indicated that inclusion of up to one spin label molecule per micelle had little influence on the spatial organization of the micelles. Furthermore, the location and environment of the spin labels in the dodecylphosphocholine micelles were not noticeably affected by the addition of glucagon and the ¹H-NMR spectra observed for glucagon in mixed spin label/deuterated dodecylphosphocholine/glucagon micelles showed that the different spin labels had essentially no effect on the conformation of glucagon. Approximate spatial locations within the micelle for the nitroxide moieties of the different spin labels were determined from the NMR relaxation rates observed for different nuclei of dodecylphosphocholine. On this basis, the line broadening of individually assigned glucagon ¹H-NMR lines by the different spin labels was used to determine the approximate orientation of the polypeptide chain with respect to the micelle surface. Overall, the data indicate that the glucagon backbone runs roughly parallel to the micelle surface, with the depth of immersion adjusted so that polar and apolar side chains can be oriented towards the surface or interior of the micelle, respectively.     

Hydrogen exchange of monomeric alpha-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

Amide proton NMR signals from the N-terminal domain of monomeric α-synuclein (αS) are lost when the sample temperature is raised from 10°C to 35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in α-helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of αS than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N- and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. Cα chemical shift data point to <10% residual α-helix structure at 10°C and 35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°C in ¹H-¹⁵N HSQC spectra of αS encapsulated within living Escherichia coli cells. Our finding that temperature effects on αS NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that αS remains predominantly unfolded at physiological temperature and pH—an important conclusion for mechanistic models of the association of αS with membranes and fibrils.     

Membrane Damage by an α-Helical Pore-forming Protein,Equinatoxin II,Proceeds through a Succession of Ordered Steps

Actinoporin equinatoxin II (EqtII) is an archetypal example of α-helical pore-forming toxins that porate cellular membranes by the use of α-helices. Previous studies proposed several steps in the pore formation: binding of monomeric protein onto the membrane, followed by oligomerization and insertion of the N-terminal α-helix into the lipid bilayer. We studied these separate steps with an EqtII triple cysteine mutant. The mutant was engineered to monitor the insertion of the N terminus into the lipid bilayer by labeling Cys-18 with a fluorescence probe and at the same time to control the flexibility of the N-terminal region by the disulfide bond formed between cysteines introduced at positions 8 and 69. The insertion of the N terminus into the membrane proceeded shortly after the toxin binding and was followed by oligomerization. The oxidized, non-lytic, form of the mutant was still able to bind to membranes and oligomerize at the same level as the wild-type or the reduced form. However, the kinetics of the N-terminal helix insertion, the release of calcein from erythrocyte ghosts, and hemolysis of erythrocytes was much slower when membrane-bound oxidized mutant was reduced by the addition of the reductant. Results show that the N-terminal region needs to be inserted in the lipid membrane before the oligomerization into the final pore and imply that there is no need for a stable prepore formation. This is different from β-pore-forming toxins that often form β-barrel pores via a stable prepore complex.     

Residual Structure of Unfolded Ubiquitin as Revealed by Hydrogen/Deuterium-Exchange 2D NMR

The characterization of residual structures persistent in unfolded proteins in concentrated denaturant solution is currently an important issue in studies of protein folding because the residual structure present, if any, in the unfolded state may form a folding initiation site and guide the subsequent folding reactions. Here, we studied the hydrogen/deuterium (H/D)-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride. We employed a dimethylsulfoxide (DMSO)-quenched H/D-exchange NMR technique with the use of spin desalting columns, which allowed us to perform a quick medium exchange from 6 M guanidinium chloride to a quenching DMSO solution. Based on the backbone resonance assignment of ubiquitin in the DMSO solution, we successfully investigated the H/D-exchange kinetics of 60 identified peptide amide groups in the ubiquitin sequence. Although a majority of these amide groups were not protected, certain amide groups involved in a middle helix (residues 23–34) and an N-terminal β-hairpin (residues 2–16) were significantly protected with a protection factor of 2.1–4.2, indicating that there were residual structures in unfolded ubiquitin and that these amide groups were more than 52% hydrogen bonded in the residual structures. We show that the hydrogen-bonded residual structures in the α-helix and the β-hairpin are formed even in 6 M guanidinium chloride, suggesting that these residual structures may function as a folding initiation site to guide the subsequent folding reactions of ubiquitin.     

A Toxin-based Probe Reveals Cytoplasmic Exposure of Golgi Sphingomyelin

Although sphingomyelin is an important cellular lipid, its subcellular distribution is not precisely known. Here we use a sea anemone cytolysin, equinatoxin II (EqtII), which specifically binds sphingomyelin, as a new marker to detect cellular sphingomyelin. A purified fusion protein composed of EqtII and green fluorescent protein (EqtII-GFP) binds to the SM rich apical membrane of Madin-Darby canine kidney (MDCK) II cells when added exogenously, but not to the SM-free basolateral membrane. When expressed intracellularly within MDCK II cells, EqtII-GFP colocalizes with markers for Golgi apparatus and not with those for nucleus, mitochondria, endoplasmic reticulum or plasma membrane. Colocalization with the Golgi apparatus was confirmed by also using NIH 3T3 fibroblasts. Moreover, EqtII-GFP was enriched in cis-Golgi compartments isolated by gradient ultracentrifugation. The data reveal that EqtII-GFP is a sensitive probe for membrane sphingomyelin, which provides new information on cytosolic exposure, essential to understand its diverse physiological roles.     

NMR Structural Studies of Antimicrobial Peptides: LPcin Analogs

Lactophoricin (LPcin), a component of proteose peptone (113–135) isolated from bovine milk, is a cationic amphipathic antimicrobial peptide consisting of 23 amino acids. We designed a series of N- or C-terminal truncated variants, mutated analogs, and truncated mutated analogs using peptide-engineering techniques. Then, we selected three LPcin analogs of LPcin-C8 (LPcin-YK1), LPcin-T2WT6W (LPcin-YK2), and LPcin-T2WT6W-C8 (LPcin-YK3), which may have better antimicrobial activities than LPcin, and successfully expressed them in E. coli with high yield. We elucidated the 3D structures and topologies of the three LPcin analogs in membrane environments by conducting NMR structural studies. We investigated the purity of the LPcin analogs and the α-helical secondary structures by performing ¹H-¹⁵N 2D HSQC and HMQC-NOESY liquid-state NMR spectroscopy using protein-containing micelle samples. We measured the 3D structures and tilt angles in membranes by conducting ¹⁵N 1D and 2D ¹H-¹⁵N SAMMY type solid-state NMR spectroscopy with an 800 MHz in-house-built ¹H-¹⁵N double-resonance solid-state NMR probe with a strip-shield coil, using protein-containing large bicelle samples aligned and confirmed by molecular-dynamics simulations. The three LPcin analogs were found to be curved α-helical structures, with tilt angles of 55–75° for normal membrane bilayers, and their enhanced activities may be correlated with these topologies.     

Conformational changes in phospholipase A2 upon binding to micellar interfaces in the absence and presence of competitive inhibitors. A 1H and 15N NMR study.

An NMR study has been made of porcine pancreatic phospholipase A2 (PLA) in three environments: free in solution, in a binary complex with dodecylphosphocholine micelles, and in a ternary complex with a micelle and the substrate-like inhibitor (R)-1-octyl-2-(N-dodecanoylamino)-2-deoxyglycero-3-phosph oglycol. 1H and 15N chemical shifts, amide exchange rates, and NOE intensities are compared for the enzyme in different environments. From these data, structural differences are found for the N-terminal part, the end of the surface loop at residues Tyr69 and Thr70, and the active site residue His48, and also for the Ca-binding loop (residues 28-32). Specifically, when binding to a micelle, the side chains of residues Ala1, Trp3, and Tyr69, as well as all protons of Thr70, are found to be closer together. After subsequent introduction of the competitive inhibitor, further changes are found for these residues. The N-terminus is flexible in PLA free in solution, in contrast with the crystal structures where it adopts an alpha-helical conformation. According to the NMR data, this helix is rigidly formed only in the ternary complex. Furthermore, in the ternary complex, the N-terminal amino group and the exchangeable hydrogen at N3 of the ring of His48 are observed. We propose that PLA is activated in two steps. An initial conformational change occurs upon binding to a micellar interface. The catalytically active conformation of the enzyme, which has an extensive network of hydrogen bonds, is formed only when binding a substrate or competitive inhibitor at a lipid-water interface.     

NMR Structure of a Viral Peptide Inserted in Artificial Membranes: A VIEW ON THE EARLY STEPS OF THE BIRNAVIRUS ENTRY PROCESS*

Nonenveloped virus must penetrate the cellular membrane to access the cytoplasm without the benefit of membrane fusion. For birnavirus, one of the peptides present in the virus capsid, pep46 for infectious bursal disease virus, is able to induce pores into membranes as an intermediate step of the birnavirus-penetration pathway. Using osmotic protection experiments, we demonstrate here that pep46 and its pore-forming N-terminal moiety (pep22) form pores of different diameters, 5–8 and 2–4 nm, respectively, showing that both pep46 moieties participate to pore formation. The solution structures of pep46, pep22, and pep24 (the pep46 C-terminal moiety) in different hydrophobic environments and micelles determined by ¹H NMR studies provide structural insights of the pep46 domain interaction. In CDCl₃/CD₃OH mixture and in dodecylphosphocholine micelles, the N-terminal domain of pep46 is structured in a long kinked helix, although the C terminus is structured in one or two helices depending upon the solvents used. We also show that the folding and the proline isomerization status of pep46 depend on the type of hydrophobic environment. NMR spectroscopy with labeled phospholipid micelles, differential scanning calorimetry, and plasmon waveguide resonance studies show the peptides lie parallel to the lipid-water interface, perturbing the fatty acid chain packing. All these data lead to a model in which the two domains of pep46 interact with the membrane to form pores.     

Listeriolysin O Affects the Permeability of Caco-2 Monolayer in a Pore-Dependent and Ca2+-Independent Manner

Listeria monocytogenes is a food and soil-borne pathogen that secretes a pore-forming toxin listeriolysin O (LLO) as its major virulence factor. We tested the effects of LLO on an intestinal epithelial cell line Caco-2 and compared them to an unrelated pore-forming toxin equinatoxin II (EqtII). Results showed that apical application of both toxins causes a significant drop in transepithelial electrical resistance (TEER), with higher LLO concentrations or prolonged exposure time needed to achieve the same magnitude of response than with EqtII. The drop in TEER was due to pore formation and coincided with rearrangement of claudin-1 within tight junctions and associated actin cytoskeleton; however, no significant increase in permeability to fluorescein or 3 kDa FITC-dextran was observed. Influx of calcium after pore formation affected the magnitude of the drop in TEER. Both toxins exhibit similar effects on epithelium morphology and physiology. Importantly, LLO action upon the membrane is much slower and results in compromised epithelium on a longer time scale at lower concentrations than EqtII. This could favor listerial invasion in hosts resistant to E-cadherin related infection.