Pore formation by Vibrio cholerae cytolysin requires cholesterol in both monolayers of the target membrane

Vibrio cholerae cytolysin (VCC) forms oligomeric transmembrane pores in cholesterol-rich membranes. To better understand this process, we used planar bilayer membranes. In symmetric membranes, the rate of the channel formation by VCC has a superlinear dependency on the cholesterol membrane fraction. Thus, more than one cholesterol molecule can facilitate VCC-pore formation. In asymmetric membranes, the rate of pore formation is limited by the leaflet with the lower cholesterol content. Methyl-beta-cyclodextrin, which removes cholesterol from membranes, rapidly inhibits VCC pore formation, even when it is added to the side opposite that of VCC addition. The results suggest that cholesterol in both membrane leaflets aid VCC-pore formation and that either leaflet can function as a kinetic bottleneck with respect to the rate of pore-formation.     

The action of Vibrio cholerae and Corynebacterium diphtheriae neuraminidases on the Sendai virus receptor of human erythrocytes.

Studies of the Sendai virus haemagglutinin receptor on the human erythrocyte surface have confirmed that it involves 2 leads to 3 linked sialic acid. Because the primary specificity of Vibrio cholerae neuraminidase is for this linkage, it is able to compete with the virus for the receptor, to which it adsorbs strongly at low temperatures. Corynebacterium diphtheriae neuraminidase, whose principal specificity is for a sialic acid linkage other than 2 leads to 3, does not easily remove Sendai virus receptors, nor does it adsorb to the erythrocyte surface. A new definition of the term "receptor-destroying enzyme" is given which takes both enzyme and virus specificity into account, and a modified assay method is suggested in order to overcome the problems due to enzyme adsorption.     

Vibrio cholerae cytolysin is composed of an alpha-hemolysin-like core

The enteric pathogen Vibrio cholerae secretes a water-soluble 80-kD cytolysin, Vibrio cholerae cytolysin (VCC) that assembles into pentameric channels following proteolytic activation by exogenous proteases. Until now, VCC has been placed in a unique class of pore-forming toxins, distinct from paradigms such as Staphyloccal alpha-hemolysin. However, as reported here, amino acid sequence analysis and three-dimensional structure modeling indicate that the core component of the VCC toxin is related in sequence and structure to a family of hemolysins from Staphylococcus aureus that include leukocidin F and alpha-hemolysin. Furthermore, our analysis has identified the channel-forming region of VCC and a potential lipid head-group binding site, and suggests a conserved mechanism of assembly and lysis. An additional domain in the VCC toxin is related to plant lectins, conferring additional target cell specificity to the toxin.     

Coupling of cholesterol and cone-shaped lipids in bilayers augments membrane permeabilization by the cholesterol-specific toxins streptolysin O and Vibrio cholerae cytolysin

Vibrio cholerae cytolysin (VCC) forms oligomeric pores in lipid bilayers containing cholesterol. Membrane permeabilization is inefficient if the sterol is embedded within bilayers prepared from phosphatidylcholine only but is greatly enhanced if the target membrane also contains ceramide. Although the enhancement of VCC action is stereospecific with respect to cholesterol, we show here that no such specificity applies to the two stereocenters in ceramide; all four stereoisomers of ceramide enhanced VCC activity in cholesterol-containing bilayers. A wide variety of ceramide analogs were as effective as D-erythro-ceramide, as was diacylglycerol, suggesting that the effect of ceramide exemplifies a general trend of lipids with a small headgroup to augment the activity of VCC. Incorporation of these cone-shaped lipids into cholesterol-containing bilayers also gave similar effects with streptolysin O, another cholesterol-specific but structurally unrelated cytolysin. In contrast, the activity of staphylococcal alpha-hemolysin, which does not share with the other toxins the requirement for cholesterol, was far less affected by the presence of lipids with a conical shape. The collective data indicate that sphingolipids and glycerolipids do not interact with the cytolysins specifically. Instead, lipids that have a conical molecular shape appear to effect a change in the energetic state of membrane cholesterol that in turn augments the interaction of the sterol with the cholesterol-specific cytolysins.     

Phloretin and phloretin analogs: Mode of action in planar lipid bilayers and monolayers

Summary Phloretin and other neutral phloretin-like molecules are able to decrease the electrostatic potential within neutral lipid bilayers and monolayers. The relationship between the change in the dipole potential and the aqueous concentration of the molecule is well described by a Langmuir isotherm. From the Langmuir isotherm, the apparent dissociation constants (K _D ^A ) and the maximum dipole potential change ( _max) are obtained for the different phloretin-like molecules tested. Considering the phloretin analogs as derivatives of acetophenone containing two kinds of substituents, one on the benzene ring and another on the carbon chain, it is found that (a)K _D ^A is related to the hydrophobicity of the compound and is also a function of the position of the hydroxyl substituent in the ring; (b) from the dependence ofK _D ^A on the length of the acyl chain, it is estimated that the free-energy change is 650 cal/mole CH₂; (c) _max is not a simple function of the dipole moment of the molecule but depends on the substituent on the carbon chain and on the position and number of hydroxyl groups on the benzene ring; (d) phloretin adsorption parameters are a function of membrane lipid composition. The results are discussed in terms of the effect of these compounds on chloride transport in red blood cells.     

Isolation and characterization of a cytolysin produced by Vibrio cholerae serogroup non-O1

A thermolabile toxin (molecular weight, 52 711; isoelectric point, 8.65) produced by a clinical isolate of Vibrio cholerae serogroup non-O1 was cytotoxic for Y-1 mouse adrenal cells and Chinese hamster ovary cells. The toxin lysed rabbit red blood cells and produced a hemorrhagic zone in rabbit skin. When injected intravenously into adult mice, the cytolysin was rapidly lethal and caused fluid accumulation in both 5- and 18-h rabbit ileal loops. Strains of V. cholerae that produced cytolysin but no cholerae enterotoxin were able to cause fluid accumulation in rabbit intestinal loops.     

Delta-endotoxins form cation-selective channels in planar lipid bilayers.

Delta-endotoxins CryIA(c) and CryIIIA, two members of a large family of toxic proteins from Bacillus thuringiensis, were each allowed to interact with planar lipid bilayers and were analyzed for their ability to form ion-conducting channels. Both of these toxins made clearly resolved channels in the membranes and exhibited several conductance states, which ranged from 200 pS to about 4000 pS (in 300 mM KCl). The channels formed by both toxins were highly cation-selective, but not ideally so. The permeability ratio of K+ to Cl- was about 25 for both channels. The ability of these proteins to form such channels may account for their toxic action on sensitive cells, and suggests that this family of toxins may act by a common mechanism.     

Fusion of liposomes with planar lipid bilayers

The fusion of liposomes with planar lipid bilayers was monitored by two different methods. (a) Liposomes consisting of phospholipids and cholesterol were added to the aqueous phase bathing the cholesterol-deficient planar lipid bilayers in the presence of nystatin. The resulting increase in the planar lipid bilayer's electrical conductance was considered indicative of fusion. (b) Transplanar lipid bilayer injection of ³⁵SO₄^2? trapped inside the liposomes.It is shown by both methods that fusion is specifically dependent on the presence of negatively charged phospholipids both in the liposomes and the planar lipid bilayers and on Ca²⁺ in the aqueous phase of the fusion system.     

Raman spectra of planar supported lipid bilayers

Raman scattering has been used to obtain high quality vibrational spectra of planar supported lipid bilayers (pslb's) at the silica/water interface without the use of resonance or surface enhancement. A total internal reflection geometry was used both to increase the bilayer signal and to suppress the water background. Polarization control permits the determination of four components of the Raman tensor, of which three are independent for a uniaxial film. Spectra are reported of the phospholipids DMPC, DPPC, and POPC, in the C-H stretching region and the fingerprint region. The temperature-dependent polarized spectra of POPC show only small changes over the range 14-41 °C. The corresponding spectra of DMPC and DPPC bilayers show large thermal changes consistent with a decreasing tilt angle from the surface normal and increasing chain ordering at lower temperatures. The thermal behavior of DMPC pslb's is similar to that of vesicles of the same lipid in bulk suspension. In contrast to calorimetry, which shows a sharp phase transition (L_α-L_β') with decreasing temperature, the changes in the Raman spectra occur over a temperature range of ca. 10 °C commencing at the calorimetric phase transition temperature.     

The VarA-CsrA regulatory pathway influences cell shape in Vibrio cholerae

Despite extensive studies on the curve-shaped bacterium Vibrio cholerae, the causative agent of the diarrheal disease cholera, its virulence-associated regulatory two-component signal transduction system VarS/VarA is not well understood. This pathway, which mainly signals through the downstream protein CsrA, is highly conserved among gamma-proteobacteria, indicating there is likely a broader function of this system beyond virulence regulation. In this study, we investigated the VarA-CsrA signaling pathway and discovered a previously unrecognized link to the shape of the bacterium. We observed that varA-deficient V. cholerae cells showed an abnormal spherical morphology during late-stage growth. Through peptidoglycan (PG) composition analyses, we discovered that these mutant bacteria contained an increased content of disaccharide dipeptides and reduced peptide crosslinks, consistent with the atypical cellular shape. The spherical shape correlated with the CsrA-dependent overproduction of aspartate ammonia lyase (AspA) in varA mutant cells, which likely depleted the cellular aspartate pool; therefore, the synthesis of the PG precursor amino acid meso-diaminopimelic acid was impaired. Importantly, this phenotype, and the overall cell rounding, could be prevented by means of cell wall recycling. Collectively, our data provide new insights into how V. cholerae use the VarA-CsrA signaling system to adjust its morphology upon unidentified external cues in its environment.     

Raman spectra of planar supported lipid bilayers

Raman scattering has been used to obtain high quality vibrational spectra of planar supported lipid bilayers (pslb's) at the silica/water interface without the use of resonance or surface enhancement. A total internal reflection geometry was used both to increase the bilayer signal and to suppress the water background. Polarization control permits the determination of four components of the Raman tensor, of which three are independent for a uniaxial film. Spectra are reported of the phospholipids DMPC, DPPC, and POPC, in the C-H stretching region and the fingerprint region. The temperature-dependent polarized spectra of POPC show only small changes over the range 14-41 degrees C. The corresponding spectra of DMPC and DPPC bilayers show large thermal changes consistent with a decreasing tilt angle from the surface normal and increasing chain ordering at lower temperatures. The thermal behavior of DMPC pslb's is similar to that of vesicles of the same lipid in bulk suspension. In contrast to calorimetry, which shows a sharp phase transition (L alpha-L beta') with decreasing temperature, the changes in the Raman spectra occur over a temperature range of ca. 10 degrees C commencing at the calorimetric phase transition temperature.     

Reconstitution of vacuolar ion channels into planar lipid bilayers.

Vacuolar ion channels were characterized after reconstitution into planar lipid bilayers. (1) Channel activity was observed after incorporation of tonoplast-enriched microsomal membranes, purified tonoplast membranes or of solubilized tonoplast proteins. (2) Channels of varying single-channel conductances were detected after reconstitution. In symmetrical 100 mmol l-1 KCl, conductances between 1 and 110 pS were frequently measured; the largest number of independent reconstitution events was seen for single-channel conductances of 16-25 pS (28 experiments), 30-42 pS (26), 49-56 pS (15) and 64-81 pS (15). Channel current usually increased linearly with voltage. (3) In asymmetrical solutions, cation-, non-selective and, for the first time for the tonoplast, anion-selective channels were detected. Ca(2+)-dependent regulation of channel opening was not observed in our reconstitution system. (4) Permeability was also observed for Cl-, NO3-, SO4(2-) and phosphate. (5) After fractionation of tonoplast proteins by size exclusion chromatography, ion channel activity was recovered in specific fractions. (6) Some of these fractions catalyzed sulfate transport after reconstitution into liposomes. The results suggest that different channels are active at the tonoplast membrane at a larger number than has been concluded from previous work.     

The Vibrio cholerae cytolysin promotes activation of mast cell (T helper 2) cytokine production

Many strains of Vibrio cholerae produce a cytolysin (VCC) that forms oligomeric transmembrane pores responsible for vacuolization of several cell types in culture. Here we suggest that VCC could contribute to the T helper 2 (Th2) response seen in the natural infection; acting through TLR2, VCC enhances mast cells secretion of IL-4, IL-6 and TNF-α by 330-, 290- and 550-fold respectively. Moreover, VCC-induced cytokine production is dependent on increased cytosolic Ca²⁺ and on the presence of the Src family kinases Lyn and Fyn, known to be required for FcεRI-dependent activation of mast cells. These findings strongly suggest that VCC has a pro-inflammatory activity promoting a Th2-type immune profile.     

The autophagic pathway: a cell survival strategy against the bacterial pore-forming toxin Vibrio cholerae cytolysin

Vibrio cholerae is the causative agent of cholera in humans. In addition to the criticalvirulence factors cholera toxin and toxin coregulated pilus, V. cholerae secretes V.cholerae cytolysin (VCC), a pore-forming exotoxin able to induce cell lysis and extensivevacuolation. We have shown that this vacuolation is related to the activation of autophagyin response to VCC action. Furthermore, we found that the autophagic pathway wasrequired to protect cells upon VCC intoxication. Based on additional data presented here,we propose a model aimed to explain the mechanism of cell protection. We postulatethat VCC-induced autophagic vacuoles, which display features of multivesicular bodies and enclose the toxin, are implicated in cell defense through VCC degradation involvingfusion with lysosomes.     

Identification of the membrane penetrating domain of Vibrio cholerae cytolysin as a beta-barrel structure

Vibrio cholerae cytolysin (VCC) is an oligomerizing pore-forming toxin that is related to cytolysins of many other Gram-negative organisms. VCC contains six cysteine residues, of which two were found to be present in free sulphydryl form. The positions of two intramolecular disulphide bonds were mapped, and one was shown to be essential for correct folding of protoxin. Mutations were created in which the two free cysteines were deleted, so that single cysteine substitution mutants could be generated for site-specific labelling. Employment of polarity-sensitive fluorophores identified amino acid side-chains that formed part of the pore-forming domain of VCC. The sequence commenced at residue 311, and was deduced to form a beta-barrel in the assembled oligomer with the subsequent odd-numbered residues facing the lipid bilayer and even-numbered residues facing the lumen. Pro328/Lys329 were tentatively identified as the position at which the sequence turns back into the membrane and where the antiparallel beta-strand commences. This was deduced from fluorimetric analyses combined with experiments in which the pore was reversibly occluded by derivatization of sulphydryl groups with a bulky moiety. Our data support computer-based predictions that the membrane-permeabilizing amino acid sequence of VCC is homologous to the beta-barrel-forming sequence of staphylococcal cytolysins and identify the beta-barrel as a membrane-perforating structure that is highly conserved in evolution.     

The prion peptide forms ion channels in planar lipid bilayers

One of the hypotheses concerning the pathogenic properties of the prion protein considers its influence on cellular ion homeostasis. Using the lipid bilayer technique, the influence of prion-derived peptides on the lipid bilayer conductance was characterized. To evaluate the physiological significance and possible pathological functions of the peptides, their effect on the membrane potential and respiration rate of hippocampal mitochondria was also studied. We used a peptide bearing the human prion protein sequence YSNQNNF (PrP [169-175]), and peptide SSQNNF (PrP [170-175]) bearing a naturally-occurring mutation in position 171 [N(r)S] linked to schizoaffective diseases in humans (Samaia, H.B., Mari, J.J., Vallada, H.P., Moura R.P., Simpson A.J.G., Brentani R.R. A prion-linked psychiatric disorder. Nature 390 (1997) 241). In this report, we show that PrP [170-175] N171S increases the conductance of planar lipid bilayers. Based on the conductance of single channel currents recorded in 500/500 mM KCl (cis/trans), we found a single channel conductance of 8 to 26 pS. The native prion peptide PrP [169-175] does not form ion channels in the lipid bilayer. Neither of the peptides significantly changed the membrane potential or respiration rate of isolated rat hippocampal mitochondria. We propose a possible mechanism for channel formation by aggregation of the prion-derived peptide.     

Unfolding distinguishes the Vibrio cholerae cytolysin precursor from the mature form of the toxin

Vibrio cholerae cytolysin (VCC) is a potent cytolytic toxin that induces colloid osmotic lysis of its target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. VCC is secreted by the bacteria as an inactive precursor (Pro-VCC) and is subsequently activated by proteolytic removal of an N-terminal "Pro-domain", thus generating the active form of the toxin (Mature-VCC). Earlier studies have indicated an intramolecular chaperone-like role of the Pro-domain favoring efficient secretion of the toxin from the periplasm into the extracellular space. However, the exact role of the Pro-domain in the VCC structure--function mechanism remains unclear. Here, we have compared the Pro-VCC and Mature-VCC molecules in terms of their structural and conformational properties. We have studied unfolding of the two variants of the VCC molecule in response to an array of denaturing conditions, including low-pH, chemical denaturant and heat-induced unfolding. Pro-VCC shows a more profound tendency to unfold in response to such denaturing conditions compared to Mature-VCC. Biophysical characterization of the isolated Pro-domain further suggests that the increased unfolding propensity of Pro-VCC does not arise because of an increased level of unfolding of the Pro-region itself. Altogether, our results imply that a natively folded architecture of the Pro-VCC molecule with sufficient structural and conformational plasticity presumably allows it to adopt a suitable configuration that is possibly required for its efficient secretion and subsequent proteolytic maturation under physiological conditions.     

Calcium-dependent inactivation of L-type calcium channels in planar lipid bilayers.

Intracellular Ca2+ can inhibit the activity of voltage-gated Ca channels by modulating the rate of channel inactivation. Ca(2+)-dependent inactivation of these channels may be a common negative feedback process important for regulating Ca2+ entry under physiological and pathological conditions. This article demonstrates that the inactivation of cardiac L-type Ca channels, reconstituted into planar lipid bilayers and studied in the presence of a dihydropyridine agonist, is sensitive to Ca2+. The rates and extents of inactivation, determined from ensemble averages of unitary Ba2+ currents, decreased when the calcium concentration facing the intracellular surface of the channel ([Ca2+]i) was lowered from approximately 10 microM to 20 nM by the addition of Ca2+ chelators. The rates and extents of Ba2+ current inactivation could also be increased by subsequent addition of Ca2+ raising the [Ca2+]i to 15 microM, thus demonstrating that the Ca2+ dependence of inactivation could be reversibly regulated by changes in [Ca2+]i. In addition, reconstituted Ca channels inactivated more quickly when the inward current was carried by Ca2+ than when it was carried by Ba2+, suggesting that local increases in [Ca2+]i could activate Ca(2+)-dependent inactivation. These data support models in which Ca2+ binds to the channel itself or to closely associated regulatory proteins to control the rate of channel inactivation, and are inconsistent with purely enzymatic models for channel inactivation.