Vibrio cholerae cytolysin permeabilizes animal cell membranes. Upon binding to the target lipid bilayer, the protein assembles into homo-oligomeric pores of an as yet unknown stoichiometry. Pore formation has been observed with model liposomes consisting of phosphatidylcholine and cholesterol, but the latter were much less susceptible to the cytolysin than were erythrocytes or intestinal epithelial cells. We here show that liposome permeabilization is strongly promoted if cholesterol is combined with sphingolipids, whereby the most pronounced effects are observed with monohexosylceramides and free ceramide. These two lipid species are prevalent in mammalian intestinal brush border membranes. We therefore propose that, on its natural target membranes, the cytolysin has a dual specificity for both cholesterol and ceramides. To assess the stoichiometry of the pore, we generated hybrid oligomers of two naturally occurring variants of the toxin that differ in molecular weight. On SDS-polyacrylamide gel electrophoresis, the mixed oligomers formed a pattern of six distinct bands. Ordered by decreasing electrophoretic mobility, the six oligomer species must comprise 0 to 5 subunits of the larger form; the pore thus is a pentamer. Due to both lipid specificity and pore stoichiometry, V. cholerae cytolysin represents a novel prototype in the class of bacterial pore-forming toxins.

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 ␣-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.To bacterial pore-forming cytolysins, cholesterol is a logical choice as a target molecule, because it confers specificity for animal as opposed to bacterial cell membranes. The specificity for cholesterol is shared between Vibrio cholerae cytolysin (VCC) 1 (1) and streptolysin O (SLO) (2). Otherwise, these toxins are not related, and the oligomeric pores they form are very different in size and morphology (1, 3). Although with SLO the sterol is already required in the initial event of membrane binding of the monomeric toxin (4), it only comes into play at the stage of oligomerization in the case of VCC (5, 6). When the sterol is incorporated into phosphatidylcholine (PC) bilayers at physiologically realistic concentrations (i.e. up to 40% by mol), these membranes do not become significantly sensitive to VCC. However, it was previously found that membrane susceptibility toward the cytolysin was greatly enhanced by inclusion of ceramide; free ceramide and monohexosyl ceramides proved similarly effective (7). A combined specificity for cholesterol and sphingolipids has previously been shown for the fusion protein of Semliki Forest virus. In that instance, the interaction with ceramide proved to be highly stereoselective (8 -10). Accordingly, we have examined the structural properties of the ceramide molecule responsible for the sensitization of membranes to VCC. To our surprise, no dependence on stereospecific features of ceramide could be detected. Membrane sensitization was readily achieved with a variety of synthetic ceramides...

V. cholerae El Tor cytolysin is a secreted, water-soluble protein of M(r) 60,000 that may be relevant to the pathogenesis of acute diarrhea. In this communication, we demonstrate that the toxin binds to and oligomerizes in target membranes to form SDS-stable aggregates of M(r) 200,000-250,000 that generate small transmembrane pores. Pores formed in erythrocytes were approximately 0.7 nm in size, as demonstrated by osmotic protection experiments. Binding was shown to occur in a temperature-independent manner preceding the temperature-dependent oligomerization step. Pores were also shown to be formed in L929 and HEp-2 cells, human fibroblasts and keratinocytes, albeit with highly varying efficacy. At neutral pH and in the presence of serum, human fibroblasts were able to repair a limited number of lesions. The collective data identify V. cholerae El Tor cytolysin as an oligomerizing toxin that damages cells by creating small transmembrane pores.

Vibrio cholerue cytolysin (VCC) is produced by many non-choleratoxigenic strains of V. cholerue, and possibly represents a relevant pathogenicity determinant of these bacteria. The protein is secreted as a pro-toxin that is proteolytically cleaved to yield the active toxin with a molecular mass of approximately 63 kDa. We here describe a simple procedure for preparative isolation of mature VCC from bacterial culture supernatants, and present information on its mode of binding and pore formation in biological membranes. At low concentrations, toxin monomers interact with a high-affinity binding site on highly susceptible rabbit erythrocytes. This as yet unidentified binding site is absent on human erythrocytes, which are less susceptible to the toxin action. At higher concentrations, binding of the toxin occurs to both rabbit and human erythrocytes in a non-saturable manner. Cell-bound toxin monomers oligomerize to form supramolecular structures that are seen in the electron microscope as apparently hollow funnels, and oligomerization correlates functionally with the appearance of small transmembrane pores. Osmotic protection experiments indicate that the toxin channels are of finite size with a diameter of 1-2 nm. The mode of action of VCC closely resembles that of classical pore-forming toxins such as staphylococcal a-toxin and the aerolysin of Aeromonus hydrophilu.Keywords: Vibrio cholerue pore-forming toxin ; membrane damage ; binding ; cytolysin.Two major categories of proteinaceous exotoxins are known to be produced by bacteria of the genus Vibrionaceae. The first group encompasses the intracellularly active, secretogenic toxins, with cholera toxin as the classical prototype. The second comprises cytotoxins that damage the plasma membrane. Knowledge regarding the signficance and molecular function of the latter is quite sparse; it appears that three distinct groups can be differentiated. The first is represented by the thermostable direct hemolysin of Vibrio puruhuemolyticus. This pathogen is responsible for approximately 50 % of food-borne diarrheal infections in Japan (11 and hemolysin appears to be an important virulence determinant [2]. The toxin consists of two identical subunits of 21 kDa, the structural gene of which has been sequenced [3]. The hemolytic and cytotoxic action of thermostable direct hemolysin appears to be derived from pore-forming activity [4], but details regarding this process are largely unknown. Some strains of Vibrio cholerae non-01, Vibrio mimicus, and Vibrio hollisue also produce toxins related to thermostable direct hemolysin [5, 61. A second membrane-damaging Vibrio toxin is elaborated by V. metschnikovii. This toxin is reportedly produced as a 50-kDa monomer that assembles into detergent-stable oligomers with pore-forming properties [7]. The structural gene has not been cloned; attempts to detect antigenic relatedness with other Vibrio cytotoxins have failed, so that this toxin may stand alone as a [ l l , 171 and V. mimicus [14] may contribute to the enteropathogenicity...

Escherichia coli hemolysin (HlyA) is a membrane-permeabilizing protein belonging to the family of RTX-toxins. Lytic activity depends on binding of Ca 21 to the C-terminus of the molecule. The N-terminus of HlyA harbors hydrophobic sequences that are believed to constitute the membrane-inserting domain. In this study, 13 HlyA cysteine-replacement mutants were constructed and labeled with the polarity-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan). The fluorescence emission of the label was examined in soluble and membrane-bound toxin. Binding effected a major blue shift in the emission of six residues within the N-terminal hydrophobic domain, indicating insertion of this domain into the lipid bilayer. The emission shifts occurred both in the presence and absence of Ca 21 , suggesting that Ca 21 is not required for the toxin to enter membranes. However, binding of Ca 21 to HlyA in solution effected conformational changes in both the C-terminal and N-terminal domain that paralleled activation. Our data indicate that binding of Ca 21 to the toxin in solution effects a conformational change that is relayed to the N-terminal domain, rendering it capable of adopting the structure of a functional pore upon membrane binding.Keywords: badan; calcium; conformation; Escherichia coli hemolysin, membrane.Escherichia coli hemolysin (HlyA), a 107-kDa protein of 1024 amino acids devoid of cysteine [1], belongs to the family of RTX-toxins [2] that are produced by many Gramnegative organisms. To obtain pore-forming activity, HlyA requires post-translational acylation of two lysine residues, Lys564 and Lys690 [3,4]. The protein harbors a hydrophobic domain between residues 177 and 411, which according to predictions of secondary structure [5] largely assumes helical conformation and might be involved in membrane penetration. The C-terminal half of the molecule contains 12 repeats of the consensus nonapeptide X-Leu-XGly-Gly-X-X-Gly-Asp-Asp-Asp. This repeat sequence spanning residues 739±849 represents a Ca 21 -binding domain and is essential for function [6±9]. In the presence of Ca 21 , HlyA forms pores of < 2 nm diameter in target membranes [10]. Several approaches to study structurefunction analysis of HlyA have been undertaken in the past. CD spectroscopy was used to investigate the effect of Ca 21 on HlyA, but no significant changes in secondary structure could be detected with this method [11]. On the other hand, results obtained from intrinsic fluorescence measurements and trypsin digestion were compatible with a Ca 21 -induced change in conformation [11]. Moayeri and Welch [12] studied the altered accessibility of toxin domains for monoclonal antibodies after the binding of HlyA to membranes. They reported that the far N-terminus and the region between residues 594 and 640 remain accessible in the membrane-bound toxin. No conclusions could be made for the putative transmembrane region and the C-terminal domain, as antibodies for these regions were not available.An important step toward unders...