Structural Requirements of Cholesterol for Binding to Vibrio cholerae Hemolysin

Abstract

Vibrio cholerae is the causative agent of cholera, which is characterized by severe secretory diarrhea in humans. The profuse secretory diarrhea that is characteristic of cholera is caused by a toxin produced by toxigenic V. cholerae when it colonizes the small intestine. Almost all V. cholerae, except the classical type, also produce a Vibrio cholerae hemolysin (VCH) monomer (8,13,23). It has been reported that a specific VCH with an apparent molecular mass of 65 kDa has strong cytotoxic effects (9,14,16,22,25,26,(29)(30)(31); however, these studies did not clarify the role of VCH in V. cholerae infection.The VCH monomer forms pore-forming transmembrane oligomers (9,11,12,14,16,22,25,(28)(29)(30)(31)(32) that are permeable to molecules that are smaller than 600 Da (9). Some studies have shown that VCH induces vacuolation in tissue culture cells (6,17,18). Therefore, it seems likely that the vacuolating activity is provoked by VCH-mediated pore formation. These findings demonstrate a functional link between enteric disease and VCH-mediated pore formation in small intestinal cell membranes. In a previous study, we showed that in liposome membranes, cholesterol (5-cholesten-3β-ol) is required for conversion of the VCH monomer into the oligomer (9). VCH oligomer formation increased with an increase in the concentration of cholesterol present in the liposomes (9); this indicates the possibility that cholesterol is required for the membrane assembly of ., 50(10), [751][752][753][754][755][756][757] 2006 Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid; Hepes, N-2-hydroxymethylpiperazine-N'-2-ethanesulfonic acid; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PVDF, polyvinylidene difluoride; N,tolidine-2Na-4H2O; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TEN, 50 mM Tris-HCl-1 mM EDTA-3 mM sodium azide (pH 8.0); VCH, Vibrio cholerae hemolysin; ∆, double bond.

Single point mutation in Vibrio cholerae cytolysin compromises the membrane pore‐formation mechanism of the toxin

“…In summary, the Ala425Val mutation severely affected the cholesterol-dependent mechanism of membrane interaction in the VCC protein, an alteration that also abolished formation of the functional pore by the membrane-bound toxin molecules. Earlier studies suggested that the presence of cholesterol in the membranes was a critical factor in optimal membrane binding, oligomerization, membrane insertion and pore formation by the VCC toxin [24][25][26][27][28][29]. Moreover, an optimal membrane pore-forming property of VCC was found to be tightly linked with its efficient interaction with the membrane [24][25][26][27][28][29].…”

“…Earlier studies suggested that the presence of cholesterol in the membranes was a critical factor in optimal membrane binding, oligomerization, membrane insertion and pore formation by the VCC toxin [24][25][26][27][28][29]. Moreover, an optimal membrane pore-forming property of VCC was found to be tightly linked with its efficient interaction with the membrane [24][25][26][27][28][29]. Consistent with this, and based on the data presented here, we hypothesize that the Ala425Val mutation in the VCC toxin compromised its optimal cholesteroldependent membrane interaction mechanism that, in turn, abrogated crucial mechanism(s) triggering the conformational fine-tuning in the VCC molecular structure, required for the generation of functional transmembrane pore assembly.…”

“…It is commonly believed that interactions of VCC with the membrane (or specific membrane components) play critical role(s) in triggering the multiple steps of the membrane pore‐formation process, namely: (a) membrane binding, (b) oligomerization of membrane‐bound monomers, and (c) membrane insertion of the ‘stem region’ from each monomeric subunit to generate a functional transmembrane β‐barrel pore. In particular, the involvement of membrane cholesterol has been implicated in modulating the efficacy of some of these events . Nevertheless, the detailed structural mechanism associated with the VCC–membrane interaction(s) that regulates formation of the functional transmembrane pore is only vaguely elucidated.…”

Trapping of Vibrio cholerae Cytolysin in the Membrane-bound Monomeric State Blocks Membrane Insertion and Functional Pore Formation by the Toxin

“…In particular, membrane lipid components like cholesterol have been widely implicated in regulating the membrane pore formation process (25,26). In the case of VCC, the presence of cholesterol in the membrane lipid bilayer has been shown to be an obligatory requirement for efficient membrane oligomerization and functional membrane pore formation by the toxin (27)(28)(29)(30)(31)(32)(33). Cholesterol appears to regulate the mode of action of VCC by physically interacting with the toxin molecule and not by simply altering the physicochemical environment of the target membrane (32).…”

“…In the case of VCC, the presence of cholesterol in the membrane lipid bilayer has been shown to be an obligatory requirement for efficient membrane oligomerization and functional membrane pore formation by the toxin (27)(28)(29)(30)(31)(32)(33). Cholesterol appears to regulate the mode of action of VCC by physically interacting with the toxin molecule and not by simply altering the physicochemical environment of the target membrane (32). It is important to note here that the VCC toxin molecule itself has been examined only to a limited extent, in the context of exploring its oligomerization mechanism.…”