Mode of Primary Binding to Target Membranes and Pore Formation Induced by Vibrio Cholerae Cytolysin (Hemolysin)

Abstract

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...

A Cellular Metalloproteinase Activates Vibrio cholerae Pro-cytolysin

“…Erythrocyte ghosts were produced as described previously (11). Erythrocytes (4 ϫ 10 8 cells/ml) or ghosts were incubated with pro-VCC (5 g/ml) for the indicated time at 37°C or 4°C in HBSS, washed twice, and subjected to SDS-PAGE (11).…”

“…When applicable, proteolytic activation was accomplished by incubation with trypsin (1 g/ml) at 25°C for 20 min. Mature nonrecombinant VCC was isolated according to published procedures (11).…”

“…Cells were finally solubilized with 1% SDS and electrophoresed. Western blots were incubated with rabbit polyclonal antiserum raised against mature V. cholerae cytolysin (2) and developed with a biotinylated secondary antibody and streptavidin-peroxidase, as described previously (11). Protease inhibitors used were N-ethylmaleimide (1 mM), aprotinin (100 M), pepstatin (20 M), phenylmethylsulfonyl fluoride (1 mM) (all from Sigma), Pefabloc™ (3 mM), and protease inhibitors mixture Complete (Roche), furin inhibitor decanoyl-Arg-ValLys-Arg-chloromethyl ketone (50 M) (30), 50 M metalloprotease inhibitor TNF-␣ protease inhibitor (TAPI) (31) (from Calbiochem-Novabiochem), and 50 M selective gelatinase inhibitor H-Cys-Thr-Thr-HisTrp-Gly-Phe-Thr-Leu-Cys-OH (32) from Bachem (Heidelberg, Germany).…”

“…In all cases, functionally active 65-kDa toxin is obtained (10). It was shown that the 65-kDa mature VCC binds to lipid membranes in a nonspecific, reversible fashion (11) and assembles into an SDS-resistant oligomer that creates discrete transmembrane pores. First biochemical evidence suggested a pentameric structure (12), but electron microscopic data are in favor of a heptamer (13), as is known to be the case with staphylococcal ␣-toxin (14,15) and aerolysin (16).…”

Coupling of Cholesterol and Cone-shaped Lipids in Bilayers Augments Membrane Permeabilization by the Cholesterol-specific Toxins Streptolysin O and Vibrio cholerae Cytolysin

“…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.…”