Cholera Toxin

Abstract

Summary 1. Death in several infectious diseases is caused by protein toxins secreted by invading bacteria. Cholera toxin is a simple protein secreted by Vibrio cholerae colonizing the gut; it is responsible for the massive diarrhoea that is cholera. 2. The primary action of cholera toxin is an activation of adenylate cyclase, an enzyme found on the inner membrane of eukaryotic cells that catalyses the conversion of ATP to cyclic AMP. Consequent increases in the intracellular concentration of cyclic AMP are responsible for other manifestations of cholera toxin including the diarrhoea. The toxin is active on almost all eukaryotic cells. 3. The toxin can be purified from culture filtrates of V. cholera. It has a molecular weight of 82000; and is composed of one subunit A (itself two polypeptide chains joined by a disulphide bond: AI (22000) and A2 (5000)) and five subunits B (11500). These can be separated in dissociating solvents such as detergents or 6 M guanidine hydrochloride. An amino‐acid sequence of subunit B has been published. The five B subunits (sometimes found by themselves in the filtrate and known as ‘choleragenoid’) are probably arranged in a ring with the subunit A in the middle joined to them non‐covalently by peptide A2. 4. The first action of cholera toxin on a cell is to bind to the membrane strongly and irreversibly. Several thousand molecules of toxin bind to each cell and the binding constants are of the order of 10‐10 M. The binding is rapid, but is followed by a lag phase of about an hour before the intracellular cyclic AMP concentration begins to increase. 5. Ganglioside GM1, a complex amphiphilic lipid found in cell membranes, binds tightly to the toxin which shows an enzyme‐like specificity for this particular ganglioside. Toxin that has already bound ganglioside can no longer bind to cells and is therefore inactive. This and other experiments using cells depleted of endogenous ganglioside suggest that ganglioside GM1 is the natural receptor of the toxin on the cell surface. The binding is followed by a lateral movement of the toxin‐ganglioside complex in the cell surface forming a ‘cap’ at one pole of the cell. 6. The binding of ganglioside by toxin is a function exclusively of subunit B; Subunit A does not bind and can be eluted with 8 M urea from an insolubilized toxin‐ganglioside complex. Subunit B is not by itself active, and so preincubation with B can protect cells or even whole gut from the action of toxin by occupying all the ganglioside binding sites. 7. Subunit A is responsible for activation of adenylate cyclase. Purified subunit A or just peptide AI is active by itself and this activity is not inhibited by ganglioside or by antisera to subunit B. In intact cells the activity is low and shows the characteristic lag phase but in lysed cells the subunit (or the whole toxin) is much more active and there is no lag phase. This suggests that the lag phase represents the time that subunit A takes to cross the cell membrane and get to its target. 8. Several cofactors are needed for t...

Mechanism of action of choleragen andE. coli heat-labile enterotoxin: activation of adenylate cyclase by ADP-ribosylation

“…initial experiments with crude LT were consistent with the possibility that this enterotoxin interacted with GM1 as well as with a cell surface receptor distinct from that utilized by choleragen (20,23,24,35,38,47,48,55,(248)(249)(250)(251). In further studies using ganglioside-deficient transformed fibroblasts that lack chemically detectable ganglioside GM1 (NCTC 2071), it was observed that after incubation with GM1, but not GM2 or GM3 , their sensitivity to a relatively impure LT preparation was increased; similar results were obtained with these cells using choleragen (56,60,251).…”

“…It is an oligomeric protein of--84 000 daltons, composed of different subunits, A and B. The A subunit consists of 2 peptides, A 1 and A 2, linked through a disulfide bond (for review see 20,23,24). The toxin initially binds to the cell surface, and there is considerable evidence that monosialoganglioside GMI ( Fig.…”

Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution.

“…The toxins thus must have a receptor binding activity, a membrane translocation mechanism, and an enzymatic domain. It is characteristic that the receptor binding function and the enzymatic activity reside in separate structural components of the molecules, in separate subunits of an oligomer (cholera toxin, LT, shiga toxin) (3)(4)(5), in separate proteins (anthrax system) (6), or within a single monomeric polypeptide (diphtheria toxin, exotoxin A) (7,8).…”

Induction by cholera toxin of synchronous divisions in vivo in the epidermis resulting in hyperplasia.

“…The molecular mechanisms of action of cholera toxin have been investigated extensively using intestinal and nonintestinal tissues and are now well understood (1). The toxin molecule has a molecular weight of 84,000 and consists of two types of noncovalently linked subunits, A and B.…”