Cholera toxin

Cholera toxin (sometimes abbreviated to CTX, Ctx, or CT) is a protein complex secreted by the bacterium Vibrio cholerae. CTX is responsible for the massive, watery diarrhea characteristic of cholera infection.

Structure
The cholera toxin is an oligomeric complex made up of six protein subunits: a single copy of the A subunit (part A, enzymatic), and five copies of the B subunit (part B, receptor binding). The three-dimensional structure of the toxin was determined using X-ray crystallography by Zhang et al. in 1995.

The five B subunits&mdash;each weighing 12 kDa, and all coloured blue in the accompanying figure&mdash;form a five-membered ring. The A subunit has two important segments. The A1 portion of the chain (CTA1, red) is a globular enzyme payload that ADP-ribosylates G proteins, while the A2 chain (CTA2, orange) forms an extended alpha helix which sits snugly in the central pore of the B subunit ring.

This structure is similar in shape, mechanism, and sequence to the heat-labile enterotoxin secreted by some strains of the Escherichia coli bacterium.

Pathogenesis
The toxin is an oligomeric protein composed of one A subunit and five B subunits (AB5). After entrance into intestinal epithelial cells via receptor-mediated endocytosis, the A subunit detaches and becomes activated by proteolytic cleavage, allowing it to catalyze the ADP ribosylation of the Gαs subunit of the heterotrimeric G protein resulting in constitutive cAMP production. This in turn leads to secretion of H2O, Na+, K+, Cl-, and HCO3- into the lumen of the small intestine resulting in rapid dehydration and other factors associated with cholera, including a rice-water stool.

Interestingly, the pertussis toxin (also an AB5 protein) produced by Bordetella pertussis acts in a similar manner with the exception that it ADP-ribosylates the Gαi subunit, rendering it inactive and unable to inhibit adenylyl cyclase production of cAMP (leading to constitutive production).

Origin
The gene encoding the cholera toxin is introduced into V. cholerae by horizontal gene transfer. Virulent strains of V. cholerae carry a variant of lysogenic bacteriophage called CTXf or CTX&phi;.

Working mechanism
Once secreted, the B subunit ring of CTX will bind to GM1 gangliosides on the surface of the host's cells. After binding takes place, the entire CTX complex is internalised by the cell and the CTA1 chain is released by the reduction of a disulfide bridge. In fact, the endosome is moved to the Golgi, where the A1 protein is recognized by the endoplasmic reticulum chaperon, protein disulfide isomerase, unfolded and delivered to the membrane, where the ER-oxidase - Ero1 triggers the release of the A1 protein by oxidation of protein disulfide isomerase complex. As A1 moves from the ER into the cytoplasm by the Sec61 channel, it refolds and avoids ubiquitination.

CTA1 is then free to bind with a human partner protein called ADP-ribosylation factor 6 (Arf6); binding to Arf6 drives a change in the conformation (the shape) of CTA1 which exposes its active site and enables its catalytic activity.

The CTA1 fragment catalyses ADP ribosylation from NAD to the regulatory component of adenylate cyclase, thereby activating it. Increased adenylate cyclase activity increases cyclic AMP (cAMP) synthesis causing massive fluid and electrolyte efflux, resulting in diarrhea.

Applications
Because the B subunit appears to be relatively non-toxic, researchers have found a number of applications for it in cell and molecular biology. It is routinely used as a neuronal tracer.

GM1 gangliosides are found in lipid rafts on the cell surface. B subunit complexes labelled with fluorescent tags or subsequently targeted with antibodies can be used to identify rafts.