Cholera Toxin and Enterotoxins
 
 Cholera toxin (CLT) and the closely similar heat-labile enterotoxins (LTs) are released by toxigenic strains of Vibrio cholerae and Escherichia coli, respectively, the causative agents of epidemic diarrheas (1). These bacteria attach firmly to the apical portion of the intestinal epithelium and produce several toxins, including CLT or LT, encoded by genes contained in a transferable virulence cassette (2). Five B subunits (103 residues) are secreted into the bacterial periplasm, where they assemble into a pentamer that binds the A chain. A is then proteolytically cleaved at a single point to generate the catalytic A1 chain (192 residues) linked via a disulfide bond to the A2 peptide (3, 4) (Fig. 1). The structural organization of these enterotoxins is shared by Shiga toxins (other enterotoxins, which cause bloody diarrheas and necrosis of intestinal epithelium and are produced by Shiga spp. and E. coli spp.) and pertussis toxin (3-6). The B pentamer has the shape of an irregular cylinder with a flat surface and convoluted bottom surface. The five B monomers are arranged around a 5-fold axis in the oligomer-binding fold, recently identified in a set of oligonucleotide- and oligosaccharide-binding proteins (7). Each monomer contains five b-strands in an antiparallel beta-sheet and one alpha-helix. The five helices form a central pore hosting the carboxyl-terminal segment of A2, which emerges on the other side with a Lys/Arg-Asp-Glu-Leu motif that may be important in cell penetration (8). The amino-terminal half of A2 forms a long a-helix, rising from the flat surface, involved in interaction with A1. Thus, very few protein–protein contacts exist between A1 and B. The convoluted oligomer B surface, distal from A1, forms five sugar-binding sites on the outside edge of the cylinder (1, 3, 4).

Figure 1. Structural organization of cholera toxin and related toxins, showing a cross section of the molecule of cholera toxin and the E. coli heat-labile enterotoxins (1, 3, 4). A is composed of two polypeptide chains: A1, endowed with ADP-ribosyltransferase activity, and A2, which consists of a long a-helix, involved in interaction with A1, followed by a structureless segment that penetrates the small central hole of the B pentamer. Each of the five B subunits forming the B oligomer has a binding site for the oligosaccharide portion of the ganglioside GM1. Thus, cholera toxin binds to the membrane via multiple interactions, with the catalytic A1 subunit pointing away from the membrane, with little protein–protein contact between A1 and B.
The structure of A1 shows a cleft where NAD binds and Arg-7 and Glu-112, two residues essential for activity, are located (9). Each B subunit of CLT or LTs possesses a single binding site specific for the oligosaccharide portion of a ganglioside (10), and one molecule of toxin binds five glycolipid molecules. Oligosaccharide binding affinity is rather low, but a high affinity of the toxin for the cell (K_d of the order of 10^–10) is obtained because of the pentavalent binding. This strategy of
multivalent binding to obtain a strong cell association is displayed by other bacterial toxins and viruses (11).
The target of CLT and LTs is localized on the basolateral membrane; therefore, these toxins have to transcytose through the cell to display their activity. Available evidence indicates that CLT and LTs are internalized inside apical endosomes that may recycle the glycolipid–toxin complex to the surface or deliver it to later endosomes. As a result of the binding of the KDEL sequence of the A subunit to a KDEL receptor, the toxin moves retrogradely through the Golgi cisternae, and some toxin molecules are then expected to be sorted into basolateral endosomes that fuse with the basolateral membrane (8). At some stage of this intracellular trafficking, these toxins have to be reduced and the A1 subunit has to translocate from the lumenal to the cytosolic side of the membrane to interact with their target on the cytosolic face of the basolateral membrane. Structural and membrane photolabeling data indicate that oligomer B does not penetrate the lipid bilayer (1, 10  ,12) but A1 inserts in the membrane upon reduction of the A1 A2 interchain disulfide bond. Hence, it is likely that as soon as the disulfide bridge is reduced, A1 “rolls over” oligomer B and inserts into the membrane. This is at variance from postulated mechanism of membrane penetration of other toxins, whose protomer B plays an active role in the insertion of the catalytic subunit. Neither the chemical nature of the reducing agent nor the intracellular stage at which this step takes place are known.
The A1 subunits of CLT and LTs catalyze the transfer of ADP-ribose from NAD to an Arg residue present in the LRXRVXT conserved sequence of the a subunit of the G_S, G_t, and G_olf large trimeric GTP-binding proteins involved in the coupling of cell surface receptors to the adenylate cyclase (13). Such modification results in a permanent activation of this latter enzyme and a large increase in cellular cyclic AMP (cAMP) level, which initiates a cascade of signal transduction pathways. A1 ADP-ribosylating activity is enhanced by a group of cytosolic or membrane GTP-binding proteins, termed ARF (ADP-ribosylating factors), present in eukaryotic cells (14). ARF are strongly conserved from yeast to humans and are involved in the control of membrane trafficking and protein transport inside cells (15).
 The increased cAMP level brought about by CLT or LTs in the enterocyte has a series of consequences, but the inhibition of a sodium channel and activation of a chloride channel localized on the apical membrane appear to be very relevant to diarrhea. In fact, a decreased sodium reabsorption and increased chloride secretion cause an osmosis-driven loss of water into the intestine. Also important in cholera is the activation of entero-chromaffim cells, which respond to the cAMP increase with release of VIP (vasointestinal peptide), which further lowers intestinal water reabsorption and inhibits muscle cells with an alteration of intestinal peristalsis.
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