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Entry of cholera toxin (CT) into target epithelial cells and the induction of toxicity depend on CT binding to the lipid-based receptor ganglioside G(M1) and association with detergent-insoluble membrane microdomains, a function of the toxin's B-subunit. The B-subunits of CT and related Escherichia coli toxins exhibit a highly conserved exposed peptide loop (Glu(51)-Ile(58)) that faces the cell membrane upon B-subunit binding to G(M1). Mutation of His(57) to Ala in this loop resulted in a toxin (CT-H57A) that bound G(M1) with high apparent affinity, but failed to induce toxicity. CT-H57A bound to only a fraction of the cell-surface receptors available to wild-type CT. The bulk of cell-surface receptors inaccessible to CT-H57A localized to detergent-insoluble apical membrane microdomains (lipid rafts). Compared with wild-type toxin, CT-H57A exhibited slightly lower apparent binding affinity for and less stable binding to G(M1) in vitro. Rather than being transported into the Golgi apparatus, a process required for toxicity, most of CT-H57A was rapidly released from intact cells at physiologic temperatures or degraded following its internalization. These data indicate that CT action depends on the stable formation of the CT B-subunit.G(M1) complex and provide evidence that G(M1) functions as a necessary sorting motif for the retrograde trafficking of toxin into the secretory pathway of target epithelial cells.

Cholera toxin (Ctx) and E. coli heat-labile enterotoxin (Etx) are structurally and functionally similar AB5 toxins with over 80% sequence identity. When their action in polarized human epithelial (T84) cells was monitored by measuring toxin-induced Cl- ion secretion, Ctx was found to be the more potent of the two toxins. Here, we examine the structural basis for this difference in toxicity by engineering a set of mutant and hybrid toxins and testing their activity in T84 cells. This revealed that the differential toxicity of Ctx and Etx was (i) not due to differences in the A-subunit's C-terminal KDEL targeting motif (which is RDEL in Etx), as a KDEL to RDEL substitution had no effect on cholera toxin activity; (ii) not attributable to the enzymatically active A1-fragment, as hybrid toxins in which the A1-fragment in Ctx was substituted for that of Etx (and vice versa) did not alter relative toxicity; and (iii) not due to the B-subunit, as the replacement of the B-subunit in Ctx for that of Etx caused no alteration in toxicity, thus excluding the possibility that the broader receptor specificity of EtxB is responsible for reduced activity. Remarkably, the difference in toxicity could be mapped to a 10-amino acid segment of the A2-fragment that penetrates the central pore of the B-subunit pentamer. A comparison of the in vitro stability of two hybrid toxins, differing only in this 10-amino acid segment, revealed that the Ctx A2-segment conferred a greater stability to the interaction between the A- and B-subunits than the corresponding segment from Etx A2. This suggests that the reason for the relative potency of Ctx compared with Etx stems from the increased ability of the A2-fragment of Ctx to maintain holotoxin stability during uptake and transport into intestinal epithelia.