Structural Basis for the Differential Toxicity of Cholera Toxin and Escherichia coli Heat-labile Enterotoxin

Abstract

Cholera toxin (Ctx) and E. coli heat-labile enterotoxin (Etx) are structurally and functionally similar AB 5 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.The severe, and at times fatal, diarrheal disease caused by Vibrio cholerae is due to the potent action of cholera toxin (Ctx) 1 (for a review, see Ref. 1). A structurally and functionally similar toxin, heat-labile enterotoxin (Etx), is produced by certain strains of enterotoxigenic Escherichia coli that are responsible for causing the generally milder "traveler's" diarrhea of humans and scouring in farm animals (2, 3). Both Ctx and Etx are hetero-oligomeric proteins comprised of a single A-subunit (M r 28,000) and five B-subunits (M r 12,000 each) (4 -6). The A-subunit contains two distinct structural domains linked by a disulfide bridge: an A1-fragment (residues 1-192) that displays ADP-ribosyl transferase activity and an A2-fragment (residues 193-240) that mediates interaction with the B-subunit pentamer (4, 6). The B-subunits of Ctx and Etx (CtxB and EtxB, respectively) bind to cell surface receptors, principally G M1 -ganglioside, found ubiquitously on the plasma membranes of eukaryotic cells (7). Although Ctx and Etx exhibit a remarkable degree of structural homology, with 81.6% sequence identity between CtxA and EtxA and 82.5% sequence identity between CtxB and EtxB (8 -10), there are a number of subtle physicochemical and function...

A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity

“…As a consequence, a Lys to Arg substitution was introduced at residue 237 in the mature CtxA subunit, resulting in an alteration in the C-terminal ϪKDEL sequence, to yield ϪRDEL (which is identical to the C terminus normally found in the A subunit of E. coli enterotoxin). This substitution in CtxA was demonstrated not to alter the A subunit's intrinsic ADPribosyltransferase activity or the kinetics and magnitude of toxin-induced Cl Ϫ secretion in polarized T84 epithelial cells (21).…”

“…Plasmids pCDR3 and pATA21 encoding Ctx holotoxin and Ctx holotoxin [with an H57A mutation in the B subunit (Ctx(H57A)], respectively, were electroporated into Vibrio cholerae 0395NT (which contains an engineered chromosomal ctxAB deletion), and the toxins were purified as reported (21). Plasmids pATA13 and pATA29 encoding wild-type CtxB and CtxB(H57A), respectively, were mobilized into the nonpathogenic Vibrio sp.…”

“…The resultant plasmids, pATA16 to pATA22, were confirmed by DNA sequencing to encode Ala substitutions at residues 52-58 in CtxB, respectively. Plasmid pCDR3 was constructed by subcloning the EcoRVSpeI fragment containing the entire ctxAB operon of pATA14 into the controlled expression vector pTTQ18 (21).…”

Cholera Holotoxin Assembly Requires a Hydrophobic Domain at the A-B5Interface: Mutational Analysis and Development of an In Vitro Assembly System

“…CTA and CTB ratios were determined by GM 1 enzyme-linked immunosorbent assay (ELISA), with the above CT-specific antibodies, as described previously (10). Holotoxin stability was assessed by a variation of the GM 1 ELISA in which the bound toxin was exposed to phosphate-buffered saline (PBS, pH 7.5), PBS (pH 7.5) plus 0.05% SDS, or McIlvaine buffer pH 5.5 plus 0.05% SDS for 30 min at room temperature before binding of the primary antibody, as described by Rodighiero et al (22). The GM 1 ELISAs presented are representative and were performed at least two times for each experiment.…”

Lipopolysaccharide 3-Deoxy-d-manno-octulosonic Acid (Kdo) Core Determines Bacterial Association of Secreted Toxins

“…Hypotheses regarding the processing and host cell trafficking of the toxins have been proposed to explain this difference in toxicity. For example, the C terminus of CTA has a prototypical KDEL ER retrograde trafficking signal sequence whereas LTA has an RDEL sequence; however, this difference has not been shown to affect the intracellular trafficking of the toxins inside the host (14). Further, CTA secreted from V. cholerae is activated by a hemagglutinin/ protease encoded by V. cholerae resulting in two polypeptides, A1 and A2, that remain linked by a disulfide bond until the toxin reaches the ER (15)(16)(17).…”