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In nature, cholera toxin (CT) and the structurally related E. coli heat labile toxin type I (LTI) must breech the epithelial barrier of the intestine to cause the massive diarrhea seen in cholera. This requires endocytosis of toxin-receptor complexes into the apical endosome, retrograde transport into Golgi cisternae or endoplasmic reticulum (ER), and finally transport of toxin across the cell to its site of action on the basolateral membrane. Targeting into this pathway depends on toxin binding ganglioside GM1 and association with caveolae-like membrane domains. Thus to cause disease, both CT and LTI co-opt the molecular machinery used by the host cell to sort, move, and organize their cellular membranes and substituent components.

Abstract. Vibrio cholerae and Escherichia coli heat labile toxins (CT and LT) elicit a secretory response from intestinal epithelia by binding apical receptors (ganglioside GM1 ) and subsequently activating basolateral effectors (adenylate cyclase). We have recently proposed that signal transduction in polarized cells may require transcytosis of toxin-containing membranes (Lencer, W. I., G. Strohmeier, S. Moe, S. L. Carlson, C. T. Constable, and J. L. Madara. 1995. Proc. Natl. Acad. Sci. USA. 92:10094-10098). Targeting of CT into this pathway depends initially on binding of toxin B subunits to GMI at the cell surface. The anatomical compartments in which subsequent steps of CT processing occur are less clearly defined. However, the enzymatically active A subunit of CT contains the ER retention signal KDEL (RDEL in LT). Thus if the KDEL motif were required for normal CT trafficking, movement of CT from the Golgi to ER would be implied. To test this idea, recombinant wild-type (wt) and mutant CT and LT were prepared. The COOH-terminal KDEL sequence in CT was replaced by seven unrelated amino acids: LEDERAS. In LT, a single point mutation replacing leucine with valine in RDEL was made. Wt and mutant toxins displayed similar enzymatic activities and binding affinities to GM1 immobilized on plastic. Biologic activity of recombinant toxins was assessed as a C1-secretory response elicited from the polarized human epithelial cell line T84 using standard electrophysiologic techniques. Mutations in K(R)DEL of both CT and LT delayed the time course of toxin-induced C1-secretion. At T1/2, dose dependencies for K(R)DELmutant toxins were increased ~>10-fold. KDELmutants displayed differentially greater temperature sensitivity. In direct concordance with a slower rate of signal transduction, KDEL-mutants were trafficked to the basolateral membrane more slowly than wt CT (assessed by selective cell surface biotinylation as transcytosis of B subunit). Mutation in K(R)DEL had no effect on the rate of toxin endocytosis. These data provide evidence that CT and LT interact directly with endogenous KDEL-receptors and imply that both toxins may require retrograde movement through Golgi cisternae and ER for efficient and maximal biologic activity.ETROGRADE transport through Golgi cisternae has been shown to occur for soluble and membrane proteins of the ER (49) and for certain protein toxins (6, 55). Targeting of soluble ER and some type II membrane proteins in this pathway depends on the COOH-terminal sorting signal Lys-Asp-Glu-Leu (KDEL or HDEL

Cholera toxin (Ctx) from Vibrio cholerae and its closely related homologue, heat-labile enterotoxin (Etx) from Escherichia coli have become superb tools for illuminating pathways of cellular trafficking and immune cell function. These bacterial protein toxins should be viewed as conglomerates of highly evolved, multi-functional elements equipped to engage the trafficking and signalling machineries of cells. Ctx and Etx are members of a larger family of A-B toxins of bacterial (and plant) origin that are comprised of structurally and functionally distinct enzymatically active A and receptor-binding B sub-units or domains. Intoxication of mammalian cells by Ctx and Etx involves B pentamer-mediated receptor binding and entry into a vesicular pathway, followed by translocation of the enzymatic A1 domain of the A sub-unit into the target cell cytosol, where covalent modification of intracellular targets leads to activation of adenylate cyclase and a sequence of events culminating in life-threatening diarrhoeal disease. Importantly, Ctx and Etx also have the capacity to induce a wide spectrum of remarkable immunological processes. With respect to the latter, it has been found that these toxins activate signalling pathways that modulate the immune system. This review explores the complexities of the cellular interactions that are engaged by these bacterial protein toxins, and highlights some of the new insights to have recently emerged.

The importance of receptor binding in the potent immunogenicity of Escherichia coli heat-labile enterotoxin B subunit (EtxB) was tested by comparing its immunogical properties with those of a receptor binding mutant, EtxB(G33D). Subcutaneous immunization of EtxB(G33D) resulted in 160-fold reduction in antibody titer compared with wild-type EtxB, whereas its oral delivery failed to provoke any detectable secretory or serum anti-B subunit responses. Moreover, the two proteins induced strikingly different effects on lymphocyte cultures in vitro. EtxB, in comparison with EtxB(G33D), caused an increase in the proportion of B cells, many ofwhich were activated (CD25+); the complete depletion of CD8+ T cells; an increase in the activation of CD4+ T cells; and an increase in interleukin 2 and a decrease in interferon 'y. These data indicate that EtxB exerts profound effects on immune cells, suggesting that its potent immunogenicity is dependent not only on efficient receptor-mediated uptake, but also on direct receptor-mediated immunomodulation of lymphocyte subsets.

No abstract

In this paper we study the assembly, in vivo and in vitro, of a family of hexameric, heat-labile enterotoxins produced by diarrheagenic bacteria. The toxins, which consist of an A subunit and five B subunits, are assembled by a highly coordinated process that ensures secretion of the holotoxin complex. We show that (i) oxidation of cysteine residues in the B subunits is a prerequisite step for in vivo formation of B-subunit pentamers, (ii) reduction of dissociated B subunits in vitro abolishes their ability to reassemble, (iii) the kinetics of B-pentamer assembly inl vivo can be mimicked under defined conditions in vitro, (iv) A subunits cannot associate with fully assembled B pentamers in vitro, and (v) A subunits cause an 3-fold acceleration in the rate of B-subunit pentamerization in vivo, implying that A subunits play a coordinating role in the pathway of holotoxin assembly. The last finding is likely to be of general significance, since it provides a mechanism for preferentially excluding or favoring certain intermediates in the assembly of multisubunit proteins.

A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation in E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.