Timothy R. Hirst

Protein disulphide isomerase: building bridges in protein folding

Protein disulphide isomerase (PDI) has been known for many years to assist in the folding of proteins containing disulphide bonds, but the exact mechanism by which it achieves this is only now becoming clear. The active site of PDI closely resembles that of the redox protein thioredoxin, and cDNA cloning has revealed a superfamily of proteins with related active-site sequences, in organisms ranging from bacteria to higher animals and plants. Recent mutagenesis studies are now helping to unravel the catalytic mechanism of PDI, and work in yeast and other systems is clarifying the physiological roles of the multiple PDI-related proteins.

MEMBRANE TRAFFIC AND THE CELLULAR UPTAKE OF CHOLERA TOXIN

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.

Immune modulation by the cholera-like enterotoxins: from adjuvant to therapeutic

Cholera toxin and its close relative, Escherichia coli heat-labile enterotoxin, are potent immunogens and mucosal adjuvants. The recent findings that their B subunits can promote tolerance highlights the complexity of their interactions with the immune system. Here, Neil Williams and colleagues review the mechanisms by which these molecules modulate leukocyte populations and seek to explain the paradox.

Cholera toxin: A paradigm for multi-functional engagement of cellular mechanisms (Review)

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.

A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae

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.

Modulation of B-cell activation by the B subunit of Escherichia coli enterotoxin: receptor interaction up-regulates MHC class II, B7, CD40, CD25 and ICAM-1

The B subunits of cholera toxin (CtxB) and Escherichia coli heat‐labile enterotoxin (EtxB) are non‐toxic lectins that bind and cross‐link a ubiquitous cell glycolipid receptor, ganglioside GM1, and are recognized as potent mucosal and systemic immunogens. Here we examine the role of EtxB receptor occupancy in modulating the activation of B cells, in vitro, in primary lymphocyte cultures containing B and T cells. When 48‐hr spleen cell cultures containing EtxB were compared with those in the presence of a non‐receptor binding mutant, EtxB(G33D), a marked shift in the ratio of CD4+ T cells:B cells was noted. Evidence suggested that this was the result of either enhanced survival or proliferation of B cells associated with receptor occupancy by EtxB. Investigation revealed that EtxB induced only a minimal increase in proliferation above that of EtxB(G33D), in mixed cell cultures, and failed to induce any cell division of purified B cells or T cells. In contrast, receptor‐binding by EtxB markedly up‐regulated the expression of major histocompatability complex (MHC) class II, B7, intracellular adhesion molecule‐1 (ICAM‐1), CD40 and CD25 on the B‐cell surface. These results indicate that the polyclonal effects of EtxB on B cells are not associated with wide‐scale proliferation, but more likely with maintenance of B‐cell survival by activation of molecules essential for B‐cell differentiation. The findings also highlight the essential role of GM1‐interaction with EtxB in the regulation of lymphocyte responses.

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

GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.

Current progress in the development of the B subunits of cholera toxin and Escherichia coli heat-labile enterotoxin as carriers for the oral delivery of heterologous antigens and epitopes

The development of non-living carrier systems for delivery of protective antigens or epitopes to the immune system represents both a fundamental and an applied aspect of vaccinology. A wide range of carrier systems, ranging from inert supports to proteins that exert direct immunomodulating effects on the immune response, are being studied. In this overview we describe the current progress in the development of the B-subunits of cholera toxin and Escherichia coli heat-labile enterotoxin as potential protein carriers for the oral delivery of chemically and genetically attached antigens and epitopes.

Comparison of the glycolipid-binding specificities of cholera toxin and porcineEscherichia coli heat-labile enterotoxin: identification of a receptor-active non-ganglioside glycolipid for the heat-labile toxin in infant rabbit small intestine

The binding specificities of cholera toxin andEscherichia coli heat-labile enterotoxin were investigated by binding of125I-labelled toxins to reference glycosphingolipids separated on thin-layer chromatograms and coated in microtitre wells. The binding of cholera toxin was restricted to the GM1 ganglioside. The heat-labile toxin showed the highest affinity for GM1 but also bound, though less strongly, to the GM2, GD2 and GD1b gangliosides and to the non-acid glycosphingolipids gangliotetraosylceramide and lactoneotetraosylceramide. The infant rabbit small intestine, a model system for diarrhoea induced by the toxins, was shown to contain two receptor-active glycosphingolipids for the heat-labile toxin, GM1 ganglioside and lactoneotetraosylceramide, whereas only the GM1 ganglioside was receptor-active for cholera toxin. Preliminary evidence was obtained, indicating that epithelial cells of human small intestine also contain lactoneotetraosylceramide and similar sequences. By computer-based molecular modelling, lactoneotetraosylceramide was docked into the active site of the heat-labile toxin, using the known crystal structure of the toxin in complex with lactose. Interactions which may explain the relatively high toxin affinity for this receptor were found.

Proteolytic Activation of Cholera Toxin and Escherichia coli Labile Toxin by Entry into Host Epithelial Cells SIGNAL TRANSDUCTION BY A PROTEASE-RESISTANT TOXIN VARIANT

Cholera and Escherichia coliheat-labile toxins (CT and LT) require proteolysis of a peptide loop connecting two major domains of their enzymatic A subunits for maximal activity (termed “nicking”). To test whether host intestinal epithelial cells may supply the necessary protease, recombinant rCT and rLT and a protease-resistant mutant CTR192H were prepared. Toxin action was assessed as a Cl− secretory response (Isc) elicited from monolayers of polarized human epithelial T84 cells. When applied to apical cell surfaces, wild type toxins elicited a brisk increase in Isc (80 μA/cm2). Isc was reduced 2-fold, however, when toxins were applied to basolateral membranes. Pretreatment of wild type toxins with trypsin in vitro restored the “basolateral” secretory responses to “apical” levels. Toxin entry into T84 cells via apical but not basolateral membranes led to nicking of the A subunit by a serine-type protease. T84 cells, however, did not nick CTR192H, and the secretory response elicited by CTR192H remained attenuated even when applied to apical membranes. Thus, T84 cells express a serine-type protease(s) fully sufficient for activating the A subunits of CT and LT. The protease, however, is only accessible for activation when the toxin enters the cell via the apical membrane.