p97 Is in a complex with cholera toxin and influences the transport of cholera toxin and related toxins to the cytoplasm

Certain protein toxins, including cholera toxin, ricin, and Pseudomonas aeruginosa exotoxin A, are transported to the lumen of the endoplasmic reticulum where they retro-translocate across the endoplasmic reticulum membrane to enter the cytoplasm. The mechanism of retrotranslocation is poorly understood but may involve the endoplasmic reticulum-associated degradation pathway. The AAA ATPase p97 (also called valosin-containing protein) participates in the retro-translocation of cellular endoplasmic reticulum-associated degradation substrates and is therefore a candidate to participate in the retrotranslocation of protein toxins. To investigate whether p97 functions in toxin delivery to the cytoplasm, we measured the sensitivity to toxins of cells expressing either wild-type p97 or a dominant ATPase-defective p97 mutant under control of a tetracycline-inducible promoter. The rate at which cholera toxin and related toxins entered the cytoplasm was reduced in cells expressing the ATPase-defective p97, suggesting that the toxins might interact with p97. To detect interaction, the cholera toxin A chain was immunoprecipitated from cholera toxin-treated Vero cells, and co-immunoprecipitation of p97 was assessed by immunoblotting. The immunoprecipitates contained both cholera toxin A chain and p97, evidence that the two proteins are in a complex. Altogether, these results provide functional and structural evidence that p97 participates in the transport of cholera toxin to the cytoplasm.     

Toxins A and B of Clostridium difficile

Abstract: The toxins produced by Clostridium difficile share several functional properties with other bacterial toxins, like the heat-labile enterotoxin of Escherichia coli and cholera toxin. However, functional and structural differences also exist. Like cholera toxin, their main target is the disruption of the microfilaments in the cell. However, since these effects are not reversible, as found with cholera toxin, additional mechanisms add to the cytotoxic potential of these toxins. Unlike most bacterial toxins, which are built from two structurally and functionally different small polypeptide chains, the functional and binding properties of the toxins of C. difficile are confined within one large polypeptide chain, making them the largest bacterial toxins known so far.     

Characterization of the receptor for cholera toxin and Escherichia coli heat-labile toxin in rabbit intestinal brush borders.

125I-labelled heat-labile toxin (from Escherichia coli) and 125I-labelled cholera toxin bound to immobilized ganglioside GM1 and Balb/c 3T3 cell membranes with identical specificities, i.e. each toxin inhibited binding of the other. Binding of both toxins to Balb/c 3T3 cell membranes was saturable, with 50% of maximal binding occurring at 0.3 nM for cholera toxin and 1.1 nM for heat-labile toxin, and the number of sites for each toxin was similar. The results suggest that both toxins recognize the same receptor, namely ganglioside GM1. In contrast, binding of 125I-heat-labile toxin to rabbit intestinal brush borders at 0 degree C was not inhibited by cholera toxin, although heat-labile toxin inhibited 125I-cholera toxin binding. In addition, there were 3-10-fold more binding sites for heat-labile toxin than for cholera toxin. At 37 degrees C cholera toxin, but more particularly its B-subunit, did significantly inhibit 125I-heat-labile toxin binding. Binding of 125I-cholera toxin was saturable, with 50% maximal of binding occurring at 1-2 nM, and was quantitatively inhibited by 10(-8) M unlabelled toxin or B-subunit. By contrast, binding of 125I-heat-labile toxin was non-saturable (up to 5 nM), and 2 X 10(-7) M unlabelled B-subunit was required to quantitatively inhibit binding. Neuraminidase treatment of brush borders increased 125I-cholera toxin but not heat-labile toxin binding. Extensive digestion of membranes with Streptomyces griseus proteinase or papain did not decrease the binding of either toxin. The additional binding sites for heat-labile toxin are not gangliosides. Thin-layer chromatograms of gangliosides which were overlayed with 125I-labelled toxins showed that binding of both toxins was largely restricted to ganglioside GM1. However, 125I-heat-labile toxin was able to bind to brush-border galactoproteins resolved by SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose.     

Dual role of GTP-binding proteins in the control of endothelial prostacyclin

Pretreatment of bovine aortic endothelial cells with pertussis toxin inhibited partially the accumulation of inositol phosphates in response to ATP, whereas cholera toxin had no effect. Both pertussis and cholera toxins enhanced the stimulatory effect of ATP on prostacyclin release from the same cells. This action of cholera toxin was mimicked neither by an increase of cyclic AMP nor by the dissociated subunits of the toxin. Cholera and pertussis toxins, as well as aluminum fluoride, also potentiated the release of prostacyclin induced by ionophore A23187. These results suggest that a pertussis toxin-sensitive GTP-binding protein is involved in the coupling between P2-purinergic receptors and phospholipase C. In addition, another GTP-binding protein would play a crucial role at a further step in the control of PGI2 biosynthesis.     

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.Abbreviations CT cholera toxin - CT-B B-subunits of cholera toxin - LT Escherichia coli heat-labile enterotoxin - hLT humanEscherichia coli heat-labile enterotoxin - pLT porcineEscherichia coli heat-labile enterotoxin - EI electron ionization     

Effect of bacterial toxins on the mitogen-induced increase of the Ca2+ concentration in the cytoplasm of rat thymocytes. The role of N proteins

The effect of bacterial toxins, modifying the activity of regulatory N proteins of adenylate cyclase and probably other systems, on the mitogen-induced changes of cytosolic free Ca2+ concentration ([Ca2+]i) has been studied using Ca2+ fluorescent probe quin-2. It is shown that treatment of thymocytes with cholera toxin, E. coli heat-labile (HL) toxin or pertussis toxin abolishes the concanavalin A (con A)-induced rise of [Ca2+]i. The inhibitory effect of cholera and HL toxins can be explained by the toxin-induced rise of intracellular cAMP. The effect of pertussis toxin indicates the involvement of N proteins in the action of con A receptor and in generation of Ca2+-signal during the mitogenic activation of thymocytes.     

Inhibition of oxytocin-stimulated phosphoinositide turnover in rat myometrium by pertussis and cholera toxins may involve protein kinase a activation

Both pertussis and cholera toxins inhibit oxytocin-stimulated phosphoinositide turnover in rat myometrium. The actions of pertussis and cholera toxins as well as those of CPTcAMP are reversed by H-8, an inhibitor of protein kinase A. H-8 does not have a major effect on cAMP elevation by the toxins in the presence of oxytocin. The results suggest that the stimulation by oxytocin of phosphoinositide turnover does not involve direct obligatory coupling to a pertussis toxin-sensitive GTP-binding protein. Rather, indirect effects on protein kinase A activation may contribute to the inhibitory effects of both cholera and pertussis toxins. This study suggests that caution must be exercised in interpreting inhibition of phosphoinositide turnover by pertussis toxin in whole cell experiments as indicative of direct involvement of a toxin-sensitive GTP-binding protein.     

Pertussis and cholera toxin ADP-ribosylation in Dictyostelium discoideum membranes

We report a 39 kDa substrate for cholera and pertussis toxins is present in D. discoideum membranes. This protein did not co-migrate with alpha subunits of either Gs (45 kDa and 52 kDa) or Gi (41 kDa) from control mammalian cells. The presence of GTP or its non-hydrolyzable analogs enhanced the ADP-ribosylation in response to cholera toxin, but did not significantly alter ADP-ribosylation by pertussis toxin. Divalent cations inhibited the ADP-ribosylation by both toxins. The possible association of this novel G-protein with D. discoideum adenylate cyclase may underlie some of the unique regulatory features of this enzyme. Alternatively, this G-protein may regulate one of several other cellular responses mediated by the cAMP receptor.     

A class of mutant CHO cells resistant to cholera toxin rapidly degrades the catalytic polypeptide of cholera toxin and exhibits increased endoplasmic reticulum-associated degradation

After binding to the eukaryotic cell surface, cholera toxin undergoes retrograde transport to the endoplasmic reticulum. The catalytic A1 polypeptide of cholera toxin (CTA1) then crosses the endoplasmic reticulum membrane and enters the cytosol in a process that may involve the quality control mechanism known as endoplasmic reticulum-associated degradation. Other toxins such as Pseudomonas exotoxin A and ricin are also thought to exploit endoplasmic reticulum-associated degradation for entry into the cytosol. To test this model, we mutagenized Chinese hamster ovary cells and selected clones that survived a prolonged coincubation with Pseudomonas exotoxin A and ricin. These lethal endoplasmic reticulum-translocating toxins bind different surface receptors and target different cytosolic substrates, so resistance to both would likely result from disruption of a shared trafficking or translocation event. Here we characterize two Pseudomonas exotoxin A/ricin-resistant clones that exhibited increased endoplasmic reticulum-associated degradation. Both clones acquired the following unselected traits: (i) resistance to cholera toxin; (ii) increased degradation of an endoplasmic reticulum-localized CTA1 construct; (iii) increased degradation of an established endoplasmic reticulum-associated degradation substrate, the Z variant of alpha1-antitrypsin (alpha1AT-Z); and (iv) reduced secretion of both alpha1AT-Z and the transport-competent protein alpha1AT-M. Proteosome inhibition partially rescued the alpha1AT-M secretion deficiencies. However, the mutant clones did not exhibit increased proteosomal activity against cytosolic proteins, including a second CTA1 construct that was expressed in the cytosol rather than in the endoplasmic reticulum. These results suggested that accelerated endoplasmic reticulum-associated degradation in the mutant clones produced a cholera toxin/Pseudomonas exotoxin A/ricin-resistant phenotype by increasing the coupling efficiency between toxin translocation and toxin degradation.     

Retrograde transport of protein toxins through the Golgi apparatus

A number of protein toxins from plants and bacteria take advantage of transport through the Golgi apparatus to gain entry into the cytosol where they exert their action. These toxins include the plant toxin ricin, the bacterial Shiga toxins, and cholera toxin. Such toxins bind to lipids or proteins at the cell surface, and they are endocytosed both by clathrin-dependent and clathrin-independent mechanisms. Sorting to the Golgi and retrograde transport to the endoplasmic reticulum (ER) are common to these toxins, but the exact mechanisms turn out to be toxin and cell-type dependent. In the ER, the enzymatically active part is released and then transported into the cytosol, exploiting components of the ER-associated degradation system. In this review, we will discuss transport of different protein toxins, but we will focus on factors involved in entry and sorting of ricin and Shiga toxin into and through the Golgi apparatus.     

Neutrophil activation by surface bound IgG: pertussis toxin insensitive activation

Surface bound IgG induces neutrophil degranulation and production of superoxide radicals by a mechanism that is not inhibited by either pertussis toxin or cholera toxin, whereas these functions induced by soluble mediators such as FMLP and soluble aggregates of IgG are profoundly inhibited by pertussis toxin. Interaction of neutrophils with surface bound IgG triggers the loss of 32P labeled PIP2 and PIP and the influx of extracellular calcium. Neither of these cellular events when induced by surface bound IgG is inhibited by pertussis toxin. These observations suggest that neutrophil activation induced by surface bound IgG proceeds along a pathway which is not regulated by proteins which are inhibited by either pertussis or cholera toxins.     

ADP-ribosylation of bovine S-antigen by cholera toxin

The S-antigen (alias 48K protein or arrestin) of bovine rod photoreceptors contains two stretches of amino acid sequence homologous to the ADP-ribosylation sites of the alpha subunit of transducin (Ta). We have found that cholera toxin transfers the ADP-ribosyl group from NAD to purified bovine S-antigen as well as to S-antigen in rod outer segment membranes, while Bordetella pertussis toxin is unable to catalyze the transfer reaction efficiently. Under the same conditions, both toxins catalyzed ADP-ribosylation of Ta in rod outer segments. The ADP-ribosylation of S-antigen by cholera toxin indicates that S-antigen not only exhibits sequence homology with the ADP-ribosylation sites of Ta, but it must also resemble Ta in the tertiary structure of the domain which determines the susceptibility of S-antigen to the catalytic action of cholera toxin. These results suggest that S-antigen may function as a competitor of Ta in some stage of the cGMP cascade of visual transduction.     

Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins

Computer analysis of the three-dimensional structure of ADP-ribosylating toxins showed that in all toxins the NAD-binding site is located in a cavity. This cavity consists of 16 contiguous amino acids that form an a-helix bent over β-strand. The tertiary folding of this structure is strictly conserved despite the differences in the amino acid sequence. Catalysis is supported by two spatially conserved amino acids, each flanking the NAD-binding site. These are: a glutamic acid that is conserved in all toxins, and a nucleophillc residue, which is a histidine in the diphtheria toxin and Pseudomonas exotoxin A, and an arginine in the cholera toxin, the Escherichia coli heat-labile enterotoxins, the pertussis toxin and the mosquitocidal toxin of Bacillus sphaericus. The latter group of toxins presents an additional histidine that appears important for catalysis. This structure suggests a general mechanism of ADP-ribosylation evolved to work on different target proteins.     

Studies of microbial toxins in Xenopus laevis oocytes

Xenopus laevis oocytes have been incubated or microinjected with cholera and diphtheria holotoxins or their respective isolated fragments A and B. Effects on progesterone-induced maturation, protein synthesis and cAMP levels were observed. Xenopus laevis oocytes were highly susceptible to cholera toxin upon incubation as evidenced by the increase of cAMP (two-fold increase in cAMP with 0.1 nM cholera toxin) and the blockade of progesterone-induced maturation. When isolated cholera toxin fragments A or B were incubated with oocytes, no activity could be detected. However, microinjection of cholera toxin fragment A into oocyte was able to mimic the effects of incubated holotoxin. Microinjection of cholera toxin B fragment was only effective at very high concentrations, probably due to trace contaminations by the A fragment. On the other hand, Xenopus laevis oocytes were very resistant to diphtheria toxin action upon incubation, a result attributable to lack of specific membrane receptors since, after microinjection of diphtheria toxin A fragment into oocytes, inhibition of protein synthesis was demonstrated. By simultaneous microinjection of highly radioactive adenine-labelled NAD and diphtheria toxin fragment A into oocytes, radioactive ADP ribosylation of the elongation factor 2 (EF₂) was observed. It is proposed that Xenopus laevis oocytes provide a new experimental approach for studying the mechanisms of action of microbial toxins.     

Liposome fluidity alters interactions between the ganglioside GM1 and cholera toxin B subunit

Cholera toxin is a complex protein with a biologically active protein (A subunit) and a cell targeting portion (B subunit). The B subunit is responsible for specific cell binding and entry of the A subunit. One way to limit potential toxicity of the toxin after exposure is to introduce cellular decoys to bind the toxin before it can enter cells. In this study the ganglioside GM1, a natural ligand for cholera toxin, was incorporated into liposomes and the interaction between fluorescent B subunit and the liposome determined. Liposome membrane fluidity was determined to play a major role in the binding between liposomes and the cholera toxin B subunit. Liposomes with lower fluidity demonstrated greater binding with the B subunit. The findings from this study could have important implications on formulation strategies for liposome decoys of toxins.     

Liposome Fluidity Alters Interactions Between the Ganglioside GM1 and Cholera Toxin B Subunit

Cholera toxin is a complex protein with a biologically active protein (A subunit) and a cell targeting portion (B subunit). The B subunit is responsible for specific cell binding and entry of the A subunit. One way to limit potential toxicity of the toxin after exposure is to introduce cellular decoys to bind the toxin before it can enter cells. In this study the ganglioside GM1, a natural ligand for cholera toxin, was incorporated into liposomes and the interaction between fluorescent B subunit and the liposome determined. Liposome membrane fluidity was determined to play a major role in the binding between liposomes and the cholera toxin B subunit. Liposomes with lower fluidity demonstrated greater binding with the B subunit. The findings from this study could have important implications on formulation strategies for liposome decoys of toxins.     

Guanine nucleotide regulation of the pertussis and cholera toxin substrates of rat glioma C6 BU1 cells

Rat glioma C6 BU1 cells contain a pertussis toxin substrate of 40 kDa which does not appear to be identical with Gi,Go or transducin. The GTP analogue, GTP[gamma S], inhibited the rate of pertussis toxin-catalysed ADPribosylation of this protein, while the GDP analogue GDP[beta S] stimulated this reaction. A protein of the same kDa value was ADPribosylated by cholera toxin in the absence of added guanine nucleotides. It is suggested that this 40 kDa protein can be a substrate for both cholera and pertussis toxins under appropriate conditions.     

Transport of protein toxins into cells: pathways used by ricin,cholera toxin and Shiga toxin

Ricin, cholera, and Shiga toxin belong to a family of protein toxins that enter the cytosol to exert their action. Since all three toxins are routed from the cell surface through the Golgi apparatus and to the endoplasmic reticulum (ER) before translocation to the cytosol, the toxins are used to study different endocytic pathways as well as the retrograde transport to the Golgi and the ER. The toxins can also be used as vectors to carry other proteins into the cells. Studies with protein toxins reveal that there are more pathways along the plasma membrane to ER route than originally believed.     

Detection of bacterial toxins with monosaccharide arrays

A large number of bacterial toxins, viruses and bacteria target carbohydrate derivatives on the cell surface to attach and gain entry into the cell. We report here the use of a monosaccharide-based array to detect protein toxins. The array-based technique provides the capability to perform simultaneous multianalyte analyses. Arrays of N-acetyl galactosamine (GalNAc) and N-acetylneuraminic acid (Neu5Ac) derivatives were immobilized on the surface of a planar waveguide and were used as receptors for protein toxins. These arrays were probed with fluorescently labeled bacterial cells and protein toxins. While Salmonella typhimurium, Listeria monocytogenes, Escherichia coli and staphylococcal enterotoxin B (SEB) did not bind to either of the monosaccharides, both cholera toxin and tetanus toxin bound to GalNAc and Neu5Ac. The results show that the binding of the toxins to the carbohydrates is density dependent and semi-selective. Both toxins were detectable at 100 ng/ml.     

Distribution of virulence-associated genes in Vibrio mimicus isolates from clinical and environmental origins

Distribution of virulence-associated genes in Vibrio mimicus was studied including the toxin genes ctxA, tdh, st and vmh and the genes necessary for regulation of toxin production, toxR, toxS, toxT, tcpA and tcpP. Approximately half of clinical V. mimicus isolates possessed one or more genes encoding V. cholerae enterotoxic factors such as ctxA, tdh and st. All of the clinical and environmental isolates possessed vmh encoding V. mimicus hemolysin (VMH). The ctxA encoding cholera toxin was detected in only 2 strains, 5% of the clinical isolates. Furthermore, there were very few strains possessing tcpP and toxT needed for the expression of ctxA. These results may suggest that VMH is a more important pathogenic factor than well recognized toxins such as cholera toxin (CT) in V. mimicus infection.