The hydrophobicities of cholera toxin, tetanus toxin and their components.
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ABSTRACT:
SUBMITTER: Ward WH
PROVIDER: S-EPMC1163391 | biostudies-other | 1981 Nov
REPOSITORIES: biostudies-other
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Project description:Cholera is a diarrheal disease caused by a protein toxin released by Vibrio cholera in the host's intestine. The toxin enters intestinal epithelial cells after binding to specific carbohydrates on the cell surface. Over recent years, considerable effort has been invested in developing inhibitors of toxin adhesion that mimic the carbohydrate ligand, with particular emphasis on exploiting the multivalency of the toxin to enhance activity. In this review we introduce the structural features of the toxin that have guided the design of diverse inhibitors and summarise recent developments in the field.
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Project description:The molecular mechanisms involved in host pathogen interactions at mucosal surfaces needs to be better understood in order to develop immune-mediated methods of protection against pathogens. Cholera toxin (CT) has the rare ability for a protein of inducing robust mucosal immunity in the gut and is therefore an excellent model with which to determine mechanisms of adjuvanticity and immunogenicity at intestinal mucosal surfaces. Jejunal epithelial cells are one of the first sites of antigen encounter. Therefore a porcine intestinal epithelial cell line, IPEC-J2, was cultured in 6-well transwell plates in the presence or absence of 50 ng/ml cholera toxin for up to 8 hours and the cell layer was harvested for gene expression analysis using the Affymetrix porcine genome array and real-time PCR analysis. Affymetrix analysis identified, and real-time PCR analysis of 15 genes confirmed, an increase in gene expression for 59 genes and a decrease in gene expression for 14 genes under CT treatment. An 8 hour time course of expression revealed that by 2-4 hours after CT treatment, all 10 upregulated genes were differentially expressed and by 4-6 hours after CT treatment 3 of the 5 downregulated genes were differentially expressed. These data suggest that the potent mucosal adjuvanticity and immunogenicity of CT derives from rapid alterations in gene expression at the site of first antigen encounter with the immune system. Characterization of early immune gene expression may elucidate potential biological mechanisms for mucosal immune induction leading to the development of effective vaccines against enteric pathogens. Keywords: Treatment comparison, cholera toxin vs untreated at 8h time point
2008-04-01 | GSE10314 | GEO
Project description:Cholera toxin (CTx) is an AB5 cytotonic protein that has medical relevance in cholera and as a novel mucosal adjuvant. Here, we report an analysis of the noncovalent homopentameric complex of CTx B chain (CTx B5) using electrospray ionization triple quadrupole mass spectrometry and tandem mass spectrometry and the analysis of the noncovalent hexameric holotoxin usingelectrospray ionization time-of-flight mass spectrometry over a range of pH values that correlate with those encountered by this toxin after cellular uptake. We show that noncovalent interactions within the toxin assemblies were maintained under both acidic and neutral conditions in the gas phase. However, unlike the related Escherichia coli Shiga-like toxin B5 pentamer (SLTx B), the CTx B5 pentamer was stable at low pH, indicating that additional interactions must be present within the latter. Structural comparison of the CTx B monomer interface reveals an additional alpha-helix that is absent in the SLTx B monomer. In silico energy calculations support interactions between this helix and the adjacent monomer. These data provide insight into the apparent stabilization of CTx B relative to SLTx B.
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Project description:Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (LeX) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the LeX glycan in vitro when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to LeX. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.
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Project description:The five B-subunits (CTB5) of the Vibrio cholerae (cholera) toxin can bind to the intestinal cell surface so the entire AB5 toxin can enter the cell. Simultaneous binding can occur on more than one of the monosialotetrahexosylganglioside (GM1) units present on the cell surface. Such simultaneous binding arising from the toxins multivalency is believed to enhance its affinity. Thus, blocking the initial attachment of the toxin to the cell surface using inhibitors with GM1 subunits has the potential to stop the disease. Previously we showed that tetravalent GM1 molecules were sub-nanomolar inhibitors of CTB5. In this study, we synthesized a pentavalent version and compared the binding and potency of penta- and tetravalent cholera toxin inhibitors, based on the same scaffold, for the first time. The pentavalent geometry did not yield major benefits over the tetravalent species, but it was still a strong inhibitor, and no major steric clashes occurred when binding the toxin. Thus, systems which can adopt more geometries, such as those described here, can be equally potent, and this may possibly be due to their ability to form higher-order structures or simply due to more statistical options for binding.
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Project description:During the past forty years there has been an inexplicable increase in chronic inflammatory disorders, including obesity. One theory, the 'hygiene hypothesis', involves dysregulated immunity arising after too few beneficial early life microbe exposures. Indeed, earlier studies have shown that gut microbe-immune interactions contribute to smoldering inflammation, adiposity, and weight gain. Here we tested a safe and well-established microbe-based immune adjuvant to restore immune homeostasis and counteract inflammation-associated obesity in animal models. We found that consuming Vibrio cholerae exotoxin subunit B (ctB) was sufficient to inhibit age-associated obesogenic outcomes in wild type mice, including reduced crown-like structures (CLS) and granulomatous necrosis histopathology in fat depots. Administration of cholera toxin reduced weight gain irrespective of age during administration; however, exposure during youth imparted greater slenderizing effects when compared with animals receiving ctB for the first time during adulthood. Beneficial effects were transplantable to other obesity-prone animals using immune cells alone, demonstrating an immune-mediated mechanism. Taken together, we concluded that oral vaccination with cholera toxin B helps stimulate health-protective immune responses that counteract age-associated obesity.
| S-EPMC6756858 | biostudies-literature
Project description:The clostridial neurotoxins (CNTs) comprise tetanus toxin (TT) and botulinum neurotoxin (BoNT [BT]) serotypes (A to G and X) and several recently identified CNT-like proteins, including BT/En and the mosquito BoNT-like toxin Pmp1. CNTs are produced as single proteins cleaved to a light chain (LC) and a heavy chain (HC) connected by an interchain disulfide bond. LC is a zinc metalloprotease (cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]), while HC contains an N-terminal translocation domain (HCN) and a C-terminal receptor binding domain (HCC). HCN-mediated LC translocation is the least understood function of CNT action. Here, ?-lactamase (?lac) was used as a reporter in discovery-based live-cell assays to characterize TT-mediated LC translocation. Directed mutagenesis identified a role for a charged loop (767DKE769) connecting ?15 and ?16 (cis-loop) within HCN in LC translocation; aliphatic substitution inhibited LC translocation but not other toxin functions such as cell binding, intracellular trafficking, or HCN-mediated pore formation. K768 was conserved among the CNTs. In molecular simulations of the HCN with a membrane, the cis-loop did not bind with the cell membrane. Taken together, the results of these studies implicate the cis-loop in LC translocation, independently of pore formation.IMPORTANCE How protein toxins translocate their catalytic domain across a cell membrane is the least understood step in toxin action. This study utilized a reporter, ?-lactamase, that was genetically fused to full-length, nontoxic tetanus toxin (?lac-TT) in discovery-based live-cell assays to study LC translocation. Directed mutagenesis identified a role for K768 in LC translocation. K768 was located between ?15 and ?16 (termed the cis-loop). Cellular assays showed that K768 did not interfere with other toxin functions, including cell binding, intracellular trafficking, and pore formation. The equivalent K768 is conserved among the clostridial neurotoxin family of proteins as a conserved structural motif. The cis-loop appears to contribute to LC translocation.
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Project description:When cholera toxin is incubated under u.v. light with NAD+ labelled in either the adenine or the nicotinamide moiety, radioactivity becomes covalently bound to the protein. The reaction is specific for cholera toxin, and is inhibited by excess unlabelled NAD+ or NAD analogues. Only the active A 1 chain of the toxin is labelled. The u.v.-absorption spectrum of the product is very similar to that of NAD+, and shows the same reaction with cyanide. The nature of the product is therefore different from that found when diphtheria toxin is photolabelled [Carroll & Collier (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3307-3311] in that the yield is lower, but both moieties of the NAD molecule become bound.
| S-EPMC1147798 | biostudies-other
Project description:1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.
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