Primary structure of cholera toxin B-subunit

The primary structure of cholera toxin B-subunit, responsible for the binding of the toxin to cell surfaces, has been elucidated. The polypeptide contains 103 amino acid residues and one intra-chain disulfide bridge between Cys 9 and Cys 86. The molecular weight is calculated to be 11,637, 15–20% higher than the values estimated by physicochemical methods. This value is consistent with a structure containing five moles of B-subunits per mole of cholera toxin.     

Pertussis toxin B-subunit-induced Ca2(+)-fluxes in Jurkat human lymphoma cells: the action of long-term pre-treatment with cholera and pertussis holotoxins

The exotoxins of Bordetella pertussis and Vibrio cholera have been used to investigate signal transduction in the human T-cell lymphoma Jurkat. Stimulation of the cells, leading to an increase in cytoplasmic free calcium, could be achieved by the anti-T-cell receptor complex antibody OKT3 and by pertussis holotoxin (PTHT), or its B-subunit (PTB), but not by cholera holotoxin (CTHT) or its B-subunit (CTB). Both holotoxins ADP-ribosylated specifically G-proteins in the plasma membrane of intact cells, while their B-subunits had no ADP-ribosyltransferase activity. Incubation of the cells with CTHT led to a state of unresponsiveness to all stimulants. CTB was without any effect, indicating that the ADP-ribosyltransferase activity of cholera toxin (located in the A-subunit of the holotoxin) was necessary for the inhibition of cellular signalling. The inhibitory effect of cholera toxin on the pertussis toxin action was not due to a blockade of pertussis toxin interaction with the cell surface, because pertussis toxin was still able to ADP-ribosylate membrane proteins in cholera toxin treated intact cells. In addition, the cholera toxin mediated inhibition was not due to elevated levels of cyclic-AMP, as forskolin (a direct activator of the adenylate cyclase) and no inhibitory effect. The stimulating effect of PTHT was independent of its ADP-ribosyltransferase activity, because it could also be obtained by the B-subunit alone. In addition, the increase of cytoplasmic free calcium after stimulation by PTHT clearly preceded the ADP-ribosylation. Pre-treatment with PTHT, PTB or OKT3, led to a long lasting increase in the level of intracellular Ca2+ in Jurkat cells, which could not, therefore, be stimulated further. Inhibition by cholera holotoxin of the stimulation by OKT3 and pertussis toxin (PTHT and PTB) imply that the mitogenic effect of pertussis toxin is perhaps mediated via the T-cell antigen receptor signalling cascade. The presented data do not support the idea that a pertussis toxin-sensitive G-protein is involved in coupling the T-cell antigen receptor to the phospholipase C.     

The ionic channels formed by cholera toxin in planar bilayer lipid membranes are entirely attributable to its B-subunit.

The interaction of cholera toxin with planar bilayer lipid membranes (BLM) at low pH results in the formation of ionic channels, the conductance of which can be directly measured in voltage-clamp experiments. It is found that the B-subunit of cholera toxin (CT-B) also is able to induce ionic channels in BLM whereas the A-subunit is not able to do it. The increase of pH inhibited the channel-forming activity of CT-B. The investigation of pH-dependences of both the conductance and the cation-anion selectivity of the CT-B channel allowed us to suggest that the water pore of this channel is confined to the B-subunit of cholera toxin. The effective diameter of the CT-B channels water pores was directly measured in BLM and is equal to 2.1 +/- 0.2 nm. The channels formed by whole toxin and its B-subunit exhibit voltage-dependent activity. We believe these channels are relevant to the mode of action of cholera toxin and especially to the endosomal pathway of the A-subunit into cells.     

Sequence of heat-labile enterotoxin of Escherichia coli pathogenic for humans.

We determined the complete nucleotide sequence of the toxB gene (375 base pairs in length), which encodes the B subunit of heat-labile enterotoxin produced from Escherichia coli pathogenic for humans (hLT). The amino acid sequence of the B subunit of hLT was deduced from the nucleotide sequence. Consequently, it has become possible to study the homology between the B subunits of three similar toxins: hLT, LT produced from E. coli pathogenic for piglets (pLT), and cholera toxin (the latter two sequences have been reported by others). The three B subunits are all 103 amino acids in length. A comparison of the toxB gene and the eltB gene, which encodes the B subunit of pLT, showed a 98% homology at the nucleotide level and a 95% homology at the amino acid (of a precursor) level, indicating the possibility that the two genes share a common ancestor. With respect to the B-subunit sequences, the homologies between hLT and pLT, between hLT and cholera toxin, and between pLT and cholera toxin were 96, 81, and 79%, respectively. Several large common sequences are conserved by the three peptides. In contrast, no sequences are present in both pLT and cholera toxin but missing in hLT.     

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.     

Vibrio cholerae: cholera toxin

The bacterial protein toxin of Vibrio cholerae, cholera toxin, is a major agent involved in severe diarrhoeal disease. Cholera toxin is a member of the AB toxin family and is composed of a catalytically active heterodimeric A-subunit linked with a homopentameric B-subunit. Upon binding to its receptor, GM0(1), cholera toxin is internalized and transported in a retrograde manner through the Golgi to the ER, where it is retrotranslocated to the cytosol. Here, cholera toxin reaches its intracellular target, the basolaterally located adenylate cyclase which becomes constitutively activated after toxin-induced mono-ADP-ribosylation of the regulating G(S)-protein. Elevated intracellular cAMP levels provoke loss of water and electrolytes which is manifested as the typical diarrhoea. The cholera toxin B-subunit displays the capacity to fortify immune responses to certain antigens, to act as a carrier and to be competent in inducing immunological tolerance. These unique features make cholera toxin a promising tool for immunologists.     

Effect of the B Subunit of the Cholera Toxin on the Raw 264.7 Murine Macrophage-Like Cell Line

Russian Journal of Bioorganic Chemistry - The 125I-labeled B-subunit of the cholera toxin ([125I]CT-B with specific activity 98 Ci/mmol) was found to be bonded to the murine macrophage-like cells...     

Thermodynamics of intersubunit interactions in cholera toxin upon binding to the oligosaccharide portion of its cell surface receptor, ganglioside GM1

The binding and the energetics of the interaction of cholera toxin with the oligosaccharide portion of ganglioside GM1 (oligo-GM1), the toxin cell surface receptor, have been studied by high-sensitivity isothermal titration calorimetry and differential scanning calorimetry. Previously, we have shown that the association of cholera toxin to ganglioside GM1 enhances the cooperative interactions between subunits in the B-subunit pentamer [Goins, B., & Freire, E. (1988) Biochemistry 27, 2046-2052]. New experiments presented in this paper reveal that the oligosaccharide portion of the receptor is by itself able to enhance the intersubunit cooperative interactions within the B pentamer. This effect is seen in the protein unfolding transition as a shift from independent unfolding of the B promoters toward a cooperative unfolding. To identify the origin of this effect, the binding of cholera toxin to oligo-GM1 has been measured calorimetrically under isothermal conditions. The binding curve at 37 degrees C is sigmoidal, indicating cooperative binding. The binding data can be described in terms of a nearest-neighbor cooperative interaction binding model. In terms of this model, the association of a oligo-GM1 molecule to a B protomer affects the association to adjacent B promoters within the pentameric ring. The measured intrinsic binding enthalpy per protomer is -22 kcal/mol and the cooperative interaction enthalpy -11 kcal/mol. The intrinsic binding constant determined calorimetrically is 1.05 x 10(6) M-1 at 37 degrees C and the cooperative Gibbs free energy equal to -850 cal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design and Study of Properties of Avirulent Genetically Modified <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Biovar El Tor Strain with Inactivated Genes of Thermolabile Hemolysin and Effective Expression of Cloned Cholera Toxin B-subunit Gene

The results of the construction of an avirulent strain of V. cholerae genovariant of the O1 serogroup of the El Tor biovar with efficient production of the cholera toxin (CT) B-subunit, which causes the formation of antitoxic immunity during cholera infection, are presented. In the beginning of the study, a modified P18899ΔCTXφchr:TnphoA (Km^RHly^–) strain was obtained via nondirectional transposon mutagenesis on the basis of a nontoxigenic strain of a P18899ΔCTXφHly⁺ genovariant. It lost the ability to produce thermolabile hemolysin, which is an additional toxin. The ctxB gene, which encodes biosynthesis of the CT B-subunit, was further introduced into cells of this strain. To accomplish this, the cointegrative recombinant plasmid pIEM3 was used. It was generated in the process of the fusion of a conjugative plasmid pIEM1 and a nonconjugative one, pCTΔ27, which is a derivative of pBR322 (which carries the cloned gene ctxB). Via restriction analysis, it was established that disjunction of a cointegrate occurred in vibrio cholera cells, followed by the preservation of a multicopy plasmid (pCTΔ27) only. As a result, avirulent clones of Km^RTc^R with a high production level (5–6 μg/mL) of secreted CT B-subunit were obtained, one of which (E99) was selected for further studies. Via real-time PCR, it was discovered that the expression of the ctxB plasmid gene in the cells of a designed strain of V. cholerae, E99, doesn’t depend on the activity of the key regulatory gene toxT, which located on the chromosome. Efficient expression of the ctxB gene in E99 cells allows their use to obtain CT B-subunit in order to produce cholera immunobiological drugs.     

Crystallization and preliminary x-ray diffraction study of cholera toxin B-subunit

Cholera toxin binds to its ganglioside GM1 receptor via its B-subunit, a pentameric assembly of identical subunits (Mr = 11,600). Diffraction quality crystals of cholera toxin B-subunit have been obtained at room temperature by vapor diffusion with polyethylene glycol in the presence of the nonionic detergent beta-octyl glucoside. The crystals have been characterized with x-radiation as monoclinic, space group P21, with unit cell dimensions a = 39.0 A, b = 94.3 A, c = 67.5 A, beta = 96.0 degrees. There are two molecules per unit cell, with one molecule (Mr = 58,000) in each asymmetric unit. Precession photographs (micron = 13 degrees) show that crystals diffract beyond 3.3-A resolution and are stable in the x-ray beam at room temperature for at least 40 h; thus, they can be used to collect three-dimensional crystallographic data.     

Conditions of two-dimensional crystallization of cholera toxin B-subunit on lipid films containing ganglioside GM1

A systematic study of the lipid-layer two-dimensional crystallization technique has been carried out on the system composed of cholera toxin B-subunit and monosialoganglioside GM1, by electron microscopy, image analysis, and lipid film surface pressure measurements. Concentrations of protein and lipid components required for two-dimensional crystallization of toxin-GM1 complexes have been determined. Crystals were only obtained in the presence of mixed lipid films, composed of GM1 and of unsaturated lipids, such as dioleoylphosphatidylcholine or dioleoylphosphatidylethanolamine, in agreement with a previous report [D. S. Ludwig et al., (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8585–8588]. Crystals were obtained with cholera toxin B-subunit concentration as low as 5 μg/ml, as well as in the presence of protein contaminants. They were obtained over a wide range of concentrations of both GM1 and unsaturated lipids. The minimal lipid amount needed for crystallization corresponded to a lipid monolayer at, or near, the maximal spreading pressure (50 mN/m). The use of an excess of lipid resulted in a stabilization of lipid monolayers and in a higher reproducibility or crystallization experiments.     

Structural organization of cholera toxin gene and its expression in an environmental non-pathogenic strain ofVibrio cholerae

Non-pathogenic, environmental strain ofVibrio cholerae, ELTOR Ogawa EW6 carries a copy of the cholera toxin gene in its chromosome. Restriction enzyme digestion followed by Southern blot analysis revealed that the structure of the cholera toxin gene in this organism is different from that found in the virulent strains. The xbaI site which has been found to be conserved in the cholera toxin of the virulent strains examined so far, is absent here. Results of the RNA dot blot analysis indicated that the cholera toxin gene in EW6 is transcribed much less efficiently compared to the cholera toxin gene present in the virulent strainVibrio cholerae classical Inaba 569B.     

Mechanisms underlying the confined diffusion of cholera toxin B-subunit in intact cell membranes

Multivalent glycolipid binding toxins such as cholera toxin have the capacity to cluster glycolipids, a process thought to be important for their functional uptake into cells. In contrast to the highly dynamic properties of lipid probes and many lipid-anchored proteins, the B-subunit of cholera toxin (CTxB) diffuses extremely slowly when bound to its glycolipid receptor GM(1) in the plasma membrane of living cells. In the current study, we used confocal FRAP to examine the origins of this slow diffusion of the CTxB/GM(1) complex at the cell surface, relative to the behavior of a representative GPI-anchored protein, transmembrane protein, and fluorescent lipid analog. We show that the diffusion of CTxB is impeded by actin- and ATP-dependent processes, but is unaffected by caveolae. At physiological temperature, the diffusion of several cell surface markers is unchanged in the presence of CTxB, suggesting that binding of CTxB to membranes does not alter the organization of the plasma membrane in a way that influences the diffusion of other molecules. Furthermore, diffusion of the B-subunit of another glycolipid-binding toxin, Shiga toxin, is significantly faster than that of CTxB, indicating that the confined diffusion of CTxB is not a simple function of its ability to cluster glycolipids. By identifying underlying mechanisms that control CTxB dynamics at the cell surface, these findings help to delineate the fundamental properties of toxin-receptor complexes in intact cell membranes.     

Blood group antigen recognition by Escherichia coli heat-labile enterotoxin

In a number of bacterial infections, such as Helicobacter pylori, Campylobacter jejuni and Vibrio cholerae infections, a correlation between the severity of disease and blood group phenotype of infected individuals has been observed. In the present investigation, we have studied the molecular basis of this effect for enterotoxigenic Escherichia coli (ETEC) infections. ETEC are non-invasive bacteria, which act through second messenger pathways to cause diarrhea. It has been suggested that the major virulence factor of ETEC from human isolates, i.e. the human heat-labile enterotoxin (hLT), recognizes certain blood group epitopes, although the molecular basis of blood group antigen recognition is unknown. The 2.5 A crystal structure of the receptor-binding B-subunit of hLT in complex with the blood group A antigen analog GalNAcalpha3(Fucalpha2)Galbeta4(Fucalpha3)Glcbeta provides evidence of a previously unknown binding site in the native toxin. The structure reveals the molecular interactions underlying blood group antigen recognition and suggests how this protein can discriminate between different blood group epitopes. These results support the previously debated role of hLT in the blood group dependence of ETEC infections. Similar observations regarding the closely related cholera toxin in V. cholera infections are also discussed.     

Cholera toxin gene-positive Vibrio cholerae O1 Ogawa and Inaba strains produce the new cholera toxin

Abstract Two strains of cholera toxin (CT) gene-positive Vibrio cholerae O1, Ogawa, isolated from patients with diarrhoea and the hypertoxigenic V. cholerae O1, Inaba (569B), were found to produce the new cholera toxin that has earlier been demonstrated to be elaborated by CT gene-negative human and environmental isolates of V. cholerae O1. The CT gene-positive strains produce the new cholera toxin simultaneously with CT, indicating that they contain the gene coding for the new cholera toxin in addition to that of CT.     

A 9 A two-dimensional projected structure of cholera toxin B-subunit-GM1 complexes determined by electron crystallography.

Highly ordered two-dimensional crystals of cholera toxin B-subunit pentamers have been grown by specific interaction with planar lipid films containing monosialoganglioside GM1. Electron diffractograms of frozen-hydrated crystals show diffraction peaks extending to beyond 4 A, while electron images diffract to 8 A. A two-dimensional projected structure of cholera toxin B-subunit-GM1 complex has been calculated at 9 A resolution by combining electron diffraction and image data. Crystals present an approximate pgg projection symmetry, with unit cell dimensions a = 119(+/- 1) A, b = 123(+/- 1) A, gamma = 90 degrees. Each pentameric assembly presents two concentric rings of electron scattering density, separated by an area of lower density. The outer and inner rings are centered at 25 A and and 11 A from the pentamer centre, respectively. The apparent projected density of the outer ring is larger than that of the inner ring. We propose that the outer and inner density rings correspond respectively to the peripheral beta-sheet arrangement and the central alpha-helix barrel, recently identified in the crystal structure of the heat-labile enterotoxin from Escherichia coli.     

Analysis of cholera toxin-ganglioside interactions by flow cytometry

Cholera toxin entry into mammalian cells is mediated by binding of the pentameric B subunit (CTB) to ganglioside GM(1) in the cell membrane. We used flow cytometry to quantitatively measure in real time the interactions of fluorescently labeled pentameric cholera toxin B-subunit (FITC-CTB) with its ganglioside receptor on microsphere-supported phospholipid membranes. A model that describes the multiple steps of this mode of recognition was developed to guide our flow cytometric experiments and extract relevant equilibrium and kinetic rate constants. In contrast to previous studies, our approach takes into account receptor cross-linking, an important feature for multivalent interactions. From equilibrium measurements, we determined an equilibrium binding constant for a single subunit of FITC-CTB binding monovalently to GM(1) presented in bilayers of approximately 8 x 10(7) M(-1) while that for binding to soluble GM(1)-pentasaccharide was found to be approximately 4 x 10(6) M(-1). From kinetic measurements, we determined the rate constant for dissociation of a single site of FITC-CTB from microsphere-supported bilayers to be (3.21 +/- 0.03) x 10(-3) s(-1), and the rate of association of a site on FITC-CTB in solution to a GM(1) in the bilayer to be (2.8 +/- 0.4) x 10(4) M(-1) s(-1). These values yield a lower estimate for the equilibrium binding constant of approximately 1 x 10(7) M(-1). We determined the equilibrium surface cross-linking constant [(1.1 +/- 0.1) x 10(-12) cm(2)] and from this value and the value for the rate constant for dissociation derived a value of approximately 3.5 x 10(-15) cm(2) s(-1) for the forward rate constant for cross-linking. We also compared the interaction of the receptor binding B-subunit with that of the whole toxin (A- and B-subunits). Our results show that the whole toxin binds with approximately 100-fold higher avidity than the pentameric B-subunit alone which is most likely due to the additional interaction of the A(2)-subunit with the membrane surface. Interaction of cholera toxin B-subunit and whole cholera toxin with gangliosides other than GM(1) revealed specific binding only to GD1(b) and asialo-GM(1). These interactions, however, are marked by low avidity and require high receptor concentrations to be observed.     

Characteristics of the B subunit of a thermolabile Escherichia coli enterotoxin produced by the A-B+ E. coli strain

The preparation and identification of B subunit of thermolabile enterotoxin produced by A-B+ gene-containing strain are described. The E. coli strain studied is shown to produce protein identical in its molecular properties and antigenic specificity to B subunit obtained from the whole thermolabile enterotoxin. Partial antigenic affinity between B subunits of thermolabile enterotoxin obtained from different sources and B subunit from cholera enterotoxin has been established in immunochemical studies. Electrophoretic and immunochemical analysis has confirmed the absence of A-subunit admixtures in B-subunit preparation obtained from /A-B+/E. coli strain.     

Thermodynamic identification of stable folding intermediates in the B-subunit of cholera toxin

The structural stability and domain structure of the pentameric B-subunit of cholera toxin have been measured as a function of different perturbants in order to assess the magnitude of the interactions within the B-subunits. For these studies, temperature, guanidine hydrochloride (GuHCl), and pH were used as perturbants, and the effects were measured by high-sensitivity differential scanning calorimetry, isothermal reaction calorimetry, fluorescence spectroscopy, and partial protease digestion. At pH 7.5 and in the absence of any additional perturbants, the thermal unfolding of the B-subunit pentamer is characterized by a single peak in the heat capacity function centered at 77 degrees C and characterized by a delta Hcal of 328 kcal/mol of B-subunit pentamer and delta Hvh/delta Hcal of 0.3. Lowering the pH down to 4 or adding GuHCl up to 2 M results in a decrease of the calorimetric enthalpy with no significant effect on the van't Hoff enthalpy. The transition enthalpy decreases in a sigmoidal fashion with pH, with an inflection point centered at pH 5.3. Isothermal titration calorimetric studies as a function of pH also report a transition centered at pH 5.3 and characterized by an enthalpy change of 27 kcal/mol of B-subunit pentamer at 27 degrees C. Below this pH, the enthalpy change for the unfolding transition is reduced to approximately 100 kcal/mol of B-subunit pentamer. Similar behavior is obtained with GuHCl. In this case, a first transition is observed at 0.5 M GuHCl and a second one at 3 M GuHCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of the epidemic significance of noncultivated forms of Vibrio cholerae by the polymerase chain reaction]

A specific method of the isolation of the cholera toxin gene by the directional amplification of DNA in the polymerase chain reaction (PCR) has been developed. The product of this reaction has a molecular weight of 440 sequence pairs and is a DNA fragment located on the A-subunit of V. cholerae gene vct. The sensitivity of the method permits the detection of one bacterial cell in the reaction mixture. The method is effective when V. cholerae purified DNA, cell lysates and the DNA of total microflora isolated from the water of natural springs are used. The study of water samples from natural water bodies by the method of PRC has revealed cholera toxin genes of V. cholerae noncultivated forms ni 5 out of 7 water samples taken from natural water bodies at the regions of Azerbaijan endemic for cholera and made it possible to evaluate the number of V. cholerae. The prospects of using PCR for the control of the epidemiological situation in regions endemic for cholera are discussed.