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

1986 ◽  
Vol 238 (2) ◽  
pp. 313-322 ◽  
Author(s):  
S L Griffiths ◽  
R A Finkelstein ◽  
D R Critchley
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Binding Sites ◽  
Cell Membranes ◽  
Ganglioside Gm1 ◽  
Neuraminidase Treatment ◽  
Toxin Binding ◽  
B Subunit ◽  
Heat Labile ◽  
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 × 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.

scholarly journals Biochemical and structural characterization of the novel sialic acid-binding site of Escherichia coli heat-labile enterotoxin LT-IIb

2016 ◽  
Vol 473 (21) ◽  
pp. 3923-3936 ◽  
Author(s):  
Dani Zalem ◽  
João P. Ribeiro ◽  
Annabelle Varrot ◽  
Michael Lebens ◽  
Anne Imberty ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Cholera Toxin ◽  
Carbohydrate Binding ◽  
Type I ◽  
Type Ii ◽  
E Coli ◽  
B Subunit ◽  
Heat Labile ◽  
B Subunits

The structurally related AB5-type heat-labile enterotoxins of Escherichia coli and Vibrio cholerae are classified into two major types. The type I group includes cholera toxin (CT) and E. coli LT-I, whereas the type II subfamily comprises LT-IIa, LT-IIb and LT-IIc. The carbohydrate-binding specificities of LT-IIa, LT-IIb and LT-IIc are distinctive from those of cholera toxin and E. coli LT-I. Whereas CT and LT-I bind primarily to the GM1 ganglioside, LT-IIa binds to gangliosides GD1a, GD1b and GM1, LT-IIb binds to the GD1a and GT1b gangliosides, and LT-IIc binds to GM1, GM2, GM3 and GD1a. These previous studies of the binding properties of type II B-subunits have been focused on ganglio core chain gangliosides. To further define the carbohydrate binding specificity of LT-IIb B-subunits, we have investigated its binding to a collection of gangliosides and non-acid glycosphingolipids with different core chains. A high-affinity binding of LT-IIb B-subunits to gangliosides with a neolacto core chain, such as Neu5Gcα3- and Neu5Acα3-neolactohexaosylceramide, and Neu5Gcα3- and Neu5Acα3-neolactooctaosylceramide was detected. An LT-IIb-binding ganglioside was isolated from human small intestine and characterized as Neu5Acα3-neolactohexaosylceramide. The crystal structure of the B-subunit of LT-IIb with the pentasaccharide moiety of Neu5Acα3-neolactotetraosylceramide (Neu5Ac-nLT: Neu5Acα3Galβ4GlcNAcβ3Galβ4Glc) was determined providing the first information for a sialic-binding site in this subfamily, with clear differences from that of CT and LT-I.

scholarly journals Escherichia coli Heat-Labile Enterotoxin B Subunit Is a More Potent Mucosal Adjuvant than Its Closely Related Homologue, the B Subunit of Cholera Toxin

2001 ◽  
Vol 69 (5) ◽  
pp. 3476-3482 ◽  
Author(s):  
Douglas G. Millar ◽  
Timothy R. Hirst ◽  
Denis P. Snider
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Lymphocyte Proliferation ◽  
Vaccine Adjuvants ◽  
B Subunit ◽  
Heat Labile ◽  
Antibody Titers ◽  
Enterotoxin B ◽  
Draining Lymph Nodes ◽  
Stimulation Of

ABSTRACT Although cholera toxin (Ctx) and Escherichia coliheat-labile enterotoxin (Etx) are known to be potent mucosal adjuvants, it remains controversial whether the adjuvanticity of the holotoxins extends to their nontoxic, receptor-binding B subunits. Here, we have systematically evaluated the comparative adjuvant properties of highly purified recombinant EtxB and CtxB. EtxB was found to be a more potent adjuvant than CtxB, stimulating responses to hen egg lysozyme when the two were coadministered to mice intranasally, as assessed by enhanced serum and secretory antibody titers as well as by stimulation of lymphocyte proliferation in spleen and draining lymph nodes. These results indicate that, although structurally very similar, EtxB and CtxB have strikingly different immunostimulatory properties and should not be considered equivalent as prospective vaccine adjuvants.

scholarly journals Differential Biological and Adjuvant Activities of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxin Hybrids

2001 ◽  
Vol 69 (3) ◽  
pp. 1528-1535 ◽  
Author(s):  
Christal C. Bowman ◽  
John D. Clements
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Cholera Toxin ◽  
The Other ◽  
Toxin Gene ◽  
Wild Type ◽  
B Subunit ◽  
Heat Labile ◽  
A Subunit ◽  
Ifn Γ

ABSTRACT Two bacterial products that have been demonstrated to function as mucosal adjuvants are cholera toxin (CT), produced by various strains of Vibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli. Although LT and CT have many features in common, they are clearly distinct molecules with biochemical and immunologic differences which make them unique. The goal of this study was to determine the basis for these biological differences by constructing and characterizing chimeric CT-LT molecules. Toxin gene fragments were subcloned to create two constructs, each expressing the enzymatically active A subunit of one toxin and the receptor binding B subunit of the other toxin. These hybrid toxins were purified, and the composition and assembly of CT A subunit (CT-A)-LT B subunit (LT-B) and LT A subunit (LT-A)-CT B subunit (CT-B) were confirmed. Hybrids were evaluated for enzymatic activity, as measured by the accumulation of cyclic AMP in Caco-2 cells, and the enterotoxicity of each toxin was assessed in a patent-mouse assay. The results demonstrated that LT-A–CT-B induces the accumulation of lower levels of cyclic AMP and has less enterotoxicity than either wild-type toxin or the other hybrid. Nonetheless, this hybrid retains adjuvant activity equivalent to or greater than that of either wild-type toxin or the other hybrid when used in conjunction with tetanus toxoid for intranasal immunization of BALB/c mice. Importantly, the ability of LT to induce a type 1 cytokine response was found to be a function of LT-A. Specifically, LT-A–CT-B was able to augment the levels of antigen-specific gamma interferon (IFN-γ) and interleukin 5 to levels comparable to those achieved with native LT, while CT-A–LT-B and native CT both produced lower levels of antigen-specific IFN-γ. Thus, these toxin hybrids possess unique biological characteristics and provide information about the basis for differences in the biological activities observed for CT and LT.

scholarly journals A Single Native Ganglioside GM1-Binding Site Is Sufficient for Cholera Toxin To Bind to Cells and Complete the Intoxication Pathway

mBio ◽  
2012 ◽  
Vol 3 (6) ◽  
Author(s):  
Michael G. Jobling ◽  
ZhiJie Yang ◽  
Wendy R. Kam ◽  
Wayne I. Lencer ◽  
Randall K. Holmes
Keyword(s):  
Binding Site ◽  
Cholera Toxin ◽  
Receptor Binding ◽  
Binding Sites ◽  
Cell Membranes ◽  
Wild Type ◽  
B Subunit ◽  
Endogenous Lipid ◽  
A Subunit ◽  
Multivalent Binding

ABSTRACT Cholera toxin (CT) from Vibrio cholerae is responsible for the majority of the symptoms of the diarrheal disease cholera. CT is a heterohexameric protein complex with a 240-residue A subunit and a pentameric B subunit of identical 103-residue B polypeptides. The A subunit is proteolytically cleaved within a disulfide-linked loop to generate the A1 and A2 fragments. The B subunit of wild-type (wt) CT binds 5 cell surface ganglioside GM1 (GM1) molecules, and the toxin-GM1 complex traffics from the plasma membrane (PM) retrograde through endosomes and the Golgi apparatus to the endoplasmic reticulum (ER). From the ER, the enzymatic A1 fragment retrotranslocates to the cytosol to cause disease. Clustering of GM1 by multivalent toxin binding can structurally remodel cell membranes in ways that may assist toxin uptake and retrograde trafficking. We have recently found, however, that CT may traffic from the PM to the ER by exploiting an endogenous glycosphingolipid pathway (A. A. Wolf et al., Infect. Immun. 76:1476–1484, 2008, and D. J. F. Chinnapen et al., Dev. Cell 23:573–586, 2012), suggesting that multivalent binding to GM1 is dispensable. Here we formally tested this idea by creating homogenous chimeric holotoxins with defined numbers of native GM1 binding sites from zero (nonbinding) to five (wild type). We found that a single GM1 binding site is sufficient for activity of the holotoxin. Therefore, remodeling of cell membranes by mechanisms that involve multivalent binding of toxin to GM1 receptors is not essential for toxicity of CT. IMPORTANCE Through multivalent binding to its lipid receptor, cholera toxin (CT) can remodel cell membranes in ways that may assist host cell invasion. We recently found that CT variants which bind no more than 2 receptor molecules do exhibit toxicity, suggesting that CT may be able to enter cells by coopting an endogenous lipid sorting pathway without clustering receptors. We tested this idea directly by using purified variants of CT with zero to five functional receptor-binding sites (BS). One BS enabled CT to intoxicate cells, supporting the conclusion that CT can enter cells by coopting an endogenous lipid-sorting pathway. Although multivalent receptor binding is not essential, it does increase CT toxicity. These findings suggest that achieving higher receptor binding avidity or affecting membrane dynamics by lipid clustering and membrane remodeling may be driving forces for evolution of AB5 subunit toxins that can bind multivalently to cell membrane lipid receptors.

scholarly journals Escherichia coli K88ac Fimbriae Expressing Heat-Labile and Heat-Stable (STa) Toxin Epitopes Elicit Antibodies That Neutralize Cholera Toxin and STa Toxin and Inhibit Adherence of K88ac Fimbrial E. coli

2010 ◽  
Vol 17 (12) ◽  
pp. 1859-1867 ◽  
Author(s):  
Chengxian Zhang ◽  
Weiping Zhang
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Broad Spectrum ◽  
Diarrheal Disease ◽  
Virulence Determinants ◽  
E Coli ◽  
B Subunit ◽  
Heat Stable ◽  
Heat Labile ◽  
Bacterial Adhesins

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Bacterial adhesins and heat-labile (LT) and heat-stable (ST) enterotoxins are the virulence determinants in ETEC diarrhea. It is believed that vaccines inducing anti-adhesin immunity to inhibit bacterial adherence and anti-toxin immunity to eliminate toxin activity would provide broad-spectrum protection against ETEC. In this study, an ETEC fimbrial adhesin was used as a platform to express LT and STa for adhesin-toxin fusion antigens to induce anti-toxin and anti-adhesin immunity. An epitope from the B subunit of LT toxin (LTP1, 8LCSEYRNTQIYTIN21) and an STa toxoid epitope (5CCELCCNPQCAGCY18) were embedded in the FaeG major subunit of E. coli K88ac fimbriae. Constructed K88ac-toxin chimeric fimbriae were harvested and used for rabbit immunization. Immunized rabbits developed anti-K88ac, anti-LT, and anti-STa antibodies. Moreover, induced antibodies not only inhibited adherence of K88ac fimbrial E. coli to porcine small intestinal enterocytes but also neutralized cholera toxin and STa toxin. Data from this study demonstrated that K88ac fimbriae expressing LT and STa epitope antigens elicited neutralizing anti-toxin antibodies and anti-adhesin antibodies and suggested that E. coli fimbriae could serve as a platform for the development of broad-spectrum vaccines against ETEC.

scholarly journals Characterization of Receptor-Mediated Signal Transduction by Escherichia coli Type IIa Heat-Labile Enterotoxin in the Polarized Human Intestinal Cell Line T84

2001 ◽  
Vol 69 (12) ◽  
pp. 7205-7212 ◽  
Author(s):  
Susan Wimer-Mackin ◽  
Randall K. Holmes ◽  
Anne A. Wolf ◽  
Wayne I. Lencer ◽  
Michael G. Jobling
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Lipid Rafts ◽  
Secretory Response ◽  
Ganglioside Gm1 ◽  
T84 Cells ◽  
B Subunit ◽  
Heat Labile ◽  
Functional Receptor

ABSTRACT Escherichia coli type IIa heat-labile enterotoxin (LTIIa) binds in vitro with highest affinity to ganglioside GD1b. It also binds in vitro with lower affinity to several other oligosialogangliosides and to ganglioside GM1, the functional receptor for cholera toxin (CT). In the present study, we characterized receptor-mediated signal transduction by LTIIa in the cultured T84 cell model of human intestinal epithelium. Wild-type LTIIa bound tightly to the apical surface of polarized T84 cell monolayers and elicited a Cl− secretory response. LTIIa activity, unlike CT activity, was not blocked by the B subunit of CT. Furthermore, an LTIIa variant with a T14I substitution in its B subunit, which binds in vitro to ganglioside GM1 but not to ganglioside GD1b, was unable to bind to intact T84 cells and did not elicit a Cl− secretory response. These findings show that ganglioside GM1 on T84 cells is not a functional receptor for LTIIa. The LTIIa receptor on T84 cells was inactivated by treatment with neuraminidase. Furthermore, LTIIa binding was blocked by tetanus toxin C fragment, which binds to gangliosides GD1b and GT1b. These findings support the hypothesis that ganglioside GD1b, or possibly a glycoconjugate with a GD1b-like oligosaccharide, is the functional receptor for LTIIa on T84 cells. The LTIIa-receptor complexes from T84 cells were associated with detergent-insoluble membrane microdomains (lipid rafts), extending the correlation between toxin binding to lipid rafts and toxin function that was previously established for CT. However, the extent of association with lipid rafts and the magnitude of the Cl− secretory response in T84 cells were less for LTIIa than for CT. These properties of LTIIa and the previous finding that enterotoxin LTIIb binds to T84 cells but does not associate with lipid rafts or elicit a Cl− secretory response may explain the low pathogenicity for humans of type II enterotoxin-producing isolates of E. coli.

scholarly journals Cloning and Characterization of Genes Encoding Homologues of the B Subunit of Cholera Toxin and the Escherichia coli Heat-Labile Enterotoxin from Clinical Isolates of Citrobacter freundii and E. coli

2002 ◽  
Vol 70 (12) ◽  
pp. 7153-7155 ◽  
Author(s):  
Tadahiro Karasawa ◽  
Hideaki Ito ◽  
Teizo Tsukamoto ◽  
Shinji Yamasaki ◽  
Hisao Kurazono ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Cholera Toxin ◽  
Dna Probes ◽  
Citrobacter Freundii ◽  
Gene Encoding ◽  
E Coli ◽  
B Subunit ◽  
Intergenic Space ◽  
Heat Labile

ABSTRACT We identified and characterized a gene encoding a homologue of the B subunits of cholera toxin (CTB) and heat-labile enterotoxin (LTB) of Escherichia coli from a clinical isolate of Citrobacter freundii that was found to produce a factor in the culture supernatant that cross-reacted with antibodies to CTB and LTB when assayed by enzyme-linked immunosorbent assay (ELISA). The gene encoding the ELISA-positive factor, cfxB, consisted of 375 nucleotides and was located downstream of an 852-nucleotide open reading frame, cfxA, with a 56-nucleotide intergenic space. The cfxB gene was predicted to encode a 125-amino-acid polypeptide, which had 73.8 and 72.8% identities with the amino acid sequences of LTB and CTB, respectively. However, the amino acid sequence of the deduced polypeptide CFXA had no homologies to those of the A subunits of CT or LT. DNA probes developed from the sequences of cfxA and cfxB were used to screen 67 C. freundii isolates and 152 E. coli isolates from diarrheal patients by colony blot hybridization. Two strains, C. freundii 48 and E. coli 176, reacted with both DNA probes under conditions of high stringency. We cloned homologues of the cfxA and cfxB genes from E. coli 176 and designated them ecxA and ecxB, respectively. The ecxA gene and the ecxB gene comprise 855 and 375 nucleotides, respectively, with a 50-nucleotide intergenic space, and encode a 285- and a 125-amino-acid residue polypeptides, respectively. The results of the present study may provide important clues to the origin and evolution of immunologically related factors sharing a common enterotoxin-like A and B subunit structures.

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