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A Donohue-Rolfe

Publications and source records attributed to A Donohue-Rolfe.

32 records · Page 2Linked to original sources
PubMed1988

Pathogenesis of shigella diarrhea: evidence for a developmentally regulated glycolipid receptor for shigella toxin involved in the fluid secretory response of rabbit small intestine.

Shigella toxin reproduces the major manifestations of shigellosis in ligated intestinal loops from adult rabbits and binds to a microvillus membrane (MVM) glycolipid receptor, globotriaosylceramide (Gb3). Because neonatal human shigellosis is uncommon, we used the animal model for obtaining MVMs from rabbits of different ages to determine the presence of toxin receptors and Gb3 and to measure the fluid secretory response to toxin in ligated ileal loops. A single class of MVM receptors for 125I-labeled shigella toxin, first detected at 20 d of age, reached adult levels by 24 d (n = 1.7-23.8 X 10(10)/micrograms of protein; K = 1.1-3.8 X 10(9) M-1). Binding was specific for toxin subunit B. A toxin binding MVM glycolipid, identified as Gb3, was detected in animals greater than or equal to 16 d of age by high-performance thin-layer chromatography and autoradiography. Fluid secretion in response to shigella toxin in ligated small bowel loops occurred in temporal relation to the appearance of Gb3, a result thus indicating the involvement of Gb3 in mediating the toxin effects.

Aging↗
PubMed1988

Synthetic peptides of Shiga toxin B subunit induce antibodies which neutralize its biological activity.

Shiga toxin B chain, the binding subunit of Shiga toxin, was recently purified; and the amino acid sequence of this 7,716-dalton polypeptide was determined (N.G. Seidah, A. Donohue-Rolfe, C. Lazure, F. Auclair, G. T. Keusch, and M. Chretien, J. Biol. Chem. 261:13928-13931, 1986). In the present study, synthetic peptides corresponding to three overlapping sequences from the N-terminal region of this subunit were prepared. The peptides synthesized consisted of residues 5 to 18, 13 to 26, and 7 to 26. This region coincides with the major peak of hydrophilicity and surface area residues predicted from a computer analysis. For the purpose of immunization, the peptides either were conjugated with a protein or synthetic carrier or were polymerized with glutaraldehyde. Antisera against these peptide derivatives raised in rabbits reacted not only with the respective homologous peptide but also to a comparable extent with the intact Shiga toxin. The anti-peptide antisera effectively neutralized the various biological activities of the Shiga toxin, namely, cytotoxicity to HeLa cells, enterotoxic activity (the fluid secretion into ligated ileal loops in rats), and neurotoxicity in mice. Furthermore, active immunization with the peptide conjugates was found to protect mice against the lethal effect of Shiga toxin.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence of the Shiga-like toxin genes of Escherichia coli.

We have determined the nucleotide sequence of the sltA and sltB genes that encode the Shiga-like toxin (SLT) produced by Escherichia coli phage H19B. The amino acid composition of the A and B subunits of SLT is very similar to that previously established for Shiga toxin from Shigella dysenteriae 1, and the deduced amino acid sequence of the B subunit of SLT is identical with that reported for the B subunit of Shiga toxin. The genes for the A and B subunits of SLT apparently constitute an operon, with only 12 nucleotides separating the coding regions. There is a 21-base-pair region of dyad symmetry overlapping the proposed promoter of the slt operon that may be involved in regulation of SLT production by iron. The peptide sequence of the A subunit of SLT is homologous to the A subunit of the plant toxin ricin, providing evidence for the hypothesis that certain prokaryotic toxins may be evolutionarily related to eukaryotic enzymes.

Amino Acid Sequence↗
PubMed1987

HeLa cell adherence and cytotoxin production by enteropathogenic Escherichia coli isolated from infants with diarrhea in Thailand.

Enteropathogenic Escherichia coli (EPEC) strains isolated from hospitalized infants with diarrhea in Thailand were examined for HeLa cell adherence and cytotoxin production. Of 101 strains examined, 56 adhered to HeLa cells in a localized pattern (LA), 27 adhered in a diffuse pattern (DA), and 18 did not adhere. All 56 LA EPEC strains were O:K serotype O119:K69. A total of 20 (83%) of 24 EPEC O86:K61 strains and 7 (38%) of 19 EPEC strains belonging to six other O:K serotypes exhibited DA. All LA EPEC strains hybridized with a DNA probe for genes encoding EPEC adherence factor, whereas none of the 27 DA or 18 nonadherent EPEC strains hybridized with EPEC adherence factor probe. Sonic extracts of 57 (58%) of 98 EPEC strains tested at a dilution of 1:100 caused greater than 25% mortality of HeLa cell monolayers. A total of 50 (88%) of 57 cytotoxic sonic extracts were inhibited to various degrees by a 1:500 dilution of polyclonal rabbit antisera to purified Shiga toxin. The mean percent inhibition of cytotoxic sonic extracts by anti-Shiga toxin was 67% (range, 29 to 89%). Fifty percent (38 of 56) of LA EPEC strains, fifty-two percent (14 of 27) of DA EPEC strains, and fifty-three percent (8 of 15) of nonadherent EPEC strains produced Shiga-like toxins. Both adherence and low levels of cell-associated cytotoxins were identified in EPEC strains from Thailand, but there did not appear to be an association between these two factors.

Bacterial Adhesion↗
PubMed1986

Complete amino acid sequence of Shigella toxin B-chain. A novel polypeptide containing 69 amino acids and one disulfide bridge.

The complete amino acid sequence of the B-chain of Shigella toxin has been determined using both liquid- and gas-phase sequenators. It reveals a 69-amino acid peptide with a single disulfide bridge, predicting a subunit molecular weight of 7691. No Asn-X-Ser(Thr) sequence was found, confirming the absence of potential N-glycosylation sites. A computer data bank search using a mutation data matrix did not detect any similarity greater than 30% with known sequences to date, indicating a novel primary structure. However, some distant homology with the 103-residue B-chain of cholera and Escherichia coli enterotoxins was revealed. Hydropathy, fractional exposure, and Chou and Fasman calculations all point to an ordered structure with a hydrophobic core spanning residues 36-52 and a hydrophilic domain between residues 10 and 20, the latter probably representing the most antigenic domain.

Amino Acid Sequence↗
PubMed1986

Pathogenesis of shigella diarrhea. XI. Isolation of a shigella toxin-binding glycolipid from rabbit jejunum and HeLa cells and its identification as globotriaosylceramide.

A glycolipid that specifically binds shigella toxin was isolated from both HeLa cells and rabbit jejunal mucosa and identified as globotriaosylceramide (Gb3) by its identical mobility on HPTLC to authentic erythrocyte Gb3. Toxin also bound to a band tentatively identified as alpha-hydroxylated Gb3. In addition, toxin bound to P1 antigen present in group B human erythrocyte glycolipid extracts. The common feature of the three binding glycolipids is a terminal Gal alpha 1----4Gal disaccharide linked beta 1----4 to either Glc or GlcNAc. Globoisotriaosylceramide, which differs from Gb3 only in possessing a Gal alpha 1----3Gal terminal disaccharide, and LacCer, which lacks the terminal Gal residue of Gb3, were incapable of binding the toxin. Binding was shown to be mediated by the B subunit by the use of isolated toxin A and B subunits and monoclonal subunit-specific antibodies. Gb3-containing liposomes competitively inhibited the binding of toxin to HeLa cell monolayers but did not inhibit toxin-induced cytotoxicity. These studies show an identical carbohydrate-specific glycolipid receptor for shigella toxin in gut and in HeLa cells. The toxin B subunit that mediates this binding has also been shown to recognize a glycoprotein receptor with different sugar specificity. Thus, we have demonstrated that the same small (Mr 6,500) B subunit polypeptide has two distinctive carbohydrate-specific binding sites. The Gal alpha 1----4Gal disaccharide of the glycolipid toxin receptor is also recognized by the Gal-Gal pilus of uropathogenic E. coli. This suggests the possibility that the pilus and toxin B subunit contain homologous sequences. If this is true, it may be possible to use the purified Gal-Gal pilus to produce toxin-neutralizing antibodies.

Animals↗
PubMed1986

Pathogenesis of shigella diarrhea: evidence for an N-linked glycoprotein shigella toxin receptor and receptor modulation by beta-galactosidase.

Pathogenic mechanisms in infectious diseases often involve specific receptor-ligand interactions of cells and soluble molecules. To further elucidate structure-function relations for shigella toxin receptors, we studied binding of purified 125I-labeled toxin and biologic response under various conditions in an experimental model using HeLa cells. Response to toxin was reversibly inhibited by treatment of cells with trypsin or tunicamycin, an inhibitor of glycoprotein synthesis that also significantly inhibited toxin binding, a result indicating that the receptor is an N-linked glycoprotein. Removal of terminal beta-linked galactose from the HeLa cell surface with beta-galactosidase increased toxin binding and activity, and it also potentiated the effects of lysozyme and wheat-germ agglutinin, which recognize oligomeric beta 1----4-linked N-acetyl-D-glucosamine and inhibit toxin activity as well. Incubation of cells with beta-N-acetylglucosaminidase, which cleaves terminal beta-linked N-acetyl-D-glucosamine, inhibited toxin activity. Effects of beta-galactosidase were reversed by readdition of galactose to cell-surface oligosaccharide acceptors. The data demonstrate that alterations of a single sugar on cell-surface glycoproteins may have a dramatic effect on receptor activity and indicate that shigella toxin is a sugar-binding protein with specificity for beta 1----4-linked N-acetyl-D-glucosamine.

Acetylglucosamine↗
PubMed1986

Pathogenesis of Shigella diarrhea: rabbit intestinal cell microvillus membrane binding site for Shigella toxin.

This study examined the binding of purified 125I-labeled shigella toxin to rabbit jejunal microvillus membranes (MVMs). Toxin binding was concentration dependent, saturable, reversible, and specifically inhibited by unlabeled toxin. The calculated number of toxin molecules bound at 4 degrees C was 7.9 X 10(10) (3 X 10(10) to 2 X 10(11))/micrograms of MVM protein or 1.2 X 10(6) per enterocyte. Scatchard analysis showed the binding site to be of a single class with an equilibrium association constant, K, of 4.7 X 10(9) M-1 at 4 degrees C. Binding was inversely related to the temperature of incubation. A total of 80% of the labeled toxin binding at 4 degrees C dissociated from MVM when the temperature was raised to 37 degrees C, but reassociated when the temperature was again brought to 4 degrees C. There was no structural or functional change of MVM due to toxin as monitored by electron microscopy or assay of MVM sucrase activity. These studies demonstrate a specific binding site for shigella toxin on rabbit MVMs. The physiological relevance of this receptor remains to be determined.

Animals↗
PubMed1986

Enzyme-linked immunosorbent assay for shigella toxin.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of shigella toxin. For the assay, a mouse monoclonal antibody against the B subunit of the toxin and a rabbit polyclonal antibody against the holotoxin were employed. The monoclonal antibody was used to coat wells of a microtiter plate, and the polyclonal antibody preparation was used as the detecting antibody. The amount of bound polyclonal antibody was determined by using a goat anti-rabbit immunoglobulin G-alkaline phosphatase conjugate and substrate. The ELISA was able to detect as little as 12 pg (0.06 ng/ml) of shigella toxin. The assay was specific for shigella toxin, not detecting a variety of other bacterial enterotoxins and lethal toxins. The ELISA values correlated well with cytotoxin activity during toxin purification. Shigella toxin was detected by ELISA and by immunoblot analysis in human fecal specimens from persons with S. dysenteriae infections, demonstrating that this toxin is produced in vivo.

Animals↗
PubMed1985

Shigella toxin and the pathogenesis of shigellosis.

Shigella dysenteriae 1 produces a periplasmic protein with multiple toxic effects in vivo and in vitro. These include neurotoxicity, cytotoxicity and enterotoxicity, as well as the ability to inhibit cell-free protein synthesis. The purified toxin is a protein of relative molecular mass (Mr) 64 000. It is composed of one catalytically active A subunit (Mr = 32 000) that inhibits protein synthesis, and a complex of five B monomers (Mr approximately 6500 each). Studies using subunit-specific antibodies demonstrate that the B subunit mediates the binding of toxin to toxin receptors in the cell membrane. In a model system in HeLa cell culture, the surface membrane receptor has been shown to be a glycoprotein, most probably asparagine-linked, and to contain oligomeric beta 1----4 linked N-acetyl-D-glucosamine. Studies with metabolic inhibitors and agents that disrupt the cytoskeleton, and/or alter the pH and function of acidic cytoplasmic vesicles, provide indirect evidence that toxin is transported from the cell surface to the cell interior. This process is probably receptor-mediated endocytosis, since it is also inhibited by amines that prevent receptor-mediated uptake of other ligands in well-characterized systems. The toxic action in the HeLa cell is due to the subsequent inhibition of protein synthesis which results from catalytic inactivation of the 60S ribosomal subunit and the cessation of polypeptide chain elongation. Inhibition of protein synthesis by toxin produced subsequent to bacterial invasion of colonic epithelial cells could explain the destructive lesions found in shigellosis. Although toxin can induce jejunal secretion in animal models, there is at present no clear explanation for the secretory response of the gut mucosa in shigella infection.

Animals↗
PubMed1985

Axonally transported Shigella cytotoxin is neuronotoxic.

Shigella dysenteriae strains produce an exotoxin (SdT) which inhibits protein synthesis in susceptible cells and is neurotoxic in some species. Intraneural microinjection of highly purified SdT into the cervical vagus nerves of rats, mice, guinea pigs and rabbits produced cytopathic changes within 24 hours in vagal sensory but not motor neurons. These changes consisted of an initial loss of Nissl substance followed by progressive cell degeneration and resulted in permanent neuronal loss. Indirect immunoperoxidase staining demonstrated SdT in nodose ganglion sensory neurons but not in vagal motor neurons of the brainstem. In rats, fatal enterotoxicity was common after intraneural injection of SdT doses that were reliably neuronotoxic; in rabbits and mice, a fatal ascending paralysis occurred at similar doses. Cytopathic changes were noted rarely in nodose sensory neurons of the uninjected side in mice suggesting systemic distribution and neuronal uptake of toxin. Injection of SdT into the tongue muscles of rats and mice failed to affect hypoglossal motor neurons within 50 hours. We conclude SdT is taken up and retrogradely transported by vagal sensory neurons with a resultant destruction of these neurons. The relationship of these findings to the reported neurotoxicity of SdT is unclear. However, SdT may prove useful in making selective lesions or as a model for some forms of neuronal degeneration.

Animals↗
PubMedDate not supplied

Shiga toxin: purification, structure, and function.

Shiga toxin is a potent toxin produced by Shigella dysenteriae type 1 strains. The toxin has three biologic activities--cytotoxicity, enterotoxicity, and neurotoxicity--and one known biochemical effect: inhibition of protein synthesis. It consists of two polypeptide chains, an A chain (molecular weight, 32,225) and a B chain (molecular weight, 7,691). These two peptides associate with a stoichiometry of one A and five B subunits to form the holotoxin. The A chain is responsible for the biochemical effect of the holotoxin: cleavage of the N-glycosidic bond of adenine at nucleotide position 4324 in the 28S rRNA of the 60S ribosomal subunit. The B chain mediates binding of toxin to cell surface receptors. Shiga toxin is the prototype of a family of toxin molecules that have been termed Shiga-like in terms of both structural and functional analysis.

Bacterial Toxins↗
PubMedDate not supplied

Shiga toxin: intestinal cell receptors and pathophysiology of enterotoxic effects.

Shiga toxin is enterotoxic in rabbit small bowel and binds to the microvillus membrane (MVM). The toxin exhibits specificity for glycolipids possessing a terminal gal-alpha 1----4gal disaccharide, including the neutral glycolipid Gb3 in MVM. Gb3, which is developmentally regulated in the rabbit small bowel, is present in very low concentration until the animals reach day 16 of life. In older animals an increase in Gb3 content is paralleled by an increase in the ability of MVM to bind toxin, which also correlates with the fluid secretion response. Shiga toxin selectively binds to villus cells, which contain Gb3, and not to crypt cells, which do not express Gb3. Targeting of the villus cell by the toxin is consistent with physiologic studies that demonstrate inhibition of villus cell Na+ absorptive pathways, with no effect on crypt cell Cl- secretory mechanisms. These effects are sufficient to account for the enterotoxicity of Shiga toxin in the rabbit model.

Animals↗