Shiga toxin: biochemistry, genetics, mode of action, and role in pathogenesis.
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Bacterial Toxins↗
Biomedical subjects
A Donohue-Rolfe
Publications and source records attributed to A Donohue-Rolfe.
Detection by ELISA of low numbers of Shiga-like toxin-producing Escherichia coli in mixed cultures after growth in the presence of mitomycin C.
Techniques currently available to detect Shiga-like toxin (SLT)-producing Escherichia coli lack sensitivity or require specialised equipment and facilities, and in some cases detect only strains belonging to serotype O157. We have used an ELISA technique, capable of detecting both SLTI and SLTII with crude P1 glycoprotein from hydatid cysts, in combination with enhancement of toxin production by culture with mitomycin C. Supernates of Tryptone Soya Broth cultures containing mitomycin C 200 ng/ml were tested for SLTII. For SLTI, cell lysates pre-treated with polymyxin B were tested. In tests with E. coli O157:H7 in mixed culture with E. coli strain C600 alone, or with E. coli C600, Proteus mirabilis and Enterococcus faecalis, SLTI could be detected when the proportion of toxigenic organisms represented 1% of the mixture, and SLTII when the proportion was 0.025%. When faecal samples with added E. coli O157:H7 were examined in this system, SLTII-producing strains were detected when they comprised less than 0.1% of the coliform population. This technique is a sensitive and specific assay for detecting low numbers of SLT-producing organisms in mixed culture such as occurs in cases of haemolytic uraemic syndrome and haemorrhagic colitis.
Bacterial Toxins↗
Antibodies to shiga holotoxin and to two synthetic peptides of the B subunit in sera of patients with Shigella dysenteriae 1 dysentery.
Acute- and convalescent-phase sera from 18 Thai patients and convalescent-phase sera from two Israeli patients and one Bangladeshi patient with Shigella dysenteriae 1 (Shiga) dysentery were tested by enzyme-linked immunosorbent assay to detect antibodies that bind S. dysenteriae lipopolysaccharide (LPS), Shiga holotoxin, or two synthetic peptides representing epitopes from the B subunit of Shiga toxin. Paired sera from 24 Maryland adults with Shigella flexneri 2a or Shigella sonnei diarrhea served as negative controls. Of the 16 paired Thai serum samples tested for immunoglobulin G LPS antibody, 10 had greater than or equal to 4-fold rises (the two subjects with the highest convalescent-phase titers exhibited toxin-neutralizing activity); acute-phase specimens from four of the remaining six individuals already had elevated Shiga LPS titers in their acute specimens ranging from 1:800 to 1:12,800. Similarly, convalescent-phase sera from the two Israeli patients and the Bangladeshi patient revealed LPS titers of 1:800 to 1:3,200. In contrast, none of the Maryland volunteers with S. flexneri or S. sonnei diarrhea manifested rises in Shiga anti-LPS (P less than 0.00001 versus 10 of 16 Thai patients). Only 4 of the 18 Thai patients had significant rise in antibody to purified Shiga toxin, while one of the two Israeli patients and the one Bangladeshi patient had elevated convalescent-phase titers. None of the sera that reacted with Shiga holotoxin had antibody that bound to the peptides. This report, which describes a search for serum antibodies that bind Shiga toxin in patients with Shiga dysentery, demonstrates such antibodies in only a minority of patients with bacteriologically confirmed disease. During Shiga dysentery, Shiga toxin may be elaborated in such small quantities in vivo that it fails to elicit an immune response in most patients even though it may exert biological effects. In this behavior Shiga toxin resembles tetanus toxin, another potent exotoxin that fails to elicit antitoxic responses in people who recover from clinical tetanus.
Adolescent↗
Crystallization of the B chain of Shiga-like toxin I from Escherichia coli.
Shiga-like toxin I (SLT-I) is produced by several pathogenic strains of Escherichia coli associated with diarrheal disease. The toxin consists of an A chain, which attacks eukaryotic ribosomes, inhibiting protein synthesis, and multiple copies of a 69 amino acid B chain. The B subunit mediates cell binding and uptake through its interactions with cell surface carbohydrate moieties. Here we report that the B chain has been crystallized in a form suitable for high-resolution X-ray analysis. The space group is P2(1)2(1)2(1), with a = 56.2 A, b = 59.9 A and c = 102.5 A. A rotation function using three-dimensional diffraction data suggests that the asymmetric unit is a tetramer.
Bacterial Toxins↗
A comparison of HEp-2 cell invasion by enteropathogenic and enteroinvasive Escherichia coli.
In order to further characterize cellular invasion by enteropathogenic Escherichia coli (EPEC), we compared invasion of HEp-2 cells by EPEC and enteroinvasive E. coli (EIEC). We used a gentamicin HEp-2 cell assay and measured bacterial recovery under conditions of varying incubation time and temperature, and in the presence or absence of inhibitors of cellular microfilaments and microtubules. We found that, unlike EIEC, EPEC did not rapidly multiply within HEp-2 cell but invaded well at 32 degrees C. While microfilament inhibitors reduced invasion by both EIEC and EPEC, microtubule inhibitors reduced invasion by EPEC only. These results suggest that EPEC and EIEC differ in their mechanisms of epithelial cell invasion.
Actin Cytoskeleton↗
Enzyme-linked immunosorbent assay for Shiga toxin and Shiga-like toxin II using P1 glycoprotein from hydatid cysts.
Shiga toxin from Shigella dysenteriae type 1 strains and Shiga-like toxins (SLT) I and II from Escherichia coli bind to terminal alpha-D-Galp-(1----4)-D-Galp containing glycolipids. Hydatid cyst fluid isolated from sheep infected with Echinococcus granulosus contains a glycoprotein (P1gp) with a terminal alpha-D-Galp-(1----4)-D-Galp disaccharide. Preparations of P1gp were shown to interact directly with Shiga toxin and to inhibit the binding and cytotoxicity of Shiga toxin to HeLa cells. A sandwich ELISA was developed using preparations of P1gp as the toxin capture molecule, which, with an appropriate polyclonal antibody, was capable of detecting as little as 80 pg/well Shiga toxin and 132 pg/well SLT-II. Thus, the P1gp-toxin interaction forms the basis for a simple antigen-capture ELISA that may be useful clinically for the rapid detection and quantitation of Shiga and Shiga-like toxins.
Animals↗
Construction and analysis of TnphoA mutants of enteropathogenic Escherichia coli unable to invade HEp-2 cells.
Enteropathogenic Escherichia coli (EPEC) strains have recently been shown to invade tissue culture cells. We describe a set of 22 Tn5 IS50L::phoA (TnphoA) insertion mutants of EPEC strain E2348-69 that are unable to invade HEp-2 cells. Each mutant was tested for the ability to adhere to and to induce the polymerization of actin in HEp-2 cells. Southern hybridization of plasmid and total DNA of each strain was performed to determine the location of each TnphoA insert, and each TnphoA insert along with flanking EPEC sequences was cloned. These studies resulted in the grouping of the mutants into five main categories. These include strains with plasmid and chromosomal insertions that alter adherence, chromosomal insertions that alter the ability to induce actin polymerization, and chromosomal insertions that do not affect adherence or actin polymerization. These studies indicate that genes affecting EPEC adherence may be located on both the plasmid and chromosome, that several genes are involved in the induction of actin polymerization in epithelial cells, and that EPEC invasion is a complex process involving multiple genetic loci.
Actin Cytoskeleton↗
A system for production and rapid purification of large amounts of the Shiga toxin/Shiga-like toxin I B subunit.
We have constructed a plasmid expression vector (pSBC32) that encodes the B subunit of Shiga toxin/Shiga-like toxin I under control of the inducible trc promoter. The encoded B subunit is transported to the periplasmic space, allowing single-step purification of milligram amounts of this protein from periplasmic extracts by using receptor analog affinity chromatography. The purified B subunit interacts normally with both polyclonal antiserum to Shiga toxin and a monoclonal antibody specific for B subunit. B subunit purified in this system is pentameric (as in native holotoxin) and biologically active in blocking binding of Shiga holotoxin to HeLa cells. This expression system may allow rapid purification of sufficient amounts of Shiga toxin B subunit to attempt crystallization or to study its efficacy as a vaccine, either by itself or coupled to an appropriate polysaccharide antigen.
Animals↗
Quantitation of the rabbit intestinal glycolipid receptor for Shiga toxin. Further evidence for the developmental regulation of globotriaosylceramide in microvillus membranes.
Shiga toxin, produced by Shigella dysenteriae 1, causes enterotoxic, cytotoxic, and neurotoxic effects, which may be mediated by a glycolipid receptor, globotriaosylceramide, Gb3. To study the relationship of this receptor and toxin effects, globotriaosylceramide was quantitated and further characterized in rabbit small intestinal microvillus membranes at various ages. Glycolipids were extracted from rabbit microvillus membranes, purified on Unisil columns, and quantitated by high-performance liquid chromatography. The major glycolipid peaks were hydroxylated fatty acid-containing glucosylceramide, lactosylceramide, and globotriaosylceramide. There was a marked increase of globotriaosylceramide levels with age, ranging from 0.02 to 16.2 pmol/micrograms microvillus membrane protein in neonates and adults, respectively. The globotriaosylceramide peak was susceptible to alpha-galactosidase treatment, which produced an elevation in the lactosylceramide peak, but markedly reduced globotriaosylceramide content in 34-day-old rabbits. Binding of iodinated Shiga toxin to globotriaosylceramide was documented on high-performance thin-layer chromatography plates by autoradiography. The glycolipid receptor for Shiga toxin in rabbit microvillus membranes is thus a hydroxylated fatty acid-containing globotriaosylceramide. This moiety is virtually absent in neonates and gradually increases with age. Quantitative differences in globotriaosylceramide may be the underlying basis for the age-specific differences in functional responsiveness of rabbit intestinal tissue to Shiga toxin.
Animals↗
Pathogenesis of Shigella diarrhea. XIV. Analysis of Shiga toxin receptors on cloned HeLa cells.
Binding kinetics of Shiga toxin to HeLa CCL-2 cells and to cell lines cloned by limiting dilutions were determined. Lines with a wide range of sensitivity to Shiga toxin were obtained. Binding data, analyzed by a computer-based Scatchard model program, revealed two classes of binding sites, one of low affinity and high capacity and one of high affinity and low capacity. The number of high affinity, but not low affinity, sites present on the clones correlated with their sensitivity to toxin. Tunicamycin-treated CCL-2 cells became resistant to Shiga toxin in parallel with a reduction in the capacity of the high-affinity site. Cell content of Gb3, the glycolipid receptor for Shiga toxin, decreased as the sensitivity of the cells diminished. These data show that a minority of Shiga toxin binding sites of HeLa cells are involved in the cytotoxic response and suggest that Gb3 is the high-affinity functional cytotoxin receptor.
Bacterial Toxins↗
Epithelial cell invasion: an overlooked property of enteropathogenic Escherichia coli (EPEC) associated with the EPEC adherence factor.
In order to investigate the ability of enteropathogenic Escherichia coli (EPEC) to invade epithelial cells, 24 strains of diarrhea-causing E. coli were studied with a HEp-2 cell-gentamicin invasion assay. Invasive ability was expressed as the percentage of the inoculum surviving gentamicin after incubation of bacteria with HEp-2 cells. Geometric mean survival of EPEC strains possessing the EPEC adherence factor (EAF+ EPEC) was 5.177%, which was significantly greater than survival of enteroinvasive E. coli (EIEC) strains (1.871%). EPEC strains lacking EAF (EAF-EPEC), enterotoxigenic E. coli (ETEC), and enterohemorrhagic E. coli (EHEC) were significantly less invasive (geometric mean survival, 0.032%, 0.013%, and 0.009%, respectively). The variation in bacterial recovery was not due to differences in the number of HEp-2 cells remaining attached to the plates, as measured by the retention of crystal violet stain in parallel assays. Transmission electron microscopy confirmed the presence of many intracellular EAF+ EPEC and EIEC, whereas EAF- EPEC, EHEC, and ETEC were found primarily outside the cells. Epithelial cell invasion is an overlooked property of EAF+ EPEC of potential relevance in disease pathogenesis.
Bacterial Adhesion↗
Isolation and characterization of functional Shiga toxin subunits and renatured holotoxin.
Shiga toxin is a protein toxin produced by Shigella dysenteriae type I strains. In this report we present a procedure for the separation of functionally intact toxin A and B chains and for their reconstitution to form biologically active molecules. In agreement with the findings of others, the isolated A chain was shown to be a potent in vitro inhibitor of eukaryotic protein synthesis. The isolated B chain bound to HeLa cells and competitively inhibited the binding and cytotoxic activity of holotoxin. These findings show that the functional role of the B chain is to recognize cell surface functional receptors. By labelling the B subunit alone, prior to renaturation of holotoxin, the polypeptide chains were shown to associate noncovalently with a stoichiometry of one A chain and five B chains.
Bacterial Toxins↗
Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized P1 glycoprotein and production of cross-reactive monoclonal antibodies.
Shiga toxin from Shigella dysenteriae 60R was purified to homogeneity by a novel one-step receptor analog affinity chromatography method. The method was based on the binding affinity of Shiga toxin for a specific disaccharide, Gal alpha 1----4Gal, which was also present in glycoproteins with P1 blood group seroreactivity produced in hydatid cysts from sheep infected with Echinococcus granulosus. Having shown that cyst fluid P1 glycoprotein bound Shiga toxin on a solid phase, a P1 glycoprotein affinity column was made by coupling P1-active substance to Sepharose 4B. Shiga toxin was purified by this method in large quantities (5 to 10 mg/20-liter batch) with a consistently good yield (greater than 80% of starting toxin). Shiga-like toxins I and II (SLT-I and -II, respectively) from Escherichia coli were also purified by the same method. A preparation containing SLT-II and SLT-I purified by receptor analog affinity chromatography was used to raise four monoclonal antibodies (MAbs) that were reactive with SLT-II by enzyme-linked immunosorbent assay. Three of these antibodies also reacted with Shiga toxin, which was the first clear demonstration of cross-reactivity between these toxins. One MAb, 4D1, which was specific for the B subunit of SLT-II and Shiga toxin, neutralized both toxins in a HeLa cell cytotoxicity assay. Two MAbs recognized the A subunit of both SLT-II and Shiga toxin by Western blot (immunoblot) analysis but were unable to neutralize either toxin. In addition, one B-subunit-specific MAb neutralized SLT-II alone, and a previously described Shiga toxin B-subunit-specific MAb was shown to be specific for Shiga toxin but not SLT-II.
Animals↗
Pathogenesis of Shigella diarrhea. XVI. Selective targetting of Shiga toxin to villus cells of rabbit jejunum explains the effect of the toxin on intestinal electrolyte transport.
To examine the mechanism by which Shiga toxin alters intestinal water and electrolyte transport, ligated loops of rabbit jejunum were incubated in vivo with purified toxin and then studied in vivo by single pass perfusion and in vitro by the Ussing chamber voltage-clamp technique. Toxin exposure led to accumulation of water in the jejunal lumen, associated with decreased active basal NaCl absorption. Glucose- and alanine-stimulated Na absorption were also reduced, while toxin had no effect on either basal short-circuit current or the secretory response to theophylline. These observations suggest that Shiga toxin selectively inhibits NaCl absorption without significantly altering active anion secretion. To localize the cellular site of toxin action, populations of villus and crypt cells from rabbit jejunum were isolated and studied. Villus cells had a greater content of the glycolipid Shiga toxin receptor, Gb3, had more toxin binding sites than did crypt cells, and were much more sensitive than crypt cells to toxin-induced inhibition of protein synthesis. These experiments demonstrate that purified Shiga toxin inhibits jejunal fluid absorption without affecting active fluid secretion by a preferential effect on villus cells. The results suggest that this is due to the differential distribution of toxin receptors on villus compared to crypt cells.
Absorption↗
Cancer-associated hemolytic uremic syndrome: a possible role of mitomycin in relation to Shiga-like toxins.
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Bacterial Toxins↗
Shiga toxin: production and purification.
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Bacterial Toxins↗
Shiga toxin as inhibitor of protein synthesis.
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Animals↗
Selective destruction of vagal sensory neurons using Shigella cytotoxin.
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Animals↗