Showing papers on "Shigella dysenteriae published in 1983"

Purification and characterization of a Shigella dysenteriae 1-like toxin produced by Escherichia coli.

Abstract: A toxin from an enteropathogenic strain of Escherichia coli (E. coli H30) was purified to apparent homogeneity from cell lysates. The steps used to isolate the E. coli H30 toxin included French pressure-cell disruption of bacteria grown in iron-depleted media. Affi-Gel Blue chromatography, chromatofocusing, and anti-Shiga toxin affinity chromatography. The mobilities of the subunits of radioiodinated E. coli H30 toxin and Shiga toxin observed after the two toxins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis were identical. In the absence of 2-mercaptoethanol, a narrow band was seen at Mr 31,500 (+/- 1,000), and a wide heavy band was observed between Mr 4,000 and 15,000. In the presence of 2-mercaptoethanol, bands were seen at Mr 31,500 (+/- 1,000), 27,000, and 4,000 to 15,000. Other similarities between purified E. coli H30 and Shiga 60R toxins included identical isoelectric points (7.03 +/- 0.02); comparable biological activities, i.e., cytotoxicity, lethality for mice, and enterotoxicity; and the same relative heat stabilities (up to 65 degrees C for 30 min). Nevertheless, the two toxins had apparently different molecular weights as determined by sucrose gradient analysis, by gel filtration, and by cross-linking experiments with dimethyl suberimidate. The Mr of native E. coli H30 toxin estimated from cross-linking studies was 48,000, whereas the estimated Mr of Shiga 60R toxin was 58,000. These results suggest that like the cholera-E. coli-heat-labile toxin family, a family of Shiga-like toxins exists.

Molecular comparison of virulence plasmids in Shigella and enteroinvasive Escherichia coli.

Abstract: Virulent isolates of Shigella dysenteriae and Shigella boydii harboured a 140 Mdal plasmid which was either absent or deleted in spontaneously avirulent strains. Together with previous data concerning S. sonnei, S. flexneri and enteroinvasive Escherichia coli, the present results established the general role of extrachromosomal elements in the virulence of such enteroinvasive species. Among different species, these virulence plasmids showed unrelated endonuclease cleavage patterns, whereas hybridization experiments showed that homologous sequences were present throughout the molecules. These plasmids may therefore have derived from a common ancestor molecule which overcame evolutionary alterations in restriction sites. Furthermore, intraspecies and intraserotype comparison of these plasmids by endonuclease cleavage demonstrated highly conserved sequences. The consequences of these data for evolution, epidemiology and diagnosis of Shigella and enteroinvasive E. coli are discussed.

Animal toxicity of Shigella dysenteriae cytotoxin: evidence that the neurotoxic, enterotoxic, and cytotoxic activities are due to one toxin.

Abstract: The lethal effect to rabbits and mice of Shigella dysenteriae toxin and the ability of the toxin to induce fluid accumulation in rabbit ileal loops were studied in relation to the cytotoxic activity. The relative concentrations of the three activities were approximately the same in a crude toxin preparation and in purified, electrophoretically homogenous toxin. The cytotoxic and lethal activities eluted identically from a high pressure liquid chromatography column and migrated at the same rate in polyacrylamide gel electrophoresis under nondenaturing conditions. The cytotoxic, lethal, and enterotoxic activities were inactivated to essentially the same extent upon incubation for few minutes at 80 degrees C and upon treatment with urea. Graded precipitation of Shigella toxin with different amounts of an antiserum to Shigella toxin in each case removed essentially the same fraction of the cytotoxic, the lethal, and the enterotoxic activity. The data indicate that one molecular entity is responsible for the three biologic effects of Shigella toxin studied. After i.v. injection, the LD50 dose was estimated to be 2.2 ng/kg in rabbits and 450 ng/kg in mice.

Shigella dysenteriae 1 cytotoxin: periplasmic protein releasable by polymyxin B and osmotic shock.

Abstract: Treatment of Shigella dysenteriae 1 either with the antibiotic polymyxin B or by osmotic shock resulted in the release of 80 to 90% of the cytotoxin activity of the organism. Under the conditions employed, the release of toxin activity was accompanied by the appearance of a periplasmic enzyme, 5'-nucleotidase. There was no significant release of cytoplasmic contents, assessed by measurement of glucose-6-phosphate dehydrogenase activity. The release of cytotoxin and 5'-nucleotidase by polymyxin B were both dependent on the duration of incubation with, and the concentration of, the antibiotic. In terms of specific activity (cytotoxin activity per milligram of protein), the polymyxin B and osmotic shock extracts were 20- to 30-fold more active than crude toxin preparation derived from a whole-cell lysate. The data strongly support a periplasmic location for Shiga cytotoxin and the utility of the polymyxin B extraction to obtain starting material for toxin purification.

Shigella infections in the United States, 1974-1980

Abstract: During the seven-year period 1974-1980, 93,516 Shigella isolates from humans were reported to the Centers for Disease Control, Atlanta, through a nationwide surveillance system. Over the past 30 years, the reported incidence of shigellosis has been declining in contrast to that reported for salmonellosis. Shigella sonnei (group D) now accounts for approximately 70% of the Shigella isolates reported, since most of the decline has been due to the increasingly less frequent infections caused by Shigella flexneri (group B); Shigella dysenteriae infections are now uncommon. The highest reported rates of shigellosis are from the western states and among children from one to five years of age. Among the total population of the United States, the ratio of S. flexneri to S. sonnei isolates was 0.28 among persons from one to 19 years of age and 0.45 among persons greater than or equal to 20 years of age; among American Indians the proportion of S. flexneri was much greater, the corresponding ratios being 2.1 and 2.9, respectively; in both groups the ratios for children younger than one year of age resembled those obtaining for adults. Age-related acquisition of immunity to S. sonnei may explain these observations.

Antigenic relationships between the enteroinvasive Escherichia coli O antigens O28ac, O112ac, O124, O136, O143, O144, O152, and O164 and Shigella O antigens.

Abstract: Enteroinvasive Escherichia coli strains have been found in the following O serogroups: O28ac, O112ac, O124, O136, O143, O144, O152, and O164. The biochemical reactions of these enteroinvasive E. coli strains are often similar to those of Shigella strains, and identification may be difficult. The problem of differentiation and identification is further complicated by the sharing of antigenic components. The antigenic relationships between these O antigens and those of all the established and provisional Shigella serovars were examined. O antigen identity was demonstrated between E. coli O124 and Shigella dysenteriae 3 and between E. coli O152 and provisional Shigella serovar 3341:55. Reciprocal relationships were found between E. coli O112ac and S. dysenteriae 2, S. boydii 1, S. boydii 15, between E. coli O136 and S. dysenteriae 3, and between E. coli O164 and S. dysenteriae 3. A one-way relationship was demonstrated between E. coli O136 and S. boydii 1. The remaining enteroinvasive E. coli O serogroups showed no significant antigenic relationships with any Shigella serovar.

Entry of Shigella dysenteriae toxin into HeLa cells.

Abstract: The rate of shigella toxin entry into the cytosol of HeLa S3 cells was estimated from the toxin-induced reduction in protein synthesis. Whereas high toxin concentrations strongly reduced protein synthesis within 30 min, lower concentrations required longer times. The major part of the cell-bound toxin entered only after several hours. Toxin entered cells after incubation at 25 degrees C but not at 20 degrees C, although toxin binding was the same at the two temperatures. Increasing the KCl concentration to 0.2 M protected against toxin. The toxin entry was strongly reduced when the level of ATP in the cells was reduced by incubation with metabolic inhibitors. Lysosomotrophic agents such as NH4Cl and chloroquine had little or no protective effect, but the protonophores carbonyl cyanide p-trifluoromethoxyphenylhydrazone and carbonyl cyanide m-chlorophenylhydrazone and the ionophore monensin protected cells against the toxin. Cells were also protected when the pH was reduced to 6.4. The entry of shigella toxin is discussed in relation to that of other protein toxins with intracellular sites of action.

Release of Shiga Toxin from Shigella dysenteriae 1 by Polymyxin B

Abstract: Release of Shiga toxin from Shigella dysenteriae 1 was found to occur after exposure to polymyxin B. The amount of toxin released was dependent on both the polymyxin concentration and time of incubation. An immunoblot characterization of the Shiga toxin released by polymyxin treatment demonstrated that it is electrophoretically similar to purified Shiga toxin and to Shiga toxin present in crude bacterial sonicates of S. dysenteriae 1 cells.

Pathogenesis of Shigella diarrhea. VIII. Evidence for a translocation step in the cytotoxic action of Shiga toxin.

Abstract: Shigella toxin interacts with HeLa cells by binding to a glycoprotein cell surface receptor and, after a lag period, inactivates the 60S ribosomal subunit and inhibits protein synthesis. The lag period may be due to energy-dependent translocation of the toxin to the interior of the cells. This step was investigated with agents known to inhibit glucose metabolism, mitochondrial energy production, or macromolecular synthesis and with drugs that alter the cytoskeletal system or lysosomes. Metabolic inhibitors reduced both cytotoxicity and binding of the toxin. All agents tested except actinomycin D also diminished endocytosis. Effects on cytotoxicity were partially reversed in the presence of the membrane permeabilizer dimethylsulfoxide. Various cytochalasins, colchicine, vinca alkaloids, chloroquine, and steroids also reduced the activity of the toxin. These diverse agents are probably acting on a common pathway affecting the internalization and/or intracellular processing of shigella toxin. Translocation of this toxin apparently occurs by endocytosis from the cell surface.

Isolation and characterization of monoclonal antibodies to Shiga toxin.

Abstract: Hybridoma cell lines which produce monoclonal antibodies to Shiga toxin from Shigella dysenteriae 1 were prepared. The monoclonal antibodies were all of the immunoglobulin G1 isotype and differed in their ability to neutralize cytotoxicity and to bind to Shiga toxin in a solid-phase radioimmunoassay. When used for immunoblot analysis, these antibodies were able to identify specifically both nicked and unnicked Shiga toxin in crude lysates of S. dysenteriae.

Rapid Method for Detection, Identification, and Susceptibility Testing of Enteric Pathogens

Abstract: Three hundred and seven colonies believed to be enteric pathogens were selected from primary plates of MacConkey, xylose desoxycholate, or salmonella-shigella agar for inoculation to lactose-sucrose broth, urea-41 motility medium, modified Andrade glucose broth with inverted Durham tube, pregrowth broth, triple sugar iron agar, lysine iron agar (LIA), and Christensen urea agar. The rapid screen consisted of interpreting the lactose-sucrose, urea-41 motility, and modified Andrade glucose broth gas reactions after 4 to 6 h at 35°C. These rapid screening biochemicals plus LIA were incubated for 24 h if further interpretation was required. Reference biochemicals (triple sugar iron, LIA, and Christensen urea agars) were interpreted at 24 h. Of 307 isolates, 49 (16%) were reported as negative for enteric pathogens after 4 to 6 h because their biochemical profiles were not compatible with those for enteric pathogens. A total of 87 (28.3%) isolates produced biochemical profiles at 4 to 6 h that were presumptive for enteric pathogens. The 87 presumptive pathogens were inoculated into the AutoMicrobic system Gram-Negative General Susceptibility Card and the AutoMicrobic system Enterobacteriaceae-Plus Biochemical Card (AMS-EBC+) after 4 to 6 h of growth in pregrowth broth. Of these isolates, 63 were confirmed to be enteric pathogens, of which 61 (96.8%) were correctly identified by the AMS-EBC+. One isolate was identified as Shigella dysenteriae by AMS-EBC+ but confirmed as Shigella flexneri biotype 6 by a reference laboratory. The other isolate was identified as Arizona hinshawii by AMS-EBC+ but was confirmed as Salmonella enteritidis. Of the 307 isolates, 166 (54.1%) required further interpretation of the rapid screening biochemicals plus LIA at 24 h; 5 of these were detected as enteric pathogens. The same 68 enteric pathogens were detected by both the rapid method and the reference method. The results from the general susceptibility card agreed with agar diffusion results at 99.2%. One Salmonella enteritidis and four Shigella spp. showed minor discrepancies with tetracycline. No very major or major discrepancies were observed.