Purification and Biological Characterization of Shiga Toxin from
            <i>Shigella dysenteriae</i>
            1

Brown, J E; Griffin, D E; Rothman, Sara W.; Doctor, Bhupendra P.

doi:10.1128/iai.36.3.996-1005.1982

Cited by 79 publications

(35 citation statements)

References 21 publications

(26 reference statements)

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“…By contrast, an extract of E. coli strain 12801/0 inhibited protein synthesis in HeLa cells but was not lethal for mice or enterotoxic for rabbits, and an extract of E. coli K12 strain 1133 was negative for all three activities. It should be noted that the mouse lethality and rabbit enterotoxicity assays for purified Shiga toxin are at least 104 times less sensitive than are tests for cytotoxicity or inhibition of protein synthesis [14,32].…”

Section: Resultsmentioning

confidence: 99%

Production of Shigella dysenteriae Type 1-Like Cytotoxin by Escherichia coli

O'Brien¹,

LaVeck²,

Thompson³

et al. 1982

The Journal of Infectious Diseases

417

260

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Strains of Escherichia coli previously implicated or proven to be causes of diarrhea were examined for production of a toxin similar to that of Shigella dysenteriae type 1 (Shiga). Organisms grown in an iron-depleted broth were lysed by pressure disruption followed by ultracentrifugation. Saline-dialyzed extracts were tested for cytotoxic effects on HeLa cells that were neutralizable with antiserum to Shiga toxin. Among the 13 E. coli strains so analyzed, 11 made a Shiga-like cytotoxin in levels ranging from trace (two avirulent isolates) to amounts equivalent to S. dysenteriae type 1 (two noninvasive strains that did not make E. coli heat-labile or -stable enterotoxins but were isolated from infants with diarrhea). As with extracts of Shiga toxin, lysates of these E. coli strains that produced high levels of Shiga-like toxin were enterotoxic for rabbits, paralytic and lethal for mice, and inhibited protein synthesis in HeLa cells. Thus, these data suggest that Shiga-like toxin may be another heretofore undiscovered factor in the pathogenesis of diarrhea caused by some E. coli strains.

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Section: Resultsmentioning

confidence: 99%

Production of Shigella dysenteriae Type 1-Like Cytotoxin by Escherichia coli

O'Brien¹,

LaVeck²,

Thompson³

et al. 1982

The Journal of Infectious Diseases

417

260

View full text Add to dashboard Cite

show abstract

“…As we show in this study, treatment with butyric acid and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induced the ap-was obtained from Bureau of Biologics, FDA, (Bethesda, MD). Shiga toxin was purified as previously described (7), and was a generous gift from Dr. J. E. Brown. Conjugates of Shiga toxin and HRP were prepared by the SPDP method as previously described (46).…”

mentioning

confidence: 99%

Endocytosis and intracellular transport of the glycolipid-binding ligand Shiga toxin in polarized MDCK cells.

Sandvig

Prydz

Ryd

et al. 1991

The Journal of Cell Biology

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Abstract. The glycolipid-binding cytotoxin produced by Shigella dysenteriae 1, Shiga toxin, binds to MDCK cells (strain 1) only after treatment with short-chain fatty acids like butyric acid or with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. The induced binding sites were found to be functional with respect to endocytosis and translocation of toxin to the cytosol. Glycolipids that bind Shiga toxin appeared at both the apical and the basolateral surface of polarized MDCK cells grown on filters, and Shiga toxin was found to be endocytosed from both sides of the cells. This was demonstrated by EM of cells incubated with Shiga-HRP and by subcellular fractionation of cells incubated with t25I-labeled Shiga toxin. The data indicated that toxin molecules are endocytosed from coated pits, and that some internalized Shiga toxin is transported to the Golgi apparatus. Fractionation of polarized cells incubated with 125I-Shiga toxin showed that the transport of toxin to the Golgi apparatus was equally efficient from both poles of the cells. After 1-h incubation at 37°C ~10% of the internalized toxin was found in the Golgi fractions. The results thus suggest that glycolipids can be efficiently transported to the Golgi apparatus from both sides of polarized MDCK cell monolayers.

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“…The A subunit inhibits protein synthesis by inactivating 60S ribosomal subunits, and the activity of the free Al fragment is much higher than that of the whole toxin (35). Originally, the observation that one STX molecule is capable of catalytically inactivating more than one ribosome in the absence of cofactors led Brown et al (3) and Reisbig et al (35) to suggest that the toxin is a hydrolytic enzyme. This hypothesis was disproved in 1988, when Endo and coworkers (9) used an in vitro translation system to show that STX and SLT are N-glycosidases which cleave the N-glycosidic bond at adenine 4324 of the 28S rRNA in the 60S subunit of rat liver ribosomes.…”

mentioning

confidence: 99%

Minimum domain of the Shiga toxin A subunit required for enzymatic activity

1993

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The minimum sequence of the enzymatic (A) subunit of Shiga toxin (STX) required for activity was investigated by introducing N-terminal and C-terminal deletions in the molecule. Enzymatic activity was assessed by using an in vitro translation system. A 253-amino-acid STX A polypeptide, which is recognized as the enzymatically active portion of the 293-amino-acid A subunit, expressed less than wild-type levels of activity. In addition, alteration of the proposed nicking site between Ala-253 and Ser-254 by site-directed mutagenesis apparently prevented proteolytic processing but had no effect on the enzymatic activity of the molecule. Therefore, deletion analysis was used to identify amino acid residue 271 as the C terminus of the enzymatically active portion of the STX A subunit. STX A polypeptides with N-terminal and C-terminal deletions were released into the periplasmic space of Escherichia coli by fusion to the signal peptide and the first 22 amino acids of Shiga-like toxin type II, a member of the STX family. Although these fusion proteins expressed less than wild-type levels of enzymatic activity, they confirmed the previous finding that Tyr-77 is an active-site residue. Therefore, the minimum domain of the A polypeptide which was required for the expression of enzymatic activity was defined as StxA residues 75 to 268.

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Purification and Biological Characterization of Shiga Toxin from Shigella dysenteriae 1

Cited by 79 publications

References 21 publications

Production of Shigella dysenteriae Type 1-Like Cytotoxin by Escherichia coli

Production of Shigella dysenteriae Type 1-Like Cytotoxin by Escherichia coli

Endocytosis and intracellular transport of the glycolipid-binding ligand Shiga toxin in polarized MDCK cells.

Minimum domain of the Shiga toxin A subunit required for enzymatic activity

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