The site of action of a Vero toxin (VT2 or Shiga-like toxin 11) from enterohemorrhagic Escherichia coli and Shiga toxin from Shigella dysenteriae 1 on eukaryotic ribosomes was studied. Treatment of eukaryotic ribosomes with either toxin caused the release of a fragment of 400 nucleotides from 28s ribosomal RNA when the isolated ribosomal RNA was treated with aniline. Release of this fragment with aniline treatment was accompanied by inhibition of protein synthesis and of elongation-factor-1-dependent aminoacyl-tRNA binding to ribosomes. Analysis of the nucleotide sequence of the 3'-terminal fragment of 553 nucleotides of 28s rRNA of rat liver 60s ribosomal subunits suggested that an adenine base at position 4324 (A-4324) was absent in toxin-treated 28s rRNA. Further analysis by thin-layer chromatography demonstrated quantitative release of adenine from rat liver ribosomes on treatment with the toxins. These results indicate that both VT2 and Shiga toxin inactivate 60s ribosomal subunits by cleaving the N-glycosidic bond at A-4324 in 28s ribosomal RNA. (T. Ogasawara et al., unpublished results) were recently shown to inactivate 60s ribosomal subunits and thereby inhibit EF-1-dependent binding of aminoacyl-tRNA to ribosomes, in this study we examined whether these bacterial cytotoxins inactivate 60s ribosomal subunits by the same mechanism as that of other plant and fungal cytotoxins. MATERIALS AND METHODS Purqications of Shiga toxin and VT2Shiga toxin was isolated and purified from the culture supernatant of S. dysenteriae 1 (RIMD 3101010) by ammonium sulfate fractionation, DEAE-cellulose column chromatography and repeated chromatofocusing column chromatographies, as described previously [28, 291. VT2, which has been shown to be immunologically unrelated to the Shiga toxin, but has similar biological activities to the latter, was isolated and purified from an enterohemorrhagic E. cali 0157:H7 strain J-2 as described earlier [12]. Both purified toxins gave a single band staining for protein on conventional polyacrylamide disc gel electrophoresis and polyacrylamide gel isoelectrofocusing. Preparation of anti-toxin sera against Shiga toxin and VT2Anti-toxin sera against Shiga toxin and VT2 were prepared by immunizing rabbits with purified Shiga toxin and purified VT2 as described previously [12, 281. Immunoglobulins

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Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochrome c from mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochrome c from mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.

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