Verocytotoxin-1 induces apoptosis in vero cells

Inward, Carol; Williams, Julie M.; Chant, I.; Crocker, J; Milford, David V.; Rose, Peter; Taylor, Christopher M.

doi:10.1016/s0163-4453(95)90693-2

Cited by 85 publications

(56 citation statements)

References 14 publications

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“…Verocytotoxin and diphtheria toxin are both known to interfere with protein synthesis, leading to programmed cell death or apoptosis [35,36]. PCR-based screening of C. jejuni with verocytotoxin-specific primers was negative and confirmed the low stringency hybridisation experiments of Moore et al [31] and this suggested that the C. jejuni cytotoxic complex was distinct from verocytotoxin.…”

Section: Discussionmentioning

confidence: 61%

Identification and characterisation of a cytotoxic porin-lipopolysaccharide complex from Campylobacter jejuni

Bacon¹,

Johnson

Rodgers³

1999

Journal of Medical Microbiology

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A clinical isolate of Campylobacter jejuni, previously found to produce a toxin active in cell culture assays, was used for identification and characterisation of a cytotoxic porinlipopolysaccharide (LPS) complex. This cytotoxic complex was isolated by highperformance liquid chromatography of crude concentrated culture supernate and DEAE-anion exchange chromatography. The complex had a toxic activity of 20.1 tissue culture dose50 (TCD50)lpg of protein for HEp-2 cells, 7.49 TCD5O/pg of protein for HeLa cells and 1.87 TCD5Olpg of protein for Chinese hamster ovary cells. Analysis by SDS-PAGE revealed a single protein band of 45 kDa and a high mol. wt carbohydrate moiety. The complex gave a positive result in the Limulus amoebocyte lysate test, indicating that the co-purifying carbohydrate was LPS, and had specificity for the lectins Galanthus nivalis agglutinin, Maackia amurensis agglutinin and Datura stramonium agglutinin. The cytotoxic activity associated with the complex was heatlabile at 70°C, resistant to inactivation with trypsin and retained activity after treatment with sodium metaperiodate and the glycosidases neuraminidase and N-glycosidase F. Sequencing of the N-terminus of the protein component of the complex revealed 97% homology with the major outer-membrane porin protein from C. jejuni. The cytotoxic activity of the complex was neutralised by a polyclonal, homologous antiserum, which reacted on Western blot with the 45-kDa protein, but not by polyclonal antisera raised against a number of other bacterial toxins.

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Section: Discussionmentioning

confidence: 61%

Identification and characterisation of a cytotoxic porin-lipopolysaccharide complex from Campylobacter jejuni

Bacon¹,

Johnson

Rodgers³

1999

Journal of Medical Microbiology

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“…1A, after treatment for 48 hr, 10 pg/ml of Stx1 induced about an 80% reduction in the number of cells as assessed by MTT assay. Previous reports, including our own, have indicated that apoptosis is involved in the process of Stx-mediated cell death (6,18,34,38). As shown in Fig.…”

Section: Identification Of the Shiga-toxin 1-resistant Stock Of Vero mentioning

confidence: 57%

“…Briefly, entire lipids extracted from 1ϫ10 6 of Vero cells were separated on a TLC plate as described above and were transferred to a PVDF membrane, followed by blocking with 5% skimmed milk in PBS. After incubation with Stx1 B-subunit at 100 ng/ml for 30 min at room temperature, the membrane was washed with PBS containing 0.025% Tween 20 (Sigma), incubated with anti-Stx1 B-subunit Ab at 1 µg/ml for 30 min at room temperature, and washed again.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a Shiga‐Toxin 1‐Resistant Stock of Vero Cells

Sekino

Kiyokawa

Taguchi

et al. 2004

Microbiology and Immunology

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Shiga toxins (Stxs, also referred to as verotoxins) were first described as a novel cytotoxic activity against Vero cells. In this study, we report the characterization of an Stx1‐resistant (R‐) stock of Vero cells. (1) When the susceptibility of R‐Vero cells to Stx1 cytotoxicity was compared to that of Stx1‐sensitive (S‐) Vero cells by methylthiazolyldiphenyl‐tetrazolium bromide (MTT) assay, cell viability after 48‐hr exposure to 10 pg/ml of Stx1 was greater than 80% and less than 15%, respectively. (2) Although both a binding assay of fluorescence‐labeled Stx1 and lipid analysis indicated considerable expression of Gb3Cer, a functional receptor for Stxs, in both Vero cells, anti‐Gb3Cer monoclonal antibodies capable of binding to S‐Vero cells failed to effectively label R‐Vero cells, suggesting a conformational difference in the Gb3Cer expressed on R‐Vero cells. (3) The lipid analysis also showed that the R‐Vero cells contained significant amounts of Gb4Cer. In addition, introduction of exogenous Gb4Cer into S‐Vero cells slightly inhibited Stx1 cytotoxicity, suggesting some correlation between glycosphingolipid composition and Stx1 resistance. (4) Both butyrate treatment and serum depression eliminated the Stx1 resistance of R‐Vero cells. (5) The results of the analysis by confocal microscopy suggest a difference in intracellular transport of Stx1 between R‐Vero and S‐Vero cells. Further study of R‐Vero cells may provide a model of Stx1 resistance via distinct intracellular transport of Stx1.

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“…Cells lacking the toxin receptor were found to be resistant to the toxic effect (80). Stx can induce apoptosis in renal tubular epithelial cells (9,81) as well as in Burkitt lymphoma cells (82), intestinal epithelial 164 cells (63, 83), pulmonary epithelium-derived cells (84), and Vero cells (85). Cell death was not only induced by the holotoxin but also by high doses of the B subunit, indicating that this effect may be independent of the effect on protein synthesis (82).…”

Section: Stec Virulence Factorsmentioning

confidence: 99%