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

Abstract

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.

Globotriaosylceramide (Gb3) content in HeLa cells is correlated to Shiga toxin-induced cytotoxicity and Gb3 synthase expression

“…The high sensitivity of Gb3-rich cells toward Stxs may be may be attributable to apoptosis, as reported by Fujii et al (20). Differences in cytotoxicity of Stx in the cloned cell lines have been described in several previous studies (21,22). Accordingly, HeLa cells were cloned in this study and the correlation between Gb3 or Gb4 content and cell viability upon exposure to Stxs was assessed.…”

Shiga toxin glycosphingolipid receptors of Vero-B4 kidney epithelial cells and their membrane microdomain lipid environment

“…TLC analysis and corresponding overlay binding assays revealed the presence of Gb3Cer, the major Stx receptor, and Gb4Cer, the less effi cient receptor, in Vero cells. Heterogeneity regarding TLC separation was observed in several previous studies and orcinol as well as Stx-detected bands of receptor GSLs suggested ceramide heterogeneity owing to fatty acids with varying acyl chain lengths ( 14,15,20,23 ). We provide here the full structural elucidation of Stx receptors of Vero-B4 cells and identifi ed Gb3Cer (d18:1, C24:0/C24:1) and Gb3Cer (d18:1, C16:0) as the prevalent Gb3Cer species (see Table 1 ), as well as similar variability for the corresponding prevalent Gb4Cer species.…”

“…Since this discovery, numerous studies have shown the functional impact of globo-series GSLs as Stx-specifi c receptors and their key role regarding toxin-mediated cytotoxicity in Vero cells ( 14,15,18,20,22,23,83 ). TLC analysis and corresponding overlay binding assays revealed the presence of Gb3Cer, the major Stx receptor, and Gb4Cer, the less effi cient receptor, in Vero cells.…”

Glycosphingolipids in vascular endothelial cells: relationship of heterogeneity in Gb3Cer/CD77 receptor expression with differential Shiga toxin 1 cytotoxicity

“…Gb3Cer species with long chain fatty acids have in fact been suggested being associated with greater toxicity, because they possibly better mediate the localization of internalized toxin to the endoplasmic reticulum [61]. Although the details are unknown, the differences in the overall glycolipid composition of sensitive and resistant cells expressing compositional identical Gb3Cer species may also affect Gb3Cer recognition and the sorting route of incorporated Stx1 as shown for Vero cells [62].…”