Abstract
A one-step affinity chromatography method was developed to purify Shiga toxin 2 variants (Stx2) Stx2a, Stx2c, Stx2d and Stx2g from bacterial culture supernatants. Analysis of the purified Stx2 variants by denaturing gel electrophoresis revealed 32 kDa and 7 kDa protein bands, corresponding to the Stx2A- and B-subunits, respectively. However, native gel electrophoresis indicated that purified Stx2c and Stx2d were significantly higher in molecular weight than Stx2a and Stx2g. In a cytotoxicity assay with Hela cells, the 50% cytotoxic dose of Stx2a and Stx2g were 100 pg and 10 pg, respectively, but 1 ng each for Stx2c and Stx2d. Interestingly, analysis of the 50% inhibitory dose in a cell-free translational system from rabbit reticulocyte lysates indicated that Stx2g had a lower capacity to inhibit protein synthesis than the other Stx2 variants. The cytotoxicities in Hela cells were neutralized with an anti-Stx2B antibody and were denatured at 80 °C for 1 h. These findings demonstrated that Stx2 variants exhibited different toxicities, holotoxin structure, and stabilities using distinct systems for assessing toxin activities. The development of a simple method for purification of Stx2 variants will enable further studies of Stx2-mediated toxicity in various model systems.
Highlights
Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of severe human diseases including bloody diarrhea and hemolytic uremic syndrome (HUS) [1,2]
Results from the PCR analyses confirmed that only a single stx2 variant was detected in each Shiga toxin-producing Escherichia coli (STEC) strain that was selected for production of Shiga toxin 2 variants (Stx2) variants (Table 1)
Sequence analysis of stx2 genes of STEC strains used in this study indicates that the deduced amino acid sequence of Stx2a was identical to its reference strain [16,17,19,37] and the sequences of Stx2c, Stx2d, and Stx2g expressed by environmental STEC
Summary
Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of severe human diseases including bloody diarrhea and hemolytic uremic syndrome (HUS) [1,2]. The A-subunit has a molecular weight of 32 kDa and is an active component of the Stx and functions as an N-glycosidase It inhibits protein synthesis by cleavage of an adenine base from the 28S rRNA component of the eukaryotic ribosomal 60S subunit, resulting in cell death [5]. Each B-subunit has a molecular weight of 7.7 kDa and contains multiple binding sites for the natural Stx receptors globotriaosyl ceramide (Gb3) [6] or globotetraosyl ceramide (Gb4) [7]. These receptors located on the surface of mammalian cells are responsible for binding the Stx and its internalization by endocytosis
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