A single-step purification and molecular characterization of functional Shiga toxin 2 variants from pathogenic Escherichia coli

Authors: He, Xiaohua; Quinones, Beatriz; Patfield, Stephanie; Mandrell, Robert

Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/19/2012
Publication Date: 6/25/2012
Citation: He, X., Quinones, B., Mcmahon, S.A., Mandrell, R.E. 2012. A single-step purification and molecular characterization of functional Shiga toxin 2 variants from pathogenic Escherichia coli. Toxins. 4(7):487-504. doi:10.3390/toxins4070487

Interpretive Summary: Shiga toxin (Stx)-producing Escherichia coli (STEC) is the frequent cause of severe human diseases including bloody diarrhea and hemolytic uremic syndrome (HUS). Stxs are thought to play a prominent role in the pathogenesis of STEC infections. To elucidate mechanisms accounted for the toxicity of Stxs in humans, it requires pure toxins. However, there are very limited amounts of purified Stx2 and no Stx2 variants toxin stocks available commercially to date. We describe in this study a simple and rapid method for purification of four Stx2 variants and characterize their purity, quantity, and biological activities.

Technical Abstract: Shiga toxin (Stx) 2 variants, Stx2a, Stx2c, Stx2d and Stx2g were purified to homogeneity from bacterial culture supernatants by a one-step monoclonal anti-Stx affinity chromatography method. The method was based on the binding affinity of these Stxs for a monoclonal antibody against the Stx2 A-subunit. The final yields of Stx2a, 2c, 2d, and 2g after this affinity purification ranged from 220 to 470 ng/mL of the culture supernatant. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins from different Stx preparations revealed two protein bands, each with molecular weights of 32 and 7 kDa, corresponding to the values for the Stx2 A- and B-subunit, respectively. Electrophoresis of these proteins under native conditions, however, resulted in different protein banding patterns. The single major protein band from the Stx2a and Stx2g preparations had a MW of 72 kDa, corresponding to the predicted size of a Stx2 holotoxin. In contrast, the protein bands from the Stx2c and Stx2d preparations had a MW at least 8-fold larger; protein mobility was unchanged by the addition of a reducing agent to the samples. The change in Stx2c and Stx2d protein mobility coincided with decreases of their cytotoxicities for Hela cells. These experimental conditions corresponded to 50% cytotoxic activities of Stx2a, Stx2c, Stx2d and Stx2g for Hela cells of 100, 1000, 1000, and 10 pg, respectively. Most Stx activity was lost after heating at 80°C for one hour, but not at 60°C. Stx2g was less heat stable compared to the other Stx2 types tested. An anti-Stx2 B-subunit mouse monoclonal antibody neutralized the cytotoxicities of all Stxs for Hela cells.