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United States Department of Agriculture

Agricultural Research Service

Research Project: Technologies for Detecting and Determining the Bioavailability of Bacterial Toxins

Location: Foodborne Toxin Detection and Prevention

Title: Purification and characterization of Shiga toxin 2f, an immunologically unrelated subtype of Shiga toxin 2

Authors
item Skinner, Craig
item Patfield, Stephanie
item Rasooly, Reuven
item Carter, John
item He, Xiaohua

Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 17, 2013
Publication Date: March 26, 2013
Citation: Skinner, C.B., Mcmahon, S.A., Rasooly, R., Carter, J.M., He, X. 2013. Purification and characterization of Shiga toxin 2f, an immunologically unrelated subtype of Shiga toxin 2. PLoS One. 8(3):e59760. doi:10.1371/journal.pone.0059760.

Interpretive Summary: E. coli expressing Shiga-like toxin (STEC) are a major bacterial food contaminant, and responsible for several recent deadly outbreaks. Shiga-like toxin (Stx) is a critical virulence factor in these STEC outbreaks. There are two main classes of Stx: Stx1 and Stx2. While both Stx1 and Stx2 can aggravate the symptoms of STEC, Stx2 is associated with potentially life-threatening Hemolyic-Uremic Syndrome (HUS). There are seven subtypes of Stx2 (Stx2a through Stx2g), several of which are difficult to detect and purify, and are therefore uncharacterized. Stx2f is the most diverse of these Stx2 subtypes. In this study, we report the purification of the Stx2f subtype. Stx2f was found to be less toxic in cell-based assays than wild-type Stx2 (Sx2a) but more resistant to acid and heat treatments. The Stx2f subtype binds to a wider variety of receptors as well, which may diversify its cellular targets in vivo. This different receptor preference may also explain why Stx2f is less toxic in the cell-based assays we conducted. This study will contribute to our understanding of Shiga-like toxins, STEC, and the diseases they cause.

Technical Abstract: Background: Shiga-like toxin 2 (Stx2) is one of the most important virulence factors in enterohaemorrhagic Escherichia coli (E. coli) strains such as O157H7. Subtypes of Stx2 are diverse with respect to their sequence, toxicity, and distribution. The most diverse Stx2 subtype, Stx2f, is difficult to detect immunologically, but is becoming more frequently associated with human illness. Methods and Findings: A purification regimen was developed for the purification of Stx2f involving cation exchange, hydrophobic interaction, anion exchange, and gel filtration. The molecular weight of Stx2f B-subunit was approximately 5 kDa, which appeared significantly smaller than that of Stx2a(6 kDa)on a SDS-PAGE gel, although the size of the A subunit was similar to Stx2a (30 kDa). Stx2f was shown to be active in both cell-free and cell-based assays. The 50% cytotoxic dose in Vero cells was 3.4 or 1.7 pg (depending on the assay conditions), about 3–5 times higher than the archetypical Stx2a, while the activity of Stx2f and Stx2a in a cell-free rabbit reticulocyte system was similar. Stx2f bound to both globotriose-lipopolysaccharide(Gb3-LPS)and globotetraose-LPS(Gb4-LPS, mimics for globotriaosylceramide and globotetraosylceramide, respectively), but its ability to bind Gb4-LPS was much stronger than Stx2a. Stx2f was also much more stable at low pH and high temperature compared to Stx2a, suggesting the toxin itself may survive harsher food preparation practices. Conclusions: Here, we detail the purification, biochemical properties, and toxicity of Stx2f, from an E. coli strain isolated from a feral pigeon. Information obtained in this study will be valuable for characterizing Stx2f and explaining the differences of Stx2a and Stx2f in host specificity and cytotoxicity.

Last Modified: 4/16/2014