Shiga toxin subtypes display dramatic differences in potency

Abstract

Purified Shiga toxin (Stx) alone is capable of producing systemic complications, including hemolytic-uremic syndrome (HUS), in animal models of disease. Stx includes two major antigenic forms (Stx1 and Stx2), with minor variants of Stx2 (Stx2a to -h). Stx2a is more potent than Stx1. Epidemiologic studies suggest that Stx2 subtypes also differ in potency, but these differences have not been well documented for purified toxin. The relative potencies of five purified Stx2 subtypes, Stx2a, Stx2b, Stx2c, Stx2d, and activated (elastase-cleaved) Stx2d, were studied in vitro by examining protein synthesis inhibition using Vero monkey kidney cells and inhibition of metabolic activity (reduction of resazurin to fluorescent resorufin) using primary human renal proximal tubule epithelial cells (RPTECs). In both RPTECs and Vero cells, Stx2a, Stx2d, and elastase-cleaved Stx2d were at least 25 times more potent than Stx2b and Stx2c. In vivo potency in mice was also assessed. Stx2b and Stx2c had potencies similar to that of Stx1, while Stx2a, Stx2d, and elastase-cleaved Stx2d were 40 to 400 times more potent than Stx1.

FIG. 1.

Sequence alignments and structural models of Stx2 subtypes. Stx2 sequences were aligned using BLASTP (NCBI/BLAST), with periods indicating identity and dashes indicating absent amino acids. Numbering starts with the first amino acid of the mature peptide; numbers correspond to bold amino acids. (A) Only the C-terminal 65 amino acids, corresponding to the region of greatest variability, of the 297-amino-acid A subunit are shown. (B) Alignment of the entire B subunit. (C) The mutagenesis function of PYMOL was used to substitute amino acids of the subtypes into the Stx2a crystal structure (1R4P). The structures are oriented to display the receptor binding face and are color coded as follows: blue, amino acids of the Stx2a B pentamer predicted to mediate receptor binding, based on the crystal structure of Stx2e subtype GT3 bound to Gb3 (31); gray, amino acids not thought to participate in binding; yellow, amino acid polymorphisms in the B subunit of Stx2b to Stx2d; red, the C terminus of the A subunit; green, amino acid polymorphisms in the A subunit of Stx2b and Stx2d. Red boxes indicate the toxin amino acid variants purified for this study; the B subunits of Stx2c used in the study and the Stx2c type are identical.

FIG. 2.

Reconstructed mass spectra of Stx2d. Stx2d or elastase-cleaved Stx2d was treated with trypsin to generate the N-terminal A1 and C-terminal A2 fragments, and the A2 fragments were analyzed by LC-MS. (A) Trypsin-treated Stx2d control full-length A2 (5,227.4 Da) is seen as the major peak. (B) Elastase-treated Stx2d. The major fragment is smaller (5,042.2 Da), corresponding to A2 minus the two terminal amino acids (GE).

FIG. 3.

Stx-mediated inhibition of metabolic activity of primary human kidney cells. RPTECs from two donors were grown in the indicated medium and treated with dilutions of purified Stx as indicated. After 48 h, medium was replaced with the medium containing 10% (vol/vol) alamarBlue, cells were incubated for an additional 4 h, and fluorescence of reduced alamarBlue was measured. The graphs depict toxin-treated cells standardized to percent metabolic activity of untreated control cells. (A) RPTECs, from a 35-year-old male, that were grown in Clonetics medium. Metabolic activity was assessed at division 12 or 16 after 2 days' incubation with toxin. (B) RPTECs, from a 35-year-old male, that were grown in Lifeline medium were treated with Stx2 at division 8 and assessed for metabolic activity at division 10. (C) RPTECs, from a 7-year-old male, that were grown in Lifeline medium were treated with Stx2 at division 8 and assessed for metabolic activity at division 10. (D) RPTECs, from a 7-year-old male, that were grown in Lifeline medium were treated with Stx1 and mutants at division 8 and assessed for metabolic activity at division 10. Error bars depict standard errors of the means, which were calculated in Prism5 (GraphPad Software, La Jolla, CA).

FIG. 4.

Flow cytometry analysis of annexin V binding versus propidium iodide staining. RPTECs at division 8 were treated for 12 h as indicated with PBS (control) (A), actinomycin D (inhibition of transcription, 10 μM) (B), cycloheximide (inhibition of protein synthesis, 100 μM) (C), or Stx2a (inhibition of protein synthesis) (D). Cells were stained with annexin V and propidium iodide. Percentages of cells in each quadrant are indicated.

FIG. 5.

Mouse toxicity studies. Male CD-1 mice were injected i.p. with half-log dilutions of purified Stx ranging from 0.3 to 1,000 ng per mouse and monitored for 120 h. The LD₅₀ values are marked by a bar and are noted at the bottom of the columns.

FIG. 6.

Interactions of multiple factors contributing to the severity of Stx-mediated disease.