Reduced Toxicity of Shiga Toxin (Stx) Type 2c in Mice Compared to Stx2d Is Associated with Instability of Stx2c Holotoxin

Abstract

Shiga toxin (Stx) is an AB5 ribotoxin made by Stx-producing Escherichia coli (STEC). These organisms cause diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome. STEC make two types of Stxs, Stx1 and/or Stx2. Stx2 has one prototype (a) and six subtypes (b-g), but only STEC that make Stx2a, and/or Stx2c, or Stx2d are associated with severe disease. However, Stx2c is about 10-fold less toxic than Stx2d in vivo despite only two amino acid differences in the A subunit at positions 291 and 297. We made mutations at these two sites to create intermediate toxins between Stx2c and Stx2d, and determined the 50% cytotoxic dose on Vero cells before and after heat treatment, and the 50% lethal dose in mice of the toxins. We found that serine 291 was associated with increased toxicity in vivo and that either amino acid change from that in Stx2c to that in Stx2d increased heat stability. We also assessed the secondary structure of Stx2c and Stx2d by circular dichroism (CD) spectroscopy. The CD studies suggest that Stx2c has a less-ordered secondary structure than Stx2d. We conclude that both amino acids at positions 291 and 297 in Stx2c contribute to its decreased stability and in vivo toxicity compared to Stx2d.

Keywords: STEC; Shiga toxin; Stx2; Stx2c; Stx2d.

Figure 1

Partial amino acid sequence alignment of the C-terminus of the A subunit and B subunit from Stx2a, Stx2c/c, Stx2d/d, Stx2c/d, and Stx2d/c. Amino acid sequence alignment of a portion of the C-terminus of the A subunit that shows the F291S and K297E changes in Stx2d/d with Stx2a as the reference sequence (A); Amino acid sequence alignment of the B subunit that shows the D16N and D24A changes in Stx2c/c and Stx2d/d with Stx2a as the reference sequence (B); Amino acid sequence alignment that shows the sequence in the intermediate toxin A subunits Stx2c/d and Stx2d/c with Stx2c/c as the reference sequence and Stx2d/d for comparison (the B subunits were not altered) (C). Sequence alignments were compiled in BioEdit.

Figure 2

Binding of toxins to Gb3. An ELISA was used to assess binding of each toxin to purified Gb3. Mean and standard deviation values were plotted from triplicate values. The nonlinear curve was approximated by “One Site–Specific Binding” within GraphPad Prism (see Materials and Methods) and the r² > 0.99 for all toxins curves. An F test to compare the K_d values indicated that they were different, p < 0.0001, and pairwise comparisons gave a value of p ≤ 0.0001. The apparent K_d values (rounded to the nearest 0.01) for Stx2c/c, Stx2d/d, Stx2c/d, and Stx2d/c, were 0.30 µg/mL with a 95% CI of 0.26–0.35 µg/mL, 0.19 µg/mL with a 95% CI of 0.17–0.21 µg/mL, 0.95 µg/mL with a 95% CI of 0.82–1.1 µg/mL, and 0.15 µg/mL with a 95% CI of 0.13–0.16 µg/mL, respectively. The B_max values were similar for each toxin and ranged from 3.2 to 3.4.

Figure 3

Thermostability assay. Toxins were incubated at 70 °C for the time indicated by the points on each line and then placed on Vero cells. Data are reported as the geometric mean with 95% confidence intervals from triplicate samples. Linear approximations were done with GraphPad Prism with a resultant r² ≥ 0.92 for all toxins. Analysis of covariance showed these approximated slopes to be statistically significantly different with a p < 0.0001. Statistical differences of pairwise comparisons: Stx2c/c versus Stx2c/d, p = 0.011; Stx2c/d versus Stx2d/c, p = 0.166; and all other comparisons, p < 0.001.

Figure 4

Circular dichroism spectra of Stx2c/c and Stx2d/d. Mean residual ellipticitities (MRE) are plotted as a function of wavelength. Spectra of native Stx2c/c and Stx2d/d were acquired at 20 °C. Post exposure to heat (PEH) spectra were acquired at 40 °C. Secondary structural analyses of both native and PEH toxins were done as described in the Experimental section, and the results are presented in Table 2.

Figure 5

Crystal structure of A₂ peptide and B subunits from Stx2a. A ribbon representation of the crystal structure of the A₂ peptide shows the relative locations of the amino acids at positions 291 and 297 in comparison to positions 16 and 24 from the B subunits. The A₂ peptide is colored red with a space-filled model of phenylalanine at position 291 depicted in teal and lysine at position 297 highlighted in green. The B subunits are colored blue with a space-filled model of aspartic acid at positions 16 and 24 highlighted in grey. Part (A) shows the binding face of Stx2a; Part (B) shows a horizontal view of the toxin with the binding face at the bottom. The PDB accession number for Stx2a is 1R4P. In this representation, the A₁ portion of the structure is not shown.