Change in conformation with reduction of alpha-helix content causes loss of neutrophil binding activity in fully cytotoxic Shiga toxin 1

Abstract

Shiga toxins (Stx) play an important role in the pathogenesis of hemolytic uremic syndrome, a life-threatening renal sequela of human intestinal infection caused by specific Escherichia coli strains. Stx target a restricted subset of human endothelial cells that possess the globotriaosylceramide receptor, like that in renal glomeruli. The toxins, composed of five B chains and a single enzymatic A chain, by removing adenines from ribosomes and DNA, trigger apoptosis and the production of pro-inflammatory cytokines in target cells. Because bacteria are confined to the gut, the toxins move to the kidney through the circulation. Polymorphonuclear leukocytes (PMN) have been indicated as the carriers that "piggyback" shuttle toxins to the kidney. However, there is no consensus on this topic, because not all laboratories have been able to reproduce the Stx/PMN interaction. Here, we demonstrate that conformational changes of Shiga toxin 1, with reduction of α-helix content and exposition to solvent of hydrophobic tryptophan residues, cause a loss of PMN binding activity. The partially unfolded toxin was found to express both enzymatic and globotriaosylceramide binding activities being fully active in intoxicating human endothelial cells; this suggests the presence of a distinct PMN-binding domain. By reviewing functional and structural data, we suggest that A chain moieties close to Trp-203 are recognized by PMN. Our findings could help explain the conflicting results regarding Stx/PMN interactions, especially as the groups reporting positive results obtained Stx by single-step affinity chromatography, which could have preserved the correct folding of Stx with respect to more complicated multi-step purification methods.

FIGURE 1.

Binding of Stx1 to PMN. A, representative single histogram analysis illustrating binding of Stx1 to mature human PMN assessed by indirect flow cytometric analysis. B, representative single histogram analysis illustrating binding of Alexa Fluor® 488-Stx1 to mature human PMN assessed by direct flow cytometric analysis. Black, none; white, Stx1-batch 8; gray, Stx1-batch 9.

FIGURE 2.

Effect of Stx1 on protein synthesis by HUVEC. Human endothelial cells were challenged with different concentrations of Stx1-batch 8 (upper panel) or Stx1-batch 9 (lower panel) (n = 3). IC₅₀ was calculated by the linear regression between fractional activity and the log of toxin concentrations.

FIGURE 3.

SDS-PAGE analysis of Stx1. Left panel, lane 1, Stx1-batch 8 (1 μg). Right panel, lane 1, molecular mass markers (112 ng/protein): bovine α-lactalbumin (14 kDa), bovine trypsinogen (24 kDa), bovine carbonic anhydrase (29 kDa), rabbit glyceraldehyde-3-phosphate (36 kDa); lane 2, Stx1-batch 9 (1.15 μg). After electrophoresis, the gels were silver-stained. R_f = distance of protein migration/distance of dye migration. The equations (Stx1-batch 8, y = 1.0136× + 4.949, r = −0.99; Stx1-batch 9, y = 0.7887× + 3.8461, r = −0.99) were obtained by plotting the mobility (R_f) of calibration proteins (see Fig. 3) versus log kDa. The R_f of A and B subunits of each batch allowed calculation of the molecular masses reported under “Results.”

FIGURE 4.

Conformation of Stx1 examined by circular dichroism. The ellipticity of Stx1 (1 μm) in PBS at 22 °C in a 0.1-cm cell is shown. a, Stx1-batch 8; b, Stx1-batch 9.

FIGURE 5.

Intrinsic fluorescence of Stx1. Fluorescence spectra of Stx1 (1 μm) in PBS at 22 °C are shown. a, Stx1-batch 8; b, Stx1-batch 9. Excitation was at 295 nm.

FIGURE 6.

Ribbon diagram of the crystal structure of Shiga toxin. This figure was reproduced with permission from Ref. , with modifications. The positions of the tryptophan residues (Trp-34, Trp-203, and Trp-277) are indicated. White, A subunit; gray scale, B subunits.