Structural characteristics of the plasmid-encoded toxin from enteroaggregative Escherichia coli

Abstract

Intoxication by the plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli requires toxin translocation from the endoplasmic reticulum (ER) to the cytosol. This event involves the quality control system of ER-associated degradation (ERAD), but the molecular details of the process are poorly characterized. For many structurally distinct AB-type toxins, ERAD-mediated translocation is triggered by the spontaneous unfolding of a thermally unstable A chain. Here we show that Pet, a non-AB toxin, engages ERAD by a different mechanism that does not involve thermal unfolding. Circular dichroism and fluorescence spectroscopy measurements demonstrated that Pet maintains most of its secondary and tertiary structural features at 37 degrees C, with significant thermal unfolding only occurring at temperatures >or=50 degrees C. Fluorescence quenching experiments detected the partial solvent exposure of Pet aromatic amino acid residues at 37 degrees C, and a cell-based assay suggested that these changes could activate an ERAD-related event known as the unfolded protein response. We also found that HEp-2 cells were resistant to Pet intoxication when incubated with glycerol, a protein stabilizer. Altogether, our data are consistent with a model in which ERAD activity is triggered by a subtle structural destabilization of Pet and the exposure of Pet hydrophobic residues at physiological temperature. This was further supported by computer modeling analysis, which identified a surface-exposed hydrophobic loop among other accessible nonpolar residues in Pet. From our data it appears that Pet can promote its ERAD-mediated translocation into the cytosol by a distinct mechanism involving partial exposure of hydrophobic residues rather than the substantial unfolding observed for certain AB toxins.

Figure 1

Heat inactivation of Pet. Pet was heated for 10 min at 60°C, 50°C, or 25°C. Either toxin-free media or Pet-containing media (40 μg/ml) were then added to CHO cells for 10 h at 37°C.

Figure 2

Thermal stability of Pet secondary structure. (A) Far-UV CD spectra of Pet were measured during a stepwise increase in temperature. The change in color from blue to red corresponds to a change in temperature from 18°C to 60°C. (B) Alterations to the secondary structure of Pet ([θ]₂₁₇) were plotted as a function of temperature (filled circles). Also shown are the [θ]₂₁₇ signals for a Pet sample that was cooled to 18°C after heating to 60°C (open circles). The overall temperature dependence of [θ]₂₁₇ is fitted with two simulated curves using transition midpoint values of T_m = 37°C (blue curve) and T_m = 55°C (red curve).

Figure 3

Thermal stability of Pet tertiary structure. (A) The fluorescence spectra of Pet were measured during a stepwise increase in temperature. The change in color from blue to red corresponds to a change in temperature from 18°C to 60°C. Temperature-dependent alterations to the fluorescence intensity are shown in the inset. The initial gradual decrease in fluorescence intensity is plotted as a dashed line, and the temperature-induced sigmoidal change in the protein tertiary structure is plotted as a solid line using T_m = 46°C. (B) Alterations to the λ_max of Pet tryptophan fluorescence were plotted as a function of temperature. The circles are the data points and the solid line was simulated using T_m = 50°C. (C) Near-UV CD spectra of Pet were measured during a stepwise increase in temperature from 18°C to 60°C. Select spectra (18°C, 24°C, 37°C, 46°C, and 60°C) are shown for clarity; the colors correspond to the temperature coloring scheme used for presentation of other CD and fluorescence data. (D) Alterations to the near-UV CD bands at 250, 270, and 280 nm are plotted as functions of temperature.

Figure 4

Irreversible disordering of Pet tertiary structure. The fluorescence spectra of Pet were measured at 37°C after cooling from the indicated temperatures. One of two experiments is shown in (A); the average ± range of both experiments is shown in (B).

Figure 5

Effect of glycerol on Pet toxicity. After a 2 h exposure to 7% glycerol and/or 37 μg Pet/ml, HEp-2 cells were fixed and exposed to rhodamine-phalloidin, anti-α-fodrin antibodies, and anti-Pet antibodies. Fluorescein-labeled secondary antibodies were used to visualize α-fodrin, while CY5-labeled secondary antibodies were used to visualize Pet. (A-D) Cells were incubated with glycerol in the absence of Pet. (E-H) Cells were incubated with Pet in the absence of glycerol. (I-L) Cells were incubated with both glycerol and Pet. Colocalization of α-fodrin and Pet is indicated by the aquamarine color in the merged images.

Figure 6

Quenching of Pet tryptophan fluorescence. Fluorescence spectra were measured at each indicated temperature with two parallel samples of Pet: one in which an increasing volume of acrylamide was added to the sample (F), and another in which an increasing volume of buffer was added to the sample (F_o). The ratio of the maximum emission wavelength for each matched set of samples (F_o/F) is plotted as function of acyrlamide concentration. For the inset, K_SV values are plotted as functions of temperature.

Figure 7

Pet protease sensitivity. Pet or the reduced CTA1/CTA2 heterodimer was incubated for 1 h at the indicated temperatures in the absence or presence of α-fodrin. Toxin samples were then shifted to 4°C and exposed to thermolysin for 1 h before resolution by SDS-PAGE with Coomassie staining.

Figure 8

Pet induction of the UPR. A luciferase-based reporter assay was used to monitor UPR activation in CHO cells exposed to Pet for 10 h or to 200 nM thapsigargin (Tg) for 2 h. To calculate the extent of UPR induction, values from the experimental conditions were divided by the control value from untreated cells. The means ± standard errors of the means from 3-5 independent experiments per condition are shown.

Figure 9

Surface-exposed hydrophobic residues of Pet. A space-filling diagram highlights the Pet tryptophan residues in green and the thermolysin-susceptible hydrophobic amino acid residues in yellow. The space-filling diagram was rotated on a 20° axis to distinguish the catalytic domain from the β-helix domain in the presented ribbon structure. For both space-filling and ribbon diagrams, orange space-filling molecules denote the hydrophobic amino acid residues (F₂₉₇, L₃₀₅, L₃₀₈, F₃₀₉, and I₃₁₀) located in the unstructured region of Pet linking the β-helix domain to the catalytic domain. In the ribbon diagram, β-sheets are highlighted in blue and α-helices are highlighted in red.