Comparisons of native Shiga toxins (Stxs) type 1 and 2 with chimeric toxins indicate that the source of the binding subunit dictates degree of toxicity

Abstract

Shiga toxin (Stx)-producing E. coli (STEC) cause food-borne outbreaks of hemorrhagic colitis. The main virulence factor expressed by STEC, Stx, is an AB5 toxin that has two antigenically distinct forms, Stx1a and Stx2a. Although Stx1a and Stx2a bind to the same receptor, globotriaosylceramide (Gb3), Stx2a is more potent than Stx1a in mice, whereas Stx1a is more cytotoxic than Stx2a in cell culture. In this study, we used chimeric toxins to ask what the relative contribution of individual Stx subunits is to the differential toxicity of Stx1a and Stx2a in vitro and in vivo. Chimeric stx1/stx2 operons were generated by PCR such that the coding regions for the A2 and B subunits of one toxin were combined with the coding region for the A1 subunit of the heterologous toxin. The toxicities of purified Stx1a, Stx2a, and the chimeric Stxs were determined on Vero and HCT-8 cell lines, while polarized HCT-8 cell monolayers grown on permeable supports were used to follow toxin translocation. In all in vitro assays, the activity of the chimeric toxin correlated with that of the parental toxin from which the B subunit originated. The origin of the native B subunit also dictated the 50% lethal dose of toxin after intraperitoneal intoxication of mice; however, the chimeric Stxs exhibited reduced oral toxicity and pH stability compared to Stx1a and Stx2a. Taken together, these data support the hypothesis that the differential toxicity of the chimeric toxins for cells and mice is determined by the origin of the B subunit.

Figure 1. Native and chimeric Stx operon structure and activities.

(A) Illustration that depicts the origin of the individual subunits, A₁, A₂, or B, in the native and chimeric operons. Stx1a is in black, while Stx2a is in white. The chimerics are named such that the number represents the native toxin from which that subunit originated. (B) Specific activities of the toxin panel after intoxication of Vero and HCT-8 cells. Geometric mean of representative of seven biological replicates.

Figure 2. Binding of toxin panel to Gb3 as measured by ELISA.

All four toxins bound Gb3; however, Stx1a and 211 bound Gb3 with a significantly higher affinity than Stx2a or 122 (p<0.01). Additionally, when toxins with identical B subunits were compared, Stx1a vs 211 and Stx2a vs 122, there was no difference in binding (p>0.05). Error bars represent standard error of the mean (SEM); n = four biological replicates. Toxin-Gb3 binding was compared by Two-Way ANOVA with Tukey's adjustment for multiple comparisons.

Figure 3. Stx translocation through a polarized HCT-8 cell monolayer after 20 ng was applied to the apical chamber.

The Stx1a B subunit was associated with statistically greater toxin translocation compared to the Stx2a B subunit. A greater amount of Stx1a and 211 was recovered from the basolateral chamber as compared to Stx2a and 122 at 2.5 and 24(p<0.05). Error bars represent SEM. n = 4 biological replicates

Figure 4. Morbidity and mortality after ig intoxication with chimeric 122.

Ig intoxication of up to 15 μg 122, 5X the Stx2a LD₅₀ of 2.9 μg , did not result in morbidity or mortality. Weight loss was observed after intoxication with 35 and 130 μg 122 (A) and one animal in the 130 μg group succumbed to intoxication on day 4 (B). Error bars represent SEM; 15 μg: n = 10; 35 and 130 μg: n = 5

Figure 5. The percent of active toxin recovered from feces compared to toxin input.

The amount of active toxin detected was divided by the amount administered and multiplied by 100 to determine the percent of active Stx2a (A), 122 (B), Stx1a (C), or 211 (D) recovered from the stool. A significantly greater amount of active Stx2a was recovered than active 122 at 9, 12, 24 hours post intoxication at all concentrations fed (t-Test p<0.05). A significantly greater amount of active Stx1a was recovered than active 211 at three, nine, and 12 hours post intoxication (Students -Test p≤0.05).