Fig. 3.

RT027 C. difficile TcdB does not interact with FZD receptors but still induces stem cell death and dysfunction. (A, i) An alignment of the FZD binding domain of TcdB₁₀₄₆₃ and TcdB₀₂₇ with TcdB₁₀₄₆₃ contact residues (blue) and nonconserved TcdB₀₂₇ residues (red). (ii) MST binding curves of TcdB₁₀₄₆₃ (black), TcdB₀₂₇ (blue), and TcdB₁₀₄₆₃^GFE (red) to FZD2-CRD. The residuals with regard to the fit of the binding curve are depicted at the bottom (n = 3). ΔF_n (‰) is the change in normalized fluorescence (parts per thousand). ND, not determined. (iii) TOPFlash assay performed in HEK293 STF cells incubated with 1:5 molar ratio of WNT3a and TcdB (n = 4). (iv) Mice were given 50 µg of TcdB intrarectally and were killed after 4 h postintoxication. The descending colon from each mouse was collected and stained with hematoxylin/eosin (H&E) (n = 5). (B) Equal numbers of colonic crypts were isolated from uninfected mice and then exposed to toxin, with or without recombinant receptor prior to organoid seeding. (i) Representative images of organoids (arrowheads) cultured from crypts incubated for 4 h with 5 nM TcdB₁₀₄₆₃ or 100 nM TcdB_027. Blocking was conducted with 50 nM or 1,000 nM, respectively, of either recombinant LGR5, FZD2, FZD7, CSPG4, or NECTIN-3 (ii) with cell viability assessed via a PrestoBlue assay. n ≥ 3. Data are represented as mean + SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (Scale bars: 500 µm.) See also SI Appendix, Figs. S4 and S5.