Fig. 4. C. difficile spore internalization via fibronectin-α₅β₁ and vitronectin-α_vβ₁ integrins intestinal epithelial cells.

C. difficile spore internalization in a–d differentiated Caco-2 cells incubated for 1 h with 1, 3, or 5 µg mL⁻¹ of RGD peptide and infected with C. difficile spores preincubated for 1 h with a, c, Fn and b, d, Vn. e–h Differentiated Caco-2 monolayers were incubated for 1 h with antibodies against α_v, α_2, α₅, β₁, β₃, nonimmune IgG antibody or without antibody. They were then infected with C. difficile spores R20291 preincubated with e, f, Fn or g, h, and Vn. i–k Immunoblotting of cell lysates of CHO cells transfected with ectopic expression of i α₅ (~120 kDa); j α_v (~120 kDa); and k β₁ (~120 kDa) and alpha tubulin as a loading control (50 kDa). C. difficile spore l, n internalization or m, o adherence in CHO cells ectopically expressing α_v, α₅, β₁ integrins, of spores pretreated 1 h with l, o Fn or n, o of Vn. Data shown in each panel are normalized to the control. In bars, each dot represents one independent well from three independent experiments. Error bars indicate mean ± S.E.M. Statistical analysis was performed by two-tailed unpaired Student’s t test, ns, p > 0.05; **p < 0.01; ****p < 0.0001.