Fig. 3: C. difficile spore internalization requires Fn and Vn which are luminally accessible in the intestinal barrier.

a Internalization b and adherence of C. difficile spores preincubated with NHS in differentiated monolayers of Caco-2 in the presence of RGD peptide. c, d Differentiated Caco-2 cells were infected with C. difficile spores preincubated with DMEM, NHS, Fn, or Vn. Data shown in each panel are normalized to the control. In bars, each dot represents one independent well from a to d three independent experiments. e–g Representative confocal micrographs of fixed whole-mount of the healthy colon of n = 2 mice for e accessible Fn (acc Fn); f accessible Vn (acc Vn); and g accessible Muc2 (acc Muc2) with accessible Ecad (acc Ecad). The main figure shown a 3D projection, below magnifications, and a z-stack of representative cells with different immunostaining. The figures h–n shows the cell repartition of cell immunodetected for h acc Ecad, i acc Fn, j total acc Fn cells that were immunodetected for acc Ecad, k cells immunodetected for acc Vn, l total acc Vn cells that were immunodetected for acc Ecad, m acc Muc2, n total acc Muc2 cells that were immunodetected for acc Ecad. h–n 1000–1200 cells were counted per field in two independent mice. h Four fields per mice, n = 2 mice; i, j two fields per mice, n = 2 mice; k, l one field per mice, n = 2 mice. Acc Fn, acc Vn, and Muc2 are shown in green, acc Ecad is shown in red, and F-actin in gray (fluorophores colors were digitally reassigned for a better representation). Scale bar, 20 µm. Error bars indicate mean ± S.E.M. Statistical analysis was performed by two-tailed unpaired Student’s t test, ns, p > 0.05; ****p < 0.0001. Scale bar, 20 µm.