Fig. 2: C. difficile spores are internalized by intestinal epithelial cells.

a Representative confocal micrograph of an internalized C. difficile 630 spores in T84 cells. C. difficile spores are shown in red, F-actin is shown in green (fluorophores colors were digitally reassigned for a better representation). The images were acquired from n = 8 fields from two independent experiments. Yellow lines indicate an internalized spore. b–e TEM of differentiated monolayers of T84 cells infected with C. difficile 630 spores. Black and white arrows denote extracellular and intracellular C. difficile spores, respectively. c, d Magnifications of black squares of b. e An adhered C. difficile spore and an apical membrane extension of T84 cells surrounding C. difficile spores. f–h TEM of differentiated monolayers of Caco-2 cells infected with C. difficile R20291 spores. White arrows in f indicate internalized C. difficile spores. g, h Magnifications of black boxes in f. Micrographs are representative of n = 3 independent experiments. Internalization of C. difficile spores i strain 630 and, j R20291 preincubated with FBS or culture media in undifferentiated (2 days) and differentiated (8 days) Caco-2, T84, Vero, and HT29. k Internalization of C. difficile spores R20291 strain preincubated with serum of different mammalian species in Caco-2 cells. i–k Representative of n = 3 independent experiments. Error bars indicate the mean ± S.E.M. Statistical analysis was performed by two-tailed unpaired Student’s t test, ns, p > 0.05. Scale bars a 5 μm; c, d, 100 nm; e, 1 μm; f, 2 μm; g, 200 nm; h, 500 nm.