FIG. 4.

C. rodentium directly infects colonic goblet cells in vivo. (A) Immunofluorescence staining for the C. rodentium translocated effector Tir (red), goblet cell-specific Muc2 (green), and DNA (blue) in distal colonic tissues at day 6 p.i. Tir staining is present in Muc2-positive cells (arrowheads) and Muc2-negative cells within the surface epithelium, progressing down the length of some crypts (arrow). (B) Magnified image of Tir (red) staining (arrows) on the apical surface of cells staining strongly for Muc2 (green) in the apical compartment and exhibiting distinct goblet cell morphology. GA, goblet cell apical compartment; GN, goblet cell nucleus; E, enterocyte; L, lumen. Original magnification, ×1,000. (C to E) TEM micrographs of distal colons of C57BL/6 mice taken at day 7 (C) and day 11 (D) following infection with wild-type C. rodentium and at day 7 following infection with ΔescN C. rodentium (E). C. rodentium is in direct contact with goblet cells in panels C and D (black arrows). In panel C, effacement of microvilli (black arrows) on a goblet cell and internalization of bacteria into the apical granule mass of the goblet cell (white arrow) can be seen. In panel E, no infection of goblet cells and intact microvilli (arrow) can be seen following infection with ΔescN C. rodentium. (F) Graph showing the proportions of Muc2-positive (goblet) cells and Muc2-negative (nongoblet) cells of the surface epithelia that were positive for Tir staining at day 6 p.i. The bars indicate the averages for three mice from two independent infections. The error bars indicate the standard errors of the means. Asterisk, P < 0.05, as determined by Student's t test.