Fig. 4.

ETEC–CEACAM interactions on human small intestinal epithelia. (A) CEACAM6 is expressed on the apical surface of small intestinal enteroids. Arrows point to immunogold labeling of CEACAM6 on tips of microvilli (7,500×). (B) CEACAM6 associated with glycocalyx at tips of microvilli (15,000×). (C) ETEC (red) associates with CEACAM6 (green) on the surface of human small intestinal (Hu135D) epithelial cells (enteroids). Image prepared in Volocity from confocal image z-stacks. (D) Confocal microscopy images of “footprinting” of CEACAM6 (green) at sites of bacterial attachment (red) to small intestinal epithelia. Each panel is flanked by representations of the xz and yz planes (Bottom and Right, respectively). (E) ETEC induction of cAMP in polarized enteroid small intestinal monolayers. Shown are data for blood A (135D), B (248D), and O (235D). ø, untreated; PDEi, phosphodiesterase inhibition. (F) LT mediates up-regulation of CEACAM6 in small intestinal epithelia. Shown are experimental replicate data (n = 3) from control cells or cells treated with LT. (Inset) Immunoblot compares CEACAM6 protein expression in treated and untreated cells. (G) Heat-labile toxin promotes adhesion to small intestinal enteroids (shown are three independent experimental replicates, each with five to seven technical replicates, using enteroids derived from a donor belonging to a different ABO blood group). P values reflect nonparametric comparisons by Mann–Whitney U test. (H) CEACAM6 is required for optimal adhesion to small intestinal enteroids [**P = 0.0067 by ANOVA (Kruskal–Wallis) comparison of untreated (ø) or treatment with scramble RNA (sc) and CEACAM6 siRNA]. (Inset) Immunoblot demonstrates CEACAM6 production (top band) in enteroids by the ø, sc, si groups relative to tubulin (bottom band). (I) Human small intestinal biopsies demonstrating CEACAM6 expression on day 2, day 7, and day 30 post infection with ETEC. Control specimen is from the same patient (at day 7) processed without primary anti-CEACAM6 antibody.