Cholera toxin is an AB5 toxin secreted by the human pathogen Vibrio cholerae. Cholera toxin binds to, enters, and intoxicates human intestinal epithelial cells, causing the diarrheal disease cholera. Using a metabolically- incorporated photocrosslinking sialic acid analog developed in the prior granting period, we showed that cholera toxin interacts directly with fucosylated glycoproteins displayed on the surface of human colonic epithelial cell lines. Further, host cell surface display of fucose is critical to the ability of cholera toxin to enter and intoxicate host cells. During the upcoming granting period, we will define the fucosylated glycan structures recognized by the B subunit of cholera toxin (CTB). The results of these experiments will provide insight into the molecular basis for human variation in susceptibility to cholera. In addition, information gained in these experiments will be used to design synthetic molecules that can interfere with cholera toxin action and could potentially be used either clinically, either as prophylactics or therapeutics. We will also investigate the relative roles of the two glycan binding pockets on CTB – the canonical GM1 binding site and the novel fucosylated glycan binding site. We will determine which glycans are bound in each site, how the two binding sites contribute to CT mechanism of action, and whether there is any interplay between the glycan binding sites. Finally, we will explore how receptor identity affects the efficiency of CTB internalization, the endocytic pathway for CTB internalization, and the ability of CT to intoxicate host cells. The results of these experiments will reveal fundamental mechanisms of CT action and may provide new insight into the molecular basis of endocytic mechanism.