Abstract
Cholera toxin (CT) is a secreted bacterial toxin that binds to glycoconjugate receptors on the surface of mammalian cells, enters mammalian cells through endocytic mechanisms and intoxicates mammalian cells by activating cytosolic adenylate cyclase. CT recognizes cell surface receptors through its B subunit (CTB). While the ganglioside GM1 has been historically described as the sole receptor, CTB is also capable of binding to fucosylated glycoconjugates, and fucosylated molecules have been shown to play a functional role in host cell intoxication by CT. Here, we use colonic epithelial and respiratory epithelial cell lines to examine how two types of CT receptors—gangliosides and fucosylated glycoconjugates—contribute to CTB internalization. We show that fucosylated glycoconjugates contribute to CTB binding to and internalization into host cells, even when the ganglioside GM1 is present. The contributions of the two classes of receptors to CTB internalization depend on cell type. Additionally, in a cell line that harbours both classes of receptors, gangliosides dictate the efficiency of CTB internalization. Together, the results lend support to the idea that fucosylated glycoconjugates play a functional role in CTB internalization, and suggest that CT internalization depends on both receptor identity and cell type.
Highlights
Cholera is caused by the pathogenic bacterium Vibrio cholerae [1]
We found that fucose (Fuc) is a key recognition determinant for Cholera toxin (CT) binding to two human intestinal epithelial cell lines (T84 and Colo205): inhibition of fucosylation (using metabolic inhibitor 2-fluoro-peracetyl-fucose (2F-Fuc) [20]) dramatically reduces cholera toxin subunit B (CTB) binding to cells, largely blocks CTB entry into cells and reduces the ability of CT to raise intracellular cAMP levels, a key mechanistic step in host cell intoxication [21]
We used T84 colonic epithelial cells, which are similar to the physiological target cells of CT and contain little endogenous GM1 [21]
Summary
Cholera is caused by the pathogenic bacterium Vibrio cholerae [1]. Vibrio cholerae produces a protein toxin composed of A and B subunits, which form an AB5 complex. Cholera toxin (CT) binds to and invades host intestinal epithelial cells. In the early 1970s, the ganglioside GM1 was identified as a high-affinity binding partner for cholera toxin subunit B (CTB) [2,3]. Structural analysis by X-ray crystallography has revealed the molecular details of CTB recognition of the GM1 glycan [9,10]. Based on this body of data, GM1 has been historically recognized as the sole receptor for CT [11]. Analysis of the glycosphingolipid composition of the normal human small intestinal epithelium, the physiological target for toxin action, revealed a surprisingly small amount of GM1 [12], calling into question the idea that GM1 is the sole functional receptor for CT. B4galnt1-null mice lack GM1 but exhibit a stronger physiological response to CT than wild-type littermates, indicating that host GM1 is not required for CT action in vivo [13]
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