Fucose-Galactose Polymers Inhibit Cholera Toxin Binding to Fucosylated Structures and Galactose-Dependent Intoxication of Human Enteroids

Abstract

A promising strategy to limit cholera severity involves blockers mimicking the canonical cholera toxin ligand (CT) ganglioside GM1. However, to date the efficacies of most of these blockers have been evaluated in noncellular systems that lack ligands other than GM1. Importantly, the CT B subunit (CTB) has a noncanonical site that binds fucosylated structures, which in contrast to GM1 are highly expressed in the human intestine. Here we evaluate the capacity of norbornene polymers displaying galactose and/or fucose to block CTB binding to immobilized protein-linked glycan structures and also to primary human and murine small intestine epithelial cells (SI ECs). We show that the binding of CTB to human SI ECs is largely dependent on the noncanonical binding site, and interference with the canonical site has a limited effect while the opposite is observed with murine SI ECs. The galactose−fucose polymer blocks binding to fucosylated glycans but not to GM1. However, the preincubation of CT with the galactose−fucose polymer only partially blocks toxic effects on cultured human enteroid cells, while preincubation with GM1 completely blocks CT-mediated secretion. Our results support a model whereby the binding of fucose to the noncanonical site places CT in close proximity to scarcely expressed galactose receptors such as GM1 to enable binding via the canonical site leading to CT internalization and intoxication. Our finding also highlights the importance of complementing CTB binding studies with functional intoxication studies when assessing the efficacy inhibitors of CT.

Research in a time of enteroids and organoids: how the human gut model has transformed the study of enteric bacterial pathogens

“…Cervin et al designed polymers to target the fucosylated non-canonical binding site, finding they blocked the binding of cholera toxin to 2D jejunal enteroid monolayers; however, polymers designed to target both the canonical and non-canonical binding sites of cholera toxin were observed to block binding, intoxication, and ion secretion. 50 Using human enteroids as a tool for inhibitor potency assays surpasses animal models in the ability to model human-specific cell surface glycosylation patterns.…”

The Inhibitory Effect of Cholera Toxin B Subunit on <i>Clostridium Perfringens</i> Iota-Toxin-Induced Cytotoxicity

“…GM1 binds to the primary binding site of CTB, but the latest reports indicate that glycosylated receptors are crucial for the secondary binding site of CTB. [22][23][24][25] The primary binding site on CTB is distinct from the secondary binding site, 23,24) with the latter being situated closer to the A subunit binding site than the former. CTB is reported to bind to histo-blood group antigens (HBGAs) at a secondary binding site.…”

Synthesis and Self-Assembling Properties of Peracetylated β-1-Triazolyl Alkyl D-Glucosides and D-Galactosides

“…This compound was then treated with NaN 3 to obtain the desires azide intermediate 3 as white solid [41]. Similar procedure was followed for the synthesis of other glucose azide with three carbon spacer and galactose azides with 2 and 3 carbon spacers [42][43][44][45].…”