Unveiling Cholera Toxin Binding and Intoxication using Enteroids and Site-Specific Mutants

Abstract

The diarrheal disease of cholera affects millions each year, causing over 100 000 deaths. This is caused by the bacteria Vibrio cholerae which produces the highly efficient Cholera toxin (CT) that exerts its function in the small intestine of humans. CT has long been considered to intoxicate using the glycolipid GM1 due to its extreme binding strength, but in the past 10 years an alternative binding site has been visualized to bind glycoproteins. Through subsequent findings, it has been shown that blocking either binding site can ablate the effects of CT on human tissue. I sought to expand upon these findings for my thesis, using human enteroids as a primary model. We also aimed to investigate the CT binding ligands in humans that can be functional, in parallel to understanding the binding site these are facilitated through. Finally, we investigated the effects of the passive defense of mucins on how they might affect CT binding and internalization. Initial work in this thesis focused on blocking CT binding using inexpensive simple polymers, where we show that a combined Fucose/Galactose polymer could both block CT binding and partially prevent CT intoxication, while a GM1 oligosaccharide could completely prevent CT intoxication. Using inhibitors in combination with CT B-subunit (CTB) binding deficient mutants, we found that fucose plays a dual role as both a component of binding decoy structures for CT and on functional ligands. Further, we found that O-linked, but not N-linked, glycoproteins play a key role in binding decoy ligands. Using holotoxin binding deficient mutants, we confirmed the requirement of both binding sites for CT to exert the diarrheal response. Inducing MUC17 also provided protection against CT, preventing expressing cells from binding CT and reducing overall internalization. It is evident that the diverse glycosylation in the human small intestine can provide layers of protection from CT, but the specific ligands that can function to facilitate intoxication are yet to be determined.