Crystal structures of cholera toxin in complex with fucosylated receptors point to importance of secondary binding site

Joel B. Heim,Julie E. Heggelund,Vesna Hodnik,Gregor Anderluh,Ute Krengel

doi:10.1038/s41598-019-48579-2

Joel B. Heim, Julie E. Heggelund + Show 3 more

Open Access

https://doi.org/10.1038/s41598-019-48579-2

Copy DOI

Abstract

Cholera is a life-threatening diarrhoeal disease caused by the human pathogen Vibrio cholerae. Infection occurs after ingestion of the bacteria, which colonize the human small intestine and secrete their major virulence factor – the cholera toxin (CT). The GM1 ganglioside is considered the primary receptor of the CT, but recent studies suggest that also fucosylated receptors such as histo-blood group antigens are important for cellular uptake and toxicity. Recently, a special focus has been on the histo-blood group antigen Lewisx (Lex), however, where and how the CT binds to Lex remains unclear. Here we report the high-resolution crystal structure (1.5 Å) of the receptor-binding B-subunits of the CT bound to the Lex trisaccharide, and complementary quantitative binding data for CT holotoxins. Lex, and also l-fucose alone, bind to the secondary binding site of the toxin, distinct from the GM1 binding site. In contrast, fucosyl-GM1 mainly binds to the primary binding site due to high-affinity interactions of its GM1 core. Lex is the first histo-blood group antigen of non-secretor phenotype structurally investigated in complex with CT. Together with the quantitative binding data, this allows unique insight into why individuals with non-secretor phenotype are more prone to severe cholera than so-called ‘secretors’.

Highlights

Cholera is an acute and severe diarrhoeal disease that, if left untreated, can cause serious dehydration and death within hours[1]
Lex and l-fucose occupied the secondary binding sites of the cholera toxin (CT), while we found fucosyl-GM1os in the primary toxin binding site, binding the CT with the same two-fingered grip as GM1
We found that the CT secondary binding site variant H18A exhibits increased GM1os binding

Summary

Introduction

Cholera is an acute and severe diarrhoeal disease that, if left untreated, can cause serious dehydration and death within hours[1]. Blood group H determinants, which are characteristic for blood group O, exhibit slightly higher affinity to CT than blood group A and B determinants (still in the millimolar range); and patients with blood group O are the most likely to get severe cholera symptoms[35,36,37,38] Another group with increased risk of severe cholera are people with non-secretor phenotype. Lex, a monofucosylated HBGA expressed on human granulocytes and intestinal cells, has been shown to bind to CTB and was proposed to function as a cellular receptor[32]. Cholera intoxication in mice can be independent of GM132, and fucose-based inhibitors are potent inhibitors of CT binding and internalization of intestinal cells[33,34] It is, not clear how the CT binds to Lex and related structures. We find this unlikely since all CT-related crystal structures with HBGAs or HBGA-analogues to date feature these ligands in the secondary binding site[21,25,26]

Methods

Results

Discussion

Conclusion