Abstract
Cholera is the prime example of blood-group-dependent diseases, with individuals of blood group O experiencing the most severe symptoms. The cholera toxin is the main suspect to cause this relationship. We report the high-resolution crystal structures (1.1–1.6 Å) of the native cholera toxin B-pentamer for both classical and El Tor biotypes, in complexes with relevant blood group determinants and a fragment of its primary receptor, the GM1 ganglioside. The blood group A determinant binds in the opposite orientation compared to previously published structures of the cholera toxin, whereas the blood group H determinant, characteristic of blood group O, binds in both orientations. H-determinants bind with higher affinity than A-determinants, as shown by surface plasmon resonance. Together, these findings suggest why blood group O is a risk factor for severe cholera.
Highlights
Cholera is a severe diarrheal disease caused by the pathogen Vibrio cholerae [1]
Blood group determinants bind to the lateral side of the cholera toxin
Five X-ray crystal structures were determined in this study, revealing for the first time the atomic details of blood group antigens bound to the cholera toxin (Fig 2)
Summary
Cloning of the cCTB geneThe nucleotide sequence of the classical CTB gene (Genbank: AAC34728.1; coding for amino acid sequence 22–123) was codon-optimized for expression in Escherichia coli and synthesized by GeneArt (Life Technologies) in a standard pMA-T plasmid. After excision of the gene, it was ligated into a pET-21b(+) vector (Novagen), and transformed by heat shock into competent E. coli BL21 (DE3) cells for protein expression. The cells transformed with the pET-21b(+)-cCTB plasmid were grown in LB medium supplemented with 0.1 mg/ml ampicillin at 37°C until an OD600nm of ~0.5 was reached. The cells were harvested by centrifugation and re-suspended in ice-cold sucrose solution (20 mM Tris pH 8, 25% (w/v) sucrose, 5 mM EDTA), and incubated on ice for 15 minutes. The supernatant was removed by centrifugation at 8500 × g for 20 minutes, and the pellet was re-suspended in periplasmic extraction buffer (5 mM MgCl2, 0.1 mg/ml lysozyme). The periplasmic fraction was separated from the cell debris by centrifugation at 8500 × g for 20 minutes, and dialyzed against PBS. The protein was concentrated and applied to a Superdex size-exclusion chromatography column (GE Healthcare), where the buffer was exchanged to 20 mM Tris pH
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