Vibrio cholerae's Largest Adhesin and How to Block It

Abstract

Numerous species of pathogenic Gram-negative bacteria produce repeats-in-toxin adhesins to bind to host cells and subsequently form biofilms during a bacterial infection. These long, membrane-anchored proteins typically contain four functional regions: A small N-terminal membrane-retention module, a long repetitive extender region, followed by a ligand-binding region, and a non-cleavable C-terminal secretion signal. Flagellum-regulated hemagglutinin A protein is a repeats-in-toxin adhesin of Vibrio cholerae, the main causative agent of cholera disease. Previous gene knockout studies showed the adhesin is required to bind to chitin, erythrocytes, epithelial cells, and to form biofilms. Using bioinformatic approaches, a peptide binding domain and sugar-binding domain were identified adjacent to a domain of unknown function. The objective of this thesis project was to determine the structures and functions of the sugar-binding and unknown domains. A recombinant 42-kDa construct spanning the sugar-binding domain and unknown domain was expressed, purified, and probed by glycan micro-array screening, which showed the two-domain protein favours binding mammalian fucosylated glycans, some of which are characteristic of red blood cell markers and intestinal cell epitopes. Isothermal titration calorimetry further showed the construct can bind soluble L-fucose with a 0.7:1.0 stoichiometry and an affinity of approximately 21 µM. This recombinant protein construct lysed red blood cells on binding to their membranes. Both membrane binding and hemolysis were blocked by the addition of soluble L-fucose. Using AlphaFold, the domain boundaries of both domains were shown to be slightly shorter than previously predicted, and the unknown domain was recognized as a split Ig-like domain that can assist in projecting the ligand-binding domain towards its target. The AlphaFold-predicted structure fits well within the molecular envelope structure iii obtained from BioSAXS. A 40-kDa protein orthologue construct from Aeromonas veronii was also expressed and purified. It appeared more stable than the V. cholerae protein, had a 1:1 stoichiometry in L-fucose binding, and was predicted to fold to the same way as the V. cholerae two-domain construct. By studying the sugar-binding domain architecture and identifying glycan ligands, inhibitors targeting the adhesin can potentially be developed to block or disrupt infection by V. cholerae, as well as by other pathogenic bacteria with similar adhesins.