Characterization of Bacterial Lectin-Carbohydrate Binding and Development of Anti-Adhesion Inhibitors

University Of North Carolina Asheville, Asheville NC

Investigators

Abstract

PROJECT SUMMARY Lectins are carbohydrate-binding proteins that mediate a range of biological processes including bacterial adhesion and biofilm formation. Bacterial lectins involved in adhesion, termed adhesins, are one type of virulence factor pathogens use to infect and persist in a host. As rates of antibiotic resistance continue to rise, new strategies are needed to treat infection, including the use of anti-virulence therapies, which target bacterial virulence factors such as adhesins, to weaken resistance and facilitate treatment and clearance. This strategy is proving to be successful for a few lectins in uropathogenic Escherichia coli and Pseudomonas aeruginosa, but no effective inhibitors of adhesins in enterotoxigenic E. coli or Helicobacter pylori have been developed. This is in part due to a lack of characterization of the binding sites and structural basis for recognition in these lectins, including the F17G adhesin on enterotoxigenic E. coli which binds N-acetylglucosamine (GlcNAc) and the SabA adhesin on H. pylori which binds sialic acid and sialyl Lewis x. There is also incomplete structural data regarding the carbohydrate binding site of SabA - no crystal structure has been reported in the presence of a ligand. This project will engage undergraduate researchers in the characterization of the molecular basis for F17G and SabA binding and adhesion and the synthesis of high-affinity ligands. In Aim 1, F17G ligands will be rationally designed, harnessing structural information from molecular docking screens and protein X-ray crystal structure analysis. Small libraries of GlcNAc derivatives will be synthesized, making modifications at both the anomeric position and the amide position on the carbohydrate aimed at increasing hydrogen bonding, hydrophobic, and π-stacking interactions. Ligands will be tested in competitive binding assays using enzyme-linked immunosorbent assays (ELISA) and surface plasmon resonance spectroscopy (SPR). Effective compounds will lead to powerful probes and inhibitors of E. coli adhesion and infection. In Aim 2, the binding site of the H. pylori adhesin SabA will be interrogated using site-directed mutagenesis, X-ray crystallography, and binding assays. These experiments will lead to a clear understanding of the amino acid residues involved in binding, the first crystal structure with SabA bound to a ligand, and structural insight toward development of H. pylori adhesion inhibitors and probes. Together, the work proposed will provide structure-activity relationships and a detailed molecular basis for ligand binding of two bacterial lectins. This foundational data will lead to the development of potent lectin inhibitors which can be used to interrupt bacterial adhesion as anti-virulence treatments. The structural information generated from this work will also provide the basis for the manipulation of lectins for use as probes of lectin function and glycan structure.