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.
