Abstract

Antibiotic resistance is a rapidly growing concern, and it is becoming increasingly important to develop new ways to treat bacterial infections. Anti-virulence strategies focus on disarming bacteria rather than killing them, putting less selective pressure on the bacteria, and making it less likely bacteria will become resistant to the treatments. One anti-virulence target is the formation of biofilms, which is facilitated by lectins on the surface of bacteria binding to carbohydrates on the host cell. By inhibiting lectin-mediated adhesion, bacteria will not be able to aggregate and bind to host cells, effectively making the bacteria less virulent. In some strains of Escherichia coli, this process involves the bacterial lectins F17G and GafD binding to the carbohydrate N-acetyl-β-D-glucosamine (GlcNAc) on the host epithelial cells. To test GlcNAc derivatives as competitive inhibitors of the GafD lectin, an expression and purification protocol for GafD was performed and optimized. This purification method relies on separating the periplasm from the cytoplasm, as well as using either the expressed 6x-histidine tag on GafD for cobalt (II) affinity, or functional GlcNAc binding affinity for purification. Then, conditions for multiple types of assays were optimized to evaluate binding, including an enzyme-linked immunosorbent competitive binding assay (ELISA) and surface plasmon resonance (SPR). These tools will enable the evaluation of molecules as inhibitors of bacterial adhesion for anti-virulence and increase understanding of the GafD protein and its ligand binding.

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