PROJECT SUMMARY/ABSTRACT Urinary Tract Infections (UTIs) are common in children, the elderly, diabetics, and immunocompromised patients. UTIs have become difficult to treat due to the rise in multi-drug resistant bacteria such as the extended spectrum beta lactamase (ESBL) producing Escherichia coli and the carbapenem resistant Klebsiella pneumoniae. Therefore, there is an urgent need to develop alternative treatments, for instance, anti-adhesive molecules that block bacteria adhesion to host cells, the all important first step in initiating an infection. Pathogenic bacteria, like E. coli and K. pneumoniae establish infections in the urinary tract by adhering to host epithelial cells via adhesion proteins, such as FimH, FmlH and MrkD. The long-term objective of this proposal is to develop novel methods of inhibiting bacterial adhesion. A monoclonal antibody (mAb) that inhibits FimH-mediated bacterial adhesion has been discovered, but its mechanism of action has not been investigated. Moreover, previous studies show that this mAb is neither a competitive nor an allosteric inhibitor, indicating a novel mechanism of biomolecular inhibition. The aim of this proposal is to elucidate the mechanism by which the mAb inhibits FimH-mediated bacterial adhesion and develop molecules capable of inhibiting FimH function. Aim 1 studies will investigate the mechanism of mAb inhibition by mapping the binding site of the mAb on FimH using Nuclear Magnetic Resonance Spectroscopy (NMR) and Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) and determining the X-ray crystal structure of the FimH-mAb complex. Furthermore, NMR and HDX-MS will provide information on how mAb binding alters the conformational dynamics of FimH. These structural and dynamics studies will provide insights into the mAb mechanism of action. In Aim 2, peptides of the mAb that interact with FimH will be identified using HDX- MS and tested for binding and inhibitory function in bacterial adhesion assays. The binding and inhibitory function of the mAb peptides will be optimized using Deep Mutational Scanning (DMS) and Rosetta computational peptide design. In Aim 3, the structures, and dynamics of FmlH and MrkD will be investigated, and peptide inhibitors will be developed. Mutagenesis and NMR studies will be carried out to investigate the different structural conformations and dynamics of FmlH and MrkD. Finally, computational peptide design using Rosetta will be employed in the de novo design of peptide inhibitors against FmlH and MrkD.