PROJECT SUMMARY/ABSTRACT: The climate surrounding COVID-19 has dramatically reminded us of the dire consequences of being unprepared for health crises. One looming health crisis is the surge of antibiotic-resistant bacterial infections that are no longer sensitive to our life saving antibiotic arsenal. Thus, this project is leveraged to translate basic science into antibiotic-sparing medicines for one of the most common bacterial infections in the United States, urinary tract infections (UTIs), as well as the most common hospital-acquired infection, catheter-associated UTIs (CAUTIs). The strategy is to develop therapeutics that work equally well against carbapenem-resistant Enterobacteriaceae (CRE) and extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae, including the cause of over 80% of community-acquired UTIs, uropathogenic Escherichia coli (UPEC). CAUTIs pose a significant challenge for healthcare globally. While UPEC causes 50% of CAUTI, other pathogens, including ESBL and CRE Klebsiella, multidrug resistant (MDR) Acinetobacter and Gram-positive Enterococcus including vancomycin resistant Enterococcus (VRE), cause a significant proportion of CAUTIs. These MDR pathogens express adhesive factors required for colonization and infection in different host habitats involved in acute and chronic/recurrent UTIs as well as in CAUTIs. Small molecules and monoclonal antibodies (mAbs) will be developed that will treat and prevent disease by blocking these critical host-pathogen interactions. By acting extracellularly, these antibiotic-sparing therapeutics will be recalcitrant to intracellular mechanisms of resistance and avoid a common obstacle of cell permeability in drug discovery of small molecules. UPEC, Klebsiella and Acinetobacter express chaperone usher pathway (CUP) pili tipped with adhesins: i) FimH, FmlH, YehD, UclD (UPEC); ii) FimH (Klebsiella) and iii) CupD (Acinetobacter). These adhesins are critical for colonization of the bladder (FimH), inflamed bladder and kidney (FmlH), gut (FimH, UclD and YehD) and catheters (FimH and CupD). Further, Enterococcus faecalis express EbpA-tipped sortase-assembled pili, which are critical in CAUTI. Glycomimetics have shown great promise in neutralizing CUP adhesins in vivo to treat disease. For example, mannosides which neutralize FimH function, are potent therapeutics for treating and preventing UTI, since FimH is required by UPEC to colonize the bladder. Validating the work in this proposal, a mannoside has been selected, in collaboration with GSK, to proceed into phase 1a/ab clinical trials in humans. Also, a FimH vaccine has completed Phase 1a/1b clinical trials. The use of mAbs has revolutionized treatments for cancer, inflammatory, and neuronal disorders. Therapeutic mAbs have not yet been fully harnessed for treating infectious diseases, perhaps due to the historic success of antibiotics. In this project, we will develop therapeutic mAb designed to prevent critical host-pathogen interactions by neutralizing the adhesins described above. The combined strategies have the potential to produce transformative antibiotic-sparing therapeutics that work equally well against and antibiotic-sensitive and resistant infections.