The rise of antibacterial resistance highlights the urgent need to develop new effective strategies to combat antibiotic-resistant infections. Ubiquitously, Gram-negative bacterial pathogens assemble extracellular fibers, termed chaperone-usher pathway (CUP) pili, that are critical for the pathogen's ability to cause infections by recognizing and colonizing different host tissues and habitats. Thus, therapeutics targeting the assembly of these fibers hold promise in their potential to result in much needed alternatives for the treatment of multidrug- resistant Gram-negative pathogens. Among these pathogens are those designated as ?Urgent Threats? carbapenem-resistant Acinetobacter and carbapenem-resistant Enterobacteriaceae (CRE), as well as ?Serious Threats? drug-resistant Campylobacter, extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, multidrug-resistant Pseudomonas aeruginosa, drug-resistant Salmonella, Shigella, and Bordetella pertussis. In each CUP pilus system, a designated periplasmic chaperone and an outer-membrane (OM) usher protein work together to assemble thousands of structural subunits into each final pilus structure. Most CUP pili are also tipped by adhesins that specifically recognize receptors in host tissues. We have made considerable progress towards understanding the remarkably complex mechanisms of pilus assembly. Building on our extensive experience and expertise in CUP pilus biogenesis and in the development of rational therapies targeting CUP pili, this proposal seeks to develop novel antibiotic-sparing therapies targeting the OM ushers using multidisciplinary approaches including bacteriology, chemical biology, medicinal chemistry, structural biology and immunology. Based on the structural characterizations and the dynamic nature of these multi-domain usher proteins, we will rationally develop small molecule usher inhibitors and pore openers by trapping specific conformational states (Aim 1). Usher inhibitors will disarm bacterial virulence factors, whereas pore openers will increase permeability of existing antibiotics into bacterial outer membranes. In addition, we will develop monoclonal antibodies that inactivate usher, thus preventing pilus biogenesis and infection (Aim 2). While our first two aims will concentrate on two of the most studied pilus systems (type 1 and P pili), Aim 3 will expand our studies of ushers in Acinetobacter, Campylobacter, P. aeruginosa, Salmonella, Shigella, and B. pertussis. Collectively, we plan to develop rational therapies against multiple antibiotic-resistant Gram-negative bacterial pathogens. These developments, together with other novel strategies proposed in our multidisciplinary U19 program, will work synergistically to act as efficient antibiotic-sparing therapeutics by blocking usher and adhesin functions. Moreover, the usher pore openers developed in this proposal will increase OM permeability, further alleviating antibiotic resistance in Gram-negative pathogens and allowing us to repurpose existing drugs to enhance the current antibiotic arsenal. Thus, successful developments in these directions will be potentially transformative in combating antibiotic resistance.