The type III secretion system (T3SS) is a bacterial appendage required by dozens of pathogens to cause disease, including Salmonella, EPEC, Shigella, and Yersinia. Collectively, these pathogens cause over 200 million cases of human illness and well over half a million deaths per year. Yet these and other bacterial pathogens are developing resistance to currently available antibiotics at an alarming rate. As only two new classes of antibiotics have reached the market since 1962, new therapeutics are critically needed to support modern medicine for future generations. The T3SS is well-conserved across a large number of bacterial species yet is specifically expressed by pathogens. This makes the T3SS an optimal target for antimicrobial agents that can hinder the pathogenesis of T3SS-expressing pathogens without causing large disruptions to the microbiota. While T3SS inhibitors have been identified, few have known molecular targets and none have reached the clinic. The function of the T3SS is to inject bacterial effector proteins inside target host cells. Once inside host cells, these effector proteins manipulate normal host cell processes, to the benefit of the pathogen. We have devised a novel strategy for identifying small molecule inhibitors that can disrupt the ability of the T3SS to functionally interact with host cells. This assay relies on the ability of mammalian cells to activate the host transcription factor NF?B in response to a functional T3SS, letting host cells serve as an indicator of whether a small molecule can inhibit T3SS function. In Aim 1, we will exploit this NF?B activation assay to develop a high throughput screen (HTS) to identify T3SS inhibitors, using Yersinia pseudotuberculosis as a model T3SS-expressing pathogen. To achieve this, we will generate a stably-transfected NF?B luciferase reporter cell line that can support a robust and reproducible HTS. In addition, we will develop a rapid secondary screen to eliminate compounds that inhibit the NF?B pathway, allowing us to focus our efforts on compounds that specifically target the T3SS. In Aim 2, we will develop and vet an experimental pipeline that (i) will serve to validate the T3SS inhibitory activity of lead compounds and (ii) will allow rapid identification of the stage of type III secretion targeted by lead compounds,-i.e., T3SS assembly, T3SS pore formation on host membranes, T3SS translocation of effector proteins inside host cells, and activity of T3SS effector proteins inside host cells. Upon completion of this work, we will carry out the HTS screen and experimental pipeline described in this proposal, using our in-house natural products and synthetic compound libraries (over 55,000 compounds). In addition, we will submit our screen for Fast Track entry into the NIH Roadmap Molecular Libraries Probe Production Centers Network. This will enable identification of a suite of T3SS inhibitors that target different stages of type III secretion, whch will serve as the foundation for future development of a biochemical toolbox of T3SS inhibitors with known molecular targets that will be used as research probes and scaffolds for novel therapeutics.