Project Summary Bacterial Type 3 Secretion System (T3SS) “effector” proteins are the primary virulence factors that guide the progression of numerous Gram-negative bacterial infectious diseases. Recent studies have estimated that a single pathogen delivers up to 250 unique effector proteins directly into host cells. Collectively, these virulence factors suppress host innate immune responses and facilitate bacterial replication, dissemination, and disease progression. Therefore, determining how bacterial effector proteins control host intracellular communication pathways at the structural, biochemical, and cellular level is an ongoing challenge in infectious disease research. This proposal seeks to reveal a structural and functional understanding of these host- pathogen relationships. Prior to this proposal, we identified a class of bacterial E3-ubiquitin ligases that protects the human pathogen Shigella flexneri from the innate immune system activation and execution of bacterial lysis. Here, we will specifically examine the molecular mechanism for bacterial regulation of the newly identified Gasdermin-family of mammalian pore forming cytolysins. This includes determining how Gasdermins function to suppress Shigella flexneri at the molecular and cellular level (Aim 1). We will also examine this host-pathogen interaction at atomic level resolution by solving the effector-Gasdermin structure using X-ray crystallography (Aim 2). The resulting structure-based theories will be tested in murine models of Gasdermin function that are designed to evaluate mucosal immune protection against a broad spectrum of enteric pathogens (Aim 3). Developing new drugs that target bacterial effector – host enzyme complexes would be an innovative approach to combat emerging infectious disease. While this idea holds great potential, the paucity of mechanistic information gleaned from virulence factor structure/function studies has so far hampered their development as suitable drug targets. As a means to this end, these studies will allow us to predict new mechanisms of action for understudied Shigella effector proteins, and provide a glimpse into the structural- based evolutionary progression of a related pathogen groups.