Pseudomonas aeruginosa (PA) is an opportunistic pathogen and an important cause of disease in humans. Immunocompromised individuals such as those suffering from HIV, burn wounds, cancer chemotherapy, or illnesses that require mechanical ventilators are particularly vulnerable to acute infection. Even with medical treatment, mortality remains high and antibiotic resistance is increasingly common. In addition, PA chronically colonizes patients afflicted with Cystic Fibrosis, leading to severe pulmonary damage and death. Because multi-drug resistant isolates are increasingly reported, identification of novel bacterial therapeutic targets is increasingly important. The long-term goal of this project is to understand how the infection process is initiated at the signaling level in PA so we can develop therapies to prevent the onset of disease. PA initiates infection by using virulence factors such as the type IV pilus (TFP) to attach to host cells and injects the host with toxic effector proteins using the Type III secretion system (T3SS). TFP and T3SS production is controlled by the cyclic AMP synthesis pathway, which is activated during the infection process. The Engel lab found a novel gene, FimL that is important for the production and function of many virulence factors including TFP and the T3SS because it regulates the level of cyclic AMP in the cell. We propose to determine the mechanism by which FimL regulates cyclic AMP levels in the cell. We have discovered that FimL is polarly localized in the bacteria, as are the TFP and T3SS. We propose the hypothesis that polar localization of FimL leads to spatially restricted production of cyclic AMP and downstream components by regulating the localization of the cyclic AMP synthesis protein, CyaB. We will test our hypothesis by 1) identifying interacting partners of FimL by comprehensive and complimentary approaches. We will test if FimL interacts with CyaB and other candidate proteins using co- immunoprecipitation experiments, and in vitro protein assays. We will also perform a genetic screen to find unknown interacting players. 2) We will also determine which factors are required for subcellular localization of FimL and if FimL is required for proper subcellular localization of known virulence factors and regulators, including the TFP and T3SS. FimL and other proteins in this pathway could serve as new and promising therapeutic targets to aid in alleviating human suffering from bacterial infections. PUBLIC HEALTH RELEVANCE: The bacterial pathogen Pseudomonas aeruginosa is the leading cause of hospital-acquired infections and causes the fatal disease Cystic Fibrosis. We propose to elucidate the mechanism of how the bacteria initiates production of disease causing elements when in contact with humans.