Cystic fibrosis (CF) is the most common inheritable lethal disorder in Caucasians. The main cause of high morbidity and mortality in CF are the recurring Pseudomonas aeruginosa infections and associated inflammation. A clear connection between the genetic lesion in CF and Pseudomonas infection has not been unequivocally established. CF is caused by mutations in the CFTR gene, which encodes a chloride channel that has pleiotropic affects on transport of other ions in epithelial cells. Using a novel pH-sensitive GFP technology, we have recently reported that trans-Golgi network (TGN) is hyperacidified in CF respiratory epithelial cells. We hypothesize that dysfunction of this main cellular biosynthetic and sorting organelle in leads to defects in CF respiratory cells contributing to the initiation of bacterial infection. We hypothesize that at least one manifestation of the previously unanticipated lower than normal pH in the TGN of CF cells is the well known glycosylation defect including undersialylation of cell surface glycoconjugates which act as receptors for increased Pseudomonas aeruginosa binding. In addition, we have observed that another intracellular organelle, the cellubrevin-labeled recycling endosome, is also hyperacidified in CF respiratory epithelial cells. We hypothesize that the dysfunctional recycling endosome in CF may affect various events following bacterial adhesion, such as intoxication of host cells and bacterial uptake and elimination by host cells. In addition, a defective endosomal pathway may result in an overabundance, overstimulation, or defective downregulation of proinflammatory receptors on CF epithelial cells.
The aims of this proposal are: 1) To delineate the molecular mechanisms leading to the hyperacidification of TGN and cellubrevin endosomal compartments in CF. 2) To investigate how hyperacidification of TGN in CF affects interactions of respiratory epithelial cells with P. aeruginosa. 3) To investigate how hyperacidification of endosomal compartments in CF influences interactions of respiratory epithelial cells with P. aeruginosa and whether it plays a role in increased inflammation. In addition, as a part of all three aims, we will determine whether normalizing the pH of intracellular compartments in CF corrects interactions with P. aeruginosa and suppress inflammation. These studies are expected to establish a connection between the CFTR defect and infection and inflammation in CF, and provide a basis for development of new chemotherapies using appropriately formulated antacids or inhibitors of pumps and ion channels.
