The long-term objective of our research laboratory is to elucidate the cellular and molecular mechanisms responsible for Pseudomonas aeruginosa (PA) lung infection in cystic fibrosis (CF). In CF, the major cause of death among afflicted patients is airway obstruction due to the presence of a thick and tenacious mucus that becomes heavily infected with PA. Concomitant with PA lung infection, CF patients exhibit chronic airway inflammation characterized by leukocyte infiltration and proinflammatory cytokine production. Because PA is invariably present in both normal and CF lungs, selective infection by this microorganism among CF patients suggests the presence of a mechanism operating in the lungs of diseased patients that is not present in normal airways. To this end, a number of theories have been proposed to account for PA lung infection and chronic inflammation in CF. Our laboratory made the observation that a transmembrane mucin on the apical surface of airway epithelial cells is used as a specific adhesion site for PA through the binding of bacterial flagellin. These studies indicated that ligation of the MUC1 mucin receptor with flagellin led to intracellular signal transduction via phosphorylation of its cytoplasmic region, recruitment of adaptor proteins, and activation of MAP kinase. In the current proposal, we will test the hypothesis that interaction of MUC1 with flagellin results in endocytosis of the receptor-ligand complex that subsequently undergoes transcytosis from the apical to basolateral side of airway epithelial cells.
The specific aims are: (1) to confirm that flagellin binds to MUC1, (2) to show that flagellin-MUC1 complexes undergo endocytosis, and (3) to demonstrate apical to basolateral transcytosis of flagellin. In summary, while binding of flagellin to airway cells leads to signaling through the receptor's intracellular region, transcytosis of its extracellular domain in association with flagellin leads to activation of a proinflammatory response. In addition, this proposal will determine whether or not CF lung cells exhibit aberrant flagellin transcytosis that may account for chronic inflammation. Relevance: This application will address the question of why individuals with CF are predisposed to lung infection by bacteria. Based on previous studies of ourselves and others, we suggest that a major bacterial component (flagellin) is processed differently in CF lungs, leading to enhanced inflammatory responses. ? ? ?
