Cystic Fibrosis (CF) is the most lethal genetic disease in Caucasians and is characterized by production of excessive amounts of viscous mucus secretions in the airways of the patient. This causes airway obstruction as well as chronic bacterial infections which eventually lead to respiratory failure. Mucins provide protection to epithelia through interaction of their saccharides with bacterial adhesins. Chronic colonization with Pseudomonas aeruginosa, is considered the principal cause of death in CF patients. Our laboratory and others have shown that P. aeruginosa had considerably stronger binding affinity for CF airway mucin than normal airway mucin. These observations implicate altered glycosylation of CF mucins. Indeed, aberrant glycosylation has been reported for CF mucin. However, to date, the molecular basis of increased interaction between P. aeruginosa and CF airway mucin has not been established. We hypothesize that altered glycosylation of CF mucin is responsible for its stronger binding with P. aeruginosa. We will determine structural features of the CF mucin carbohydrate ligand(s) that provide increased binding to P. aeruginosa by preparing glycopeptides and individual saccharides from CF and control mucins. The glycopeptide(s) which show high inhibition of asialo-GM; binding to P. aeruginosa will be used to isolate 0-linked glycans for further testing of inhibitory activity and structural determination using state-of-the-art highly sensitive mass spectrometry and enzymnatic methods. Affinity gels containing selected mucin glycopeptide or mucin saccharide will be used to purify the P. aeruginosa adhesins which interact with airway mucins and glycolipids, respectively. The primary structure of the major adhesins will be determined using molecular cloning techniques. Structural characterization of major adhesins will open additional approaches to prevent the binding of P. aeruginosa to airway epithelial cells and mucins of CF patients. Information on the adhesin binding sites will permit molecular modeling, design and synthesis of potent 0-glycan inhibitors of the P. aeruginosa infection. The overall long-term goal of this study is to prevent and/or treat lung infections in CF patients.
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