Physiological and molecular investigations of cell surface structures important in the pathogenesis of Pseudomonas aeruginosa and Burkholderia cepacia

Pseudomonas aeruginosa and Burkholderia cepacia are opportunistic pathogens that infect the lungs of cystic fibrosis (CF) patients. CF arises from mutations affecting the cystic fibrosis transmembrane conductance regulator, a chloride ion transporter found at the apical surface of mammalian epithelial cells. This defect results in an increased viscosity of the airway surface fluid, inhibiting mucociliary clearance and macrophage-mediated phagocytosis. The ability of these pathogens to cause chronic bronchopuimonary infections is exacerbated by their resistance to many conventional antibiotics. Protegrin-1 (PG-1), a cationic octadecapeptide originally isolated from porcine leukocytes, is a potential candidate for treating pulmonary infections in CF patients. However, some strains of P. aeruginosa are capable of producing alginate, a viscous exopolysaccharide, which contributes to the chronic infection of the CF lung. Ironically, these strains express alginate lyase (AlgL), which catalyzes the depolymerization of alginate and is encoded by algL in the alginate biosynthetic operon.; This dissertation has two foci. The first evaluated the correlation between PG-1 sensitivity/resistance and it's binding to P. aeruginosa and B. cepacia. Antimicrobial assays demonstrated that P. aeruginosa is sensitive to PG-1 while B. cepacia was resistant. When incubated with radioiodinated PG-1, P. aeruginosa bound more protegrin molecules per cell than B. cepacia . Binding/diffusion and surface plasmon resonance assays revealed that isolated lipopolysaccharide (LPS) and lipid A from P. aeruginosa bound PG-1 more effectively than LPS and lipid A from B. cepacia. The second focus examined the role of AlgL in alginate biosynthesis. Allelic exchange of algL in P. aeruginosa with a gentamicin cassette resulted in a non-mucoid mutant (FRD-MA7) that did not produce alginate or uronic acid and could be complemented with pNLS 18 carrying algL. Site directed mutagenesis of AlgL within its active cleft generated mutant lyases that localized to the periplasm, lacked lyase activity, and could not complement FRD-MA7. These findings support our hypotheses that (1) the relative sensitivity or resistance of P. aeruginosa and B. cepacia strains to PG-1 correlates with the extent of PG-1 binding to the lipid A moiety of LPS, and (2) the enzymatic activity of AlgL is required for the production of alginate by P. aeruginosa.