Chronic lung infections with the opportunistic pathogen Pseudomonas aeruginosa remain the major cause of morbidity and mortality in patients with cystic fibrosis (CF). Initially P. aeruginosa strains that infect CF patients have a nonmucoid and smooth lipopolysaccharide (LPS) phenotype, typical of those found in the environment. As the clinical course of CF progresses, typical P. aeruginosa strains that are mucoid and have a rough LPS phenotype become predominant. The long-term objective of this proposal is to determine the bacterial and host factors responsible for the hypersusceptibility of CF patients to this infection. In vitro studies from the investigators' laboratory have shown that P. aeruginosa is internalized by human airway epithelial cells expressing wild-type CF transconductance membrane regulator (CFTR) significantly better than by epithelial cells that express the F508 mutant CFTR. Using structurally defined P. aeruginosa LPS mutants, we have identified the outer portion of the LPS core as an important ligand for this interaction. This ligand inhibited uptake of P. aeruginosa by the human airway epithelial cells and inhibited clearance of P. aeruginosa in vivo using a neonatal mouse model of pneumonia. The investigators suggest that the internalization of P. aeruginosa into normal airway epithelial cells represents an under-appreciated host defense mechanism that promotes desquamation and subsequent clearance. Further, this defense mechanism is ineffective in CF patient; thus, P. aeruginosa remains in the mucus layer and can cause a chronic infection. P. aeruginosa exacerbates this situation and evades the residual uptake mechanism of the CF cells by converting to an LPS-rough form that is even more poorly internalized.
The specific aims of this proposal attempt to further define the P. aeruginosa LPS core structures involved in the interaction with human airway epithelial cells. The investigators will test structurally defined LPS core mutants for uptake by human airway epithelial cells. They will clone and sequence the P. aeruginosa LPS core genes encoding the proteins responsible for the synthesis of this ligand. This analysis will allow them to construct and characterize genetically defined LPS core mutants of P. aeruginosa and verify that the ligand is required for internalization. They will also determine the structure of the LPS core of three different P. aeruginosa CF isolates, since poor internalization of these strains suggests a defect in LPS core. The information derived from these studies may allow the investigators to devise a strategy to increase P. aeruginosa elimination by CF airway epithelial cells, and thereby inhibit the infection caused by this opportunistic pathogen.
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