Pseudomonas aeruginosa (Pa) is an opportunistic pathogen that infects the lungs of patients with COPD, Cystic Fibrosis (CF), and is an important cause of pneumonia. Pa contributes to 5-10% of the acute exacerbations in COPD, which afflicts 24 million Americans, and is the 3rd leading cause of death in the US. Moreover, ~85% of CF patients are chronically infected with antibiotic resistant strains of Pa. In the last fundin period we demonstrated that outer membrane vesicles (OMVs) secreted by Pa fuse with lipid rafts in human bronchial epithelial (HBE) cells and decrease wt-CFTR mediated Cl secretion by delivering Cif (CFTR inhibitory factor), a virulence factor, into cells. Pa also produces another virulence factor(s) in OMVs, in addition to Cif, that inhibits delF508-CFTR Cl secretion in CF HBE cells that have been treated with the Vertex investigational drug VX-809. In this application, we demonstrate that a non-Cif factor in OMVs enhances the lysosomal degradation of delF508-CFTR in CF HBE cells by increasing tyrosine phosphorylated Lyn, a Src-tyrosine kinase. In addition, we report that disruption of lipid rafts with filipin blocks the OMV induced degradation of delF508-CFTR. Because OMVs also increase phosphorylated Lyn in mouse lung, and because Lyn phosphorylates c-Cbl, an E3 ligase that increases the ubiquitination and lysosomal degradation of wt-CFTR, we propose studies to test the hypothesis that non-Cif virulence factors in OMVs inhibit VX-809 stimulated delF508-CFTR Cl secretion in CF-HBE cells and in CF mouse lung by increasing phosphoryated Lyn, which phosphorylates c-Cbl and delF508-CFTR, thereby increasing the ubiquitination and degradation of delF508-CFTR. Studies will also be conducted to block Cif and non-Cif inhibition of delF508-CFTR Cl secretion using cyclodextrins that are in clinical trials and simvastatin (Zocor), which also disrupt lipid rafts. These studies will provide novel insight into the molecular mechanism(s) whereby OMVs reduce VX-809 stimulated delF508-CFTR Cl secretion, and thereby increase the severity of Pa lung infections, which cause considerable morbidity and mortality in the US. In addition, because filipin mitigates the effect of OMVs on delF508-CFTR Cl secretion, we anticipate that studies with cyclodextrins and simvastatin will lead to a novel treatment that, in combination with antibiotics, will ameliorate Pa infections in patients with CF as well as COPD and pneumonia.
P. aeruginosa is a Gram-negative, opportunistic pathogen that causes pneumonia and is a major cause of morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD) and Cystic Fibrosis (CF). Because all Gram-negative bacteria are highly resistant to antibiotics, and they secrete OMVs, our studies will be relevant t all Gram-negative infections, will provide novel insight into the molecular mechanism(s) whereby OMV virulence factors affect host cell biology, and we anticipate that our studies will lead to novel and effective therapeutic approaches to treat Gram-negative bacterial infections.
Showing the most recent 10 out of 51 publications
