Abstract
The lungs of patients with cystic fibrosis (CF) are typically chronically infected with Pseudomonas aeruginosa. We used an immunoproteomics approach to analyze the responses of patients to secreted P. aeruginosa proteins. Extracellular proteins from P. aeruginosa strain PAO1 that had been grown to stationary phase were separated by two-dimensional polyacrylamide gel electrophoresis and analyzed by Western blotting using sera from four chronically infected patients. Sera from all four patients detected multiple extracellular proteins. The identities of selected proteins recognized by antisera were determined. Production of at least four of these proteins (azurin and three proteases: elastase, PrpL, and PasP) is governed by quorum sensing, consistent with active bacterial quorum sensing in the lungs of CF patients. The CF lung is generally thought to be an iron-deficient environment for infecting bacteria, and growing the bacteria in the presence of an iron-chelating agent, ethylene-diamine-di(o-hydroxyphenylacetic acid), enabled detection of additional proteins that were recognized by patient sera. The sera also detected multiple proteins from cells in the logarithmic growth phase, and protein identification suggested that most of these were the result of cell lysis or secretion in membrane vesicles. Comparison with extracellular proteins from a second P. aeruginosa strain, strain Pa4, showed that many proteins recognized by patient sera are common to both strains, although there are also some strain-specific extracellular proteins. Our data show that while there are some differences in the responses of different patients to P. aeruginosa, there are also many similarities, and that an immunoproteomics approach enables the identification of proteins that are made by P. aeruginosa during infection.