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Infection and Immunity, October 2008, p. 4624-4632, Vol. 76, No. 10
0019-9567/08/$08.00+0     doi:10.1128/IAI.01707-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Immunoproteomics To Examine Cystic Fibrosis Host Interactions with Extracellular Pseudomonas aeruginosa Proteins

Hamish G. Upritchard,¹ Stuart J. Cordwell,^2, and Iain L. Lamont^1*

Department of Biochemistry and Webster Centre for Infectious Disease, University of Otago, P.O. Box 56, Dunedin, New Zealand,¹ Australian Proteome Analysis Facility, Sydney, Australia²

Received 21 December 2007/ Returned for modification 30 January 2008/ Accepted 18 July 2008

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.

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* Corresponding author. Mailing address: Department of Biochemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand. Phone: 64-3-479-7869. Fax: 64-3-479-7866. E-mail: iain.lamont{at}stonebow.otago.ac.nz

Published ahead of print on 28 July 2008.

Editor: B. A. McCormick

Present address: School of Molecular and Microbial Biosciences, The University of Sydney, Sydney, NSW 2006, Australia.

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Infection and Immunity, October 2008, p. 4624-4632, Vol. 76, No. 10
0019-9567/08/$08.00+0     doi:10.1128/IAI.01707-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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