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Infect. Immun. doi:10.1128/IAI.00015-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Pseudomonas aeruginosa LasB metalloproteinase regulates the human urokinase-type plasminogen activator receptor (uPAR, CD87) through domain-specific endoproteolysis

Inserm, U874, Paris, F-75015 France; Inst Pasteur, Unité de Défense Innée et Inflammation, Paris, F-75015 France; CNRS, IBSM-UPR9027, Marseille, F-13402 France; Inst Pasteur, Plate-Forme de Protéomique, Paris, F-75015 France

^* To whom correspondence should be addressed. Email: pidard{at}bichat.inserm.fr.

	Abstract

Pseudomonas aeruginosa is an opportunistic pathogen in human lungs, where its secretable LasB metalloproteinase can be a virulence factor. The urokinase receptor (uPAR) participates in pericellular proteolysis and adherence/migration of epithelial cells and leukocytes recruited during infection, and shows functional regulation by various proteinases via limited endoproteolysis occuring within its three domains (D1 to D3). We thus examined proteolytic activity of LasB on uPAR, using a recombinant uPAR as well as uPAR-expressing, human monocytic and bronchial epithelial cell lines. Protein immunoblotting and flow immunocytometry using a panel of domain-specific anti-uPAR antibodies, showed that LasB is able to cleave uPAR both within the sequence linking D1 to D2, and at the carboxyterminus of D3. Comparison of LasB-producing and LasB-deficient bacterial strains indicated that LasB is entirely responsible for the uPAR cleavage ability of P. aeruginosa. Based on aminoterminal protein microsequencing, and mass spectrometry analysis of the cleavage of peptides mimicking the uPAR sequences targeted by LasB, cleavage sites were determined to be Ala⁸⁴-Val⁸⁵ and Thr⁸⁶-Tyr⁸⁷ (D1-D2), and Gln²⁷⁹-Tyr²⁸⁰ (D3). Such a dual cleavage of uPAR led to both removal of aminoterminal D1 and generation of truncated D2D3 species, and shedding of D2D3 from cells. This proteolytic processing of uPAR was found to (i) drastically reduce the capacity of cells to bind urokinase, and (ii) abrogate the interaction between uPAR and the matrix adhesive protein vitronectin. The LasB proteinase is thus endowed with a high potential for alteration of uPAR expression and functioning on inflammatory cells during infections by P. aeruginosa.

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