Human Cysteine Cathepsins Are Not Reliable Markers of Infection by Pseudomonas aeruginosa in Cystic Fibrosis

Clément Naudin,Franciszek Kasprzykowski,Gérard Couetdic,Fabien Lecaille,Patrick Plésiat,Alix Joulin-Giet,Francis Gauthier,Aneta Szymanska,Gilles Lalmanach,Emilia Gorna,Stefan Wölfl

doi:10.1371/journal.pone.0025577

Abstract

Cysteine cathepsins have emerged as new players in inflammatory lung disorders. Their activities are dramatically increased in the sputum of cystic fibrosis (CF) patients, suggesting that they are involved in the pathophysiology of CF. We have characterized the cathepsins in CF expectorations and evaluated their use as markers of colonization by Pseudomonas aeruginosa. The concentrations of active cathepsins B, H, K, L and S were the same in P. aeruginosa-positive (19 Ps+) and P. aeruginosa-negative (6 Ps−) samples, unlike those of human neutrophil elastase. Also the cathepsin inhibitory potential and the cathepsins/cathepsin inhibitors imbalance remained unchanged and similar (∼2-fold) in the Ps+ and Ps− groups (p<0.001), which correlated with the breakdown of their circulating cystatin-like inhibitors (kininogens). Procathepsins, which may be activated autocatalytically, are a potential proteolytic reservoir. Immunoblotting and active-site labeling identified the double-chain cathepsin B, the major cathepsin in CF sputum, as the main molecular form in both Ps+ and Ps− samples, despite the possible release of the ∼31 kDa single-chain form from procathepsin B by sputum elastase. Thus, the hydrolytic activity of cysteine cathepsins was not correlated with bacterial colonization, indicating that cathepsins, unlike human neutrophil elastase, are not suitable markers of P. aeruginosa infection.

Highlights

Cystic fibrosis is an inherited life-threatening disorder
Stimulated monocyte-derived macrophages can release the CPs that are found in the bronchoalveolar lavage fluids (BALFs) of smokers suffering from emphysema [11]
The supernatants obtained by centrifuging the 25 Pseudomonas aeruginosa-positive and Pseudomonas aeruginosa-negative cystic fibrosis (CF) sputum samples were immediately buffered at pH 5.5 and stabilized to preserve cysteine cathepsins from inactivation at neutral pH and uncontrolled proteolysis

Summary

Introduction

Cystic fibrosis is an inherited life-threatening disorder It is associated with a mutation of the CF transmembrane glycoprotein that is involved in the transport of chloride ions [1,2]. The clinical manifestations of chronic inflammation of the respiratory epithelium (overproduction of mucus, persistent cough, wheezing, repeated lung and sinus infections), are mainly due to the release of proteolytic enzymes and the disruption of the protease-antiprotease balance. The cysteine cathepsins B, H, L, K and S are involved in a variety of proteolytic processes, such as the turnover of endocytosed proteins, prohormone processing, MHC-II antigen presentation, and extracellular matrix and basal membrane degradation. Active forms of cysteine cathepsins are present in BAL fluids from patients suffering from infiltrative inflammatory disorders like sarcoidosis and alveolar proteinosis, and silicosis [12,13,14]

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