Disruption of the Endothelial Barrier by Proteases
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Disruption of the endothelial barrier by proteases from the bacterial pathogen Pseudomonas aeruginosa: implication of matrilysis and receptor cleavage Nathalie Beaufort, Elisabeth Corvazier, Saouda Mlanaoindrou, Sophie Bentzmann, Dominique Pidard To cite this version: Nathalie Beaufort, Elisabeth Corvazier, Saouda Mlanaoindrou, Sophie Bentzmann, Dominique Pi- dard. Disruption of the endothelial barrier by proteases from the bacterial pathogen Pseudomonas aeruginosa: implication of matrilysis and receptor cleavage. PLoS ONE, Public Library of Science, 2013, 8 (9), pp.e75708. 10.1371/journal.pone.0075708. hal-01458236 HAL Id: hal-01458236 https://hal.archives-ouvertes.fr/hal-01458236 Submitted on 5 Oct 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License Disruption of the Endothelial Barrier by Proteases from the Bacterial Pathogen Pseudomonas aeruginosa: Implication of Matrilysis and Receptor Cleavage Nathalie Beaufort1,2¤☯, Elisabeth Corvazier1,2☯, Saouda Mlanaoindrou1,2, Sophie de Bentzmann3,4, Dominique Pidard1,2* 1 Inserm, U698, Paris, France, 2 Université Denis Diderot, UMR-S698, Paris, France, 3 CNRS, UMR 7255-LISM, Marseille, France, 4 Aix-Marseille University, Marseille, France Abstract Within the vasculature, uncontrolled pericellular proteolysis can lead to disruption of cell-to-cell and cell-to-matrix interactions and subsequent detachment-induced cell apoptosis, or anoikis, contributing to inflammatory vascular diseases, with the endothelium as the major target. Most studies so far have focused on endogenous proteinases. However, during bloodstream infections, bacterial proteinases may also trigger endothelial anoikis. We thus investigated the potential apoptotic activity of the proteinases secreted by the haematotropic opportunistic pathogen, Pseudomonas aeruginosa, and particularly its predominant metalloproteinase, LasB. For this, we used the secretome of the LasB-expressing pseudomonal strain, PAO1, and compared it with that from the isogenic, LasB-deficient strain (PAO1∆lasB), as well as with purified LasB. Secretomes were tested for apoptotic activity on cultured human endothelial cells derived from the umbilical vein or from the cerebral microvasculature. We found that the PAO1 secretome readily induced endothelial cell anoikis, as did secretomes of LasB-positive clinical pseudomonal isolates, while the PAO1∆lasB secretome had only a limited impact on endothelial adherence and viability. Notably, purified LasB reproduced most of the effects of the LasB-containing secretomes, and these were drastically reduced in the presence of the LasB-selective inhibitor, phosphoramidon. A precocious and extensive LasB-dependent degradation of several proteins associated with the endothelial extracellular matrix, fibronectin and von Willebrand factor, was observed by immunofluorescence and/or immunoblotting analysis of cell cultures. Moreover, the PAO1 secretome, but not that from PAO1∆lasB, specifically induced rapid endoproteolysis of two major interendothelial junction components, VE-cadherin and occludin, as well as of the anti-anoikis, integrin-associated urokinase receptor, uPAR. Taken as a prototype for exogenous haemorrhagic proteinases, pseudomonal LasB thus appears to induce endothelial anoikis not only via matrilysis, as observed for many pro-apoptotic proteinases, but also via cleavage of some essential cell-to-cell and cell-to-matrix adhesion receptors implicated in the maintenance of the endothelial barrier. Citation: Beaufort N, Corvazier E, Mlanaoindrou S, de Bentzmann S, Pidard D (2013) Disruption of the Endothelial Barrier by Proteases from the Bacterial Pathogen Pseudomonas aeruginosa: Implication of Matrilysis and Receptor Cleavage. PLoS ONE 8(9): e75708. doi:10.1371/journal.pone.0075708 Editor: Barry I Hudson, University of Miami, United States of America Received February 6, 2013; Accepted August 19, 2013; Published September 19, 2013 Copyright: © 2013 Beaufort et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a fellowship from the Fondation Lefoulon-Delalande/Institut de France (http://lefoulon-delalande.institut-de- france.fr/), Paris, France (to NB), and by Vaincre la Mucoviscidose (http://www.vaincrelamuco.org/), Paris, France, the Fondation Bettencourt-Schueller (http://www.fondationbs.org/), Paris, France, and grants from Centre National de la Recherche Scientifique (CNRS; http://www.cnrs.fr/) and Agence Nationale pour la Recherche (ANR; http://www.agence-nationale-recherche.fr/), Paris, France (to SdB). DP is Chargé de Recherche at the Institut National des Sciences du Vivant within the CNRS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: [email protected] ☯ These authors contributed equally to this work. ¤ Current address: Institute for Stroke and Dementia Research, Klinikum der Universität München, Munich, Germany Introduction proliferation and migration, and the balance between EC survival and apoptosis. These include serine-proteinases of the Proteinases associated with focal or systemic inflammation plasma coagulation and fibrinolytic systems, or secreted by in the vessel wall play a prominent role in modulating activated leukocytes, as well as metalloproteinases, either permeability of the endothelial barrier, endothelial cell (EC) secreted or membrane-associated [1,2]. A noticeable PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e75708 Pseudomonal Proteases Induce Endothelial Anoikis pathogenic activity of inflammatory proteinases on vascular Furthermore, LasB specifically and directly degrades cells, particularly ECs, is their capacity to induce the interendothelial junctional proteins such as VE-cadherin and disassembly of cell-to-cell and/or cell-to-matrix connections occludin, as well as uPAR, an important integrin-associated [3,4] that then triggers an apoptotic type of programmed cell membrane protein involved in cell adhesion to matrix and cell death called anoikis [5-8]. survival, all these proteolytic events being thus likely to The vascular endothelium is a major target for many human participate in endothelial anoikis. pathogenic bacteria and their virulence factors. Upon entering the bloodstream, they can dampen and escape from the Materials and Methods immune defenses, and, in order to disseminate into the body, they alter or even disrupt the endothelial barrier, thus inducing Reagents severe pathological events within the vasculature [9-12]. The Rabbit polyclonal and mouse monoclonal antibodies (pAb Gram-negative bacterium Pseudomonas aeruginosa is such an and mAb, respectively) were from the following sources: anti- opportunistic human pathogen, and a major agent for Fn pAb F3648, from Sigma-Aldrich (Saint-Louis, MO); anti-vWf bloodstream infections, particularly in immunocompromised pAb A0082, from DakoCytomation (Glostrup, Denmark); anti- patients, leading to focal and/or systemic severe vascular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mAb diseases [13-15]. This bacterium expresses numerous 2D4A7, from Abcam (Cambridge, UK); anti-VE-cadherin pAb virulence factors targeting several cell types, including BMS158, from BenderMedSystems (Vienna, Austria); anti- phagocytes, epithelial cells and ECs. Among these factors are occludin pAb 71-1500, and anti-claudin-5 pAb 34-1600, from several cell wall-associated adhesins and invasins, potent Invitrogen Corp. (Camarillo, CA); anti-uPAR domain 2 mAb intracellular toxins delivered into host cells through #3932, from American Diagnostica (Greenwich, CT), or a gift multimolecular secretion systems, and a panel of extracellular from Dr. Viktor Magdolen (Technische Universität Munchen, proteinases [13,15-18]. The latter are mostly Germany). Control antibodies were mAb MOPC-21 and rabbit metalloproteinases or serine-proteinases, and are considered polyclonal serum IgGs, from Sigma-Aldrich and as virulence factors in various human pseudomonal infections DakoCytomation, respectively, while horseradish peroxidase-, [15,16,19]. Particular emphasis has been placed on the or Alexa Fluor 555-conjugated IgGs were from Jackson pseudomonal elastase, LasB/pseudolysin, a member of the Immunoresearch (West Grove, PA) and Invitrogen, thermolysin/M4 superfamily of metallopeptidases that includes respectively. proteinases secreted by several common pathogenic Proteinase inhibitors phosphoramidon (PA; from haematotropic bacteria [16,20]. It is the major protein(ase) Streptomyces tanashiensis) and aprotinin (Apro; from bovine secreted by P. aeruginosa [21], is widely expressed among lung) were from Sigma-Aldrich, while batimastat (BB-94) was pseudomonal clinical