Pore-forming activity of type III system-secreted proteins leads to oncosis of Pseudomonas aeruginosa-infected macrophages

Authors

  • Denis Dacheux,

    1. Biochimie et Biophysique des Systèmes Intégrés (UMR-5092 CNRS/CEA/UJF), DBMS, CEA, 17 Avenue des Martyrs, 38054 Grenoble cedex 09, France.
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    • Julien Goure,

      1. Biochimie et Biophysique des Systèmes Intégrés (UMR-5092 CNRS/CEA/UJF), DBMS, CEA, 17 Avenue des Martyrs, 38054 Grenoble cedex 09, France.
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    • Jacqueline Chabert,

      1. Biochimie et Biophysique des Systèmes Intégrés (UMR-5092 CNRS/CEA/UJF), DBMS, CEA, 17 Avenue des Martyrs, 38054 Grenoble cedex 09, France.
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    • Yves Usson,

      1. Laboratoire DyOGen, Institut Albert Bonniot, La Tronche, France.
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    • Ina Attree

      Corresponding author
      1. Biochimie et Biophysique des Systèmes Intégrés (UMR-5092 CNRS/CEA/UJF), DBMS, CEA, 17 Avenue des Martyrs, 38054 Grenoble cedex 09, France.
      • *For correspondence. E-mail iattreedelic@cea.fr; Tel. (+33) 4 76 88 34 83; Fax (+33) 4 76 88 44 99. †Present address: Département de Bactériologie et Mycologie, Unité BMM, Laboratoire de Yersinia, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France.

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    Abstract

    The Pseudomonas aeruginosa cystic fibrosis isolate CHA induces type III secretion system-dependent but ExoU-independent oncosis of neutrophils and macrophages. Time-lapse microscopy of the infection process revealed the rapid accumulation of motile bacteria around infected cells undergoing the process of oncosis, a phenomenon we termed pack swarming. Characterization of the non-chemotactic CHAcheZ mutant showed that pack swarming is a bacterial chemotactic response to infected macrophages. A non-cytotoxic mutant, lacking the type III-secreted proteins PcrV, PopB and PopD, was able to pack swarm only in the presence of the parental strain CHA or when macrophages were pretreated with the pore-forming toxin streptolysin O. Interaction of P. aeruginosa with red blood cells (RBCs) showed that the contact-dependent haemolysis provoked by CHA requires secretion via the type III system and the PcrV, PopB/PopD proteins. The pore inserted into RBC membrane was estimated from osmoprotection experiments to be between 2.8 and 3.5 nm. CHA-infected macrophages could be protected from cell lysis with PEG3350, indicating that the pore introduced into RBC and macrophage membranes is of similar size. The time course uptake of the vital fluorescent dye, Yo-Pro-1, into infected macrophages confirmed that the formation of transmembrane pores by CHA precedes cellular oncosis. Therefore, CHA-induced macrophage death results from a pore-forming activity that is dependent on the intact pcrGVHpopBD operon.

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