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Article

  • The EMBO Journal (2006) 25, 5405 - 5413
  • doi:10.1038/sj.emboj.7601408

Published online: 2 November 2006

Cell entry and cAMP imaging of anthrax edema toxin

Federica Dal Molin1, Fiorella Tonello1, Daniel Ladant2, Irene Zornetta1, Ilaria Zamparo3,4, Giulietta Di Benedetto3,4, Manuela Zaccolo3,4,5 and Cesare Montecucco1,5

  1. Dipartimento di Scienze Biomediche ed Istituto CNR Neuroscienze, Padova, Italy
  2. Departement de Biologie Structurale et Chimie, Institut Pasteur, Paris Cedex, France
  3. Dulbecco Telethon Institute, Padova, Italy
  4. Istituto Veneto di Medicina Molecolare, Padova, Italy
  5. These authors share senior authorship

Correspondence to:

Cesare Montecucco, Dipartimento di Scienze Biomediche, Viale G Colombo 3, 35121 Padova, Italy. Tel.: +39 049 827 6058; Fax: +39 049 827 6049; E-mails: cesare.montecucco@unipd.it or manuela.zaccolo@unipd.it

Received 18 April 2006; Accepted 5 October 2006

The entry and enzymatic activity of the anthrax edema factor (EF) in different cell types was studied by monitoring EF-induced changes in intracellular cAMP with biochemical and microscopic methods. cAMP was imaged in live cells, transfected with a fluorescence resonance energy transfer biosensor based on the protein kinase A regulatory and catalytic subunits fused to CFP and YFP, respectively. The cAMP biosensor was located either in the cytosol or was membrane-bound owing to the addition of a tag determining its myristoylation/palmitoylation. Real-time imaging of cells expressing the cAMP biosensors provided the time course of EF catalytic activity and an indication of its subcellular localization. Bafilomycin A1, an inhibitor of the vacuolar ATPase proton pump, completely prevented EF activity, even when added long after the toxin. The time course of appearance of the adenylate cyclase activity and of bafilomycin A1 action suggests that EF enters the cytosol from late endosomes. EF remains associated to these compartments and its activity shows a perinuclear localization generating intracellular cAMP concentration gradients from the cell centre to the periphery.

  • Keywords:

    • anthrax,
    • cAMP,
    • cell entry,
    • edema toxin,
    • FRET imaging