Cell entry and cAMP imaging of anthrax edema toxin.
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.
Dal Molin
Federica
F
Dipartimento di Scienze Biomediche ed Istituto CNR Neuroscienze, Padova, Italy.
Tonello
Fiorella
F
Ladant
Daniel
D
Zornetta
Irene
I
Zamparo
Ilaria
I
Di Benedetto
Giulietta
G
Zaccolo
Manuela
M
Montecucco
Cesare
C
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D000942:Antigens, Bacterial; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins; D015374:Biosensing Techniques; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics; D011992:Endosomes / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D018942:Macrolides / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence; D021122:Protein Subunits / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D018942:Macrolides; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C030325:anthrax toxin; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; C040929:bafilomycin A1; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D025262:Vacuolar Proton-Translocating ATPases; D000262:Adenylyl Cyclases