Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells.
Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H(2)O(2) threefold above the endogenous H(2)O(2) production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 microM) oxidized the cytosol from a resting value of -318+/-5 mV by 48.0+/-4.6 mV within 2 h; a comparable oxidation was induced by 100 microM H(2)O(2). Whereas resting Cl(-) secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl(-) secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for DeltaF508 CFTR failed to secrete Cl(-) in response to pyocyanin or H(2)O(2), indicating that these oxidants specifically target the CFTR and not other Cl(-) conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H(2)O(2), depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA.
Christian
C
Schwarzer
Horst
H
Fischer
Eun-Jin
EJ
Kim
Katharine J
KJ
Barber
Aaron D
AD
Mills
Mark J
MJ
Kurth
Dieter C
DC
Gruenert
Jung H
JH
Suh
Terry E
TE
Machen
Beate
B
Illek
D000255:Adenosine Triphosphate / Q000378:metabolism; D001980:Bronchi / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000378:metabolism / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004847:Epithelial Cells; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects*; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects*; D011710:Pyocyanine / Q000494:pharmacology*
C505032:CFTR protein, human; D002712:Chlorides; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D011710:Pyocyanine; D006861:Hydrogen Peroxide; D005978:Glutathione