title: Effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell function and viability.
abstract: OBJECTIVE
  We determined the effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell viability and function in vitro.
  
  
  METHODS
  RT4 urothelial cells were treated with pyocyanin (1 to 100 μM) for 24 hours. After exposure the treatment effects were measured according to certain end points, including changes in urothelial cell viability, reactive oxygen species formation, caspase-3 activity, basal and stimulated adenosine triphosphate release, SA-β-gal activity and detection of acidic vesicular organelles.
  
  
  RESULTS
  The 24-hour pyocyanin treatment resulted in a concentration dependent decrease in cell viability at concentrations of 25 μM or greater, and increases in reactive oxygen species formation and caspase-3 activity at 25 μM or greater. Basal adenosine triphosphate release was significantly decreased at all tested pyocyanin concentrations while stimulated adenosine triphosphate release was significantly inhibited at pyocyanin concentrations of 12.5 μM or greater with no significant stimulated release at 100 μM. Pyocyanin treated RT4 cells showed morphological characteristics associated with cellular senescence, including SA-β-gal expression. This effect was not evident at 100 μM pyocyanin and may have been due to apoptotic cell death, as indicated by increased caspase-3 activity. An increase in acridine orange stained vesicular-like organelles was observed in RT4 urothelial cells after pyocyanin treatment.
  
  
  CONCLUSIONS
  Exposure to pyocyanin alters urothelial cell viability, reactive oxygen species production and caspase-3 activity. Treatment also results in cellular senescence, which may affect the ability of urothelium to repair during infection. The virulence factor depressed stimulated adenosine triphosphate release, which to our knowledge is a novel finding with implications for awareness of bladder filling in patients with P. aeruginosa urinary tract infection.
authors.affiliation: Department of Biomedical Science, Bond University, Queensland, Australia.
authors.forename: Catherine
authors.initials: C
authors.lastname: McDermott
authors.forename: Russ
authors.initials: R
authors.lastname: Chess-Williams
authors.forename: Gary D
authors.initials: GD
authors.lastname: Grant
authors.forename: Anthony V
authors.initials: AV
authors.lastname: Perkins
authors.forename: Amelia J
authors.initials: AJ
authors.lastname: McFarland
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Davey
authors.forename: Shailendra
authors.initials: S
authors.lastname: Anoopkumar-Dukie
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000704:Analysis of Variance; D001343:Autophagy / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D011710:Pyocyanine / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D001743:Urinary Bladder / Q000166:cytology* / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D000255:Adenosine Triphosphate; D011710:Pyocyanine; D001616:beta-Galactosidase; D053148:Caspase 3