Subcellular localization ofPseudomonaspyocyanin cytotoxicity in human lung epithelial cells

Abstract

The Pseudomonas aeruginosa secretory product pyocyanin damages lung epithelium, likely due to redox cycling of pyocyanin and resultant superoxide and H2O2 generation. Subcellular site(s) of pyocyanin redox cycling and toxicity have not been well studied. Therefore, pyocyanin's effects on subcellular parameters in the A549 human type II alveolar epithelial cell line were examined. Confocal and electron microscopy studies suggested mitochondrial redox cycling of pyocyanin and extracellular H2O2 release, respectively. Pyocyanin decreased mitochondrial and cytoplasmic aconitase activity, ATP levels, cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and mitochondrial membrane potential. These effects were transient at low pyocyanin concentrations and were linked to apparent cell-mediated metabolism of pyocyanin. Overexpression of MnSOD, but not CuZnSOD or catalase, protected cellular aconitase, but not ATP, from pyocyanin-mediated depletion. This suggests that loss of aconitase activity is not responsible for ATP depletion. How pyocyanin leads to ATP depletion, the mechanism of cellular metabolism of pyocyanin, and the impact of mitochondrial pyocyanin redox cycling on other cellular events are important areas for future study.

Redox-independent Activation of NF-κB by Pseudomonas aeruginosa Pyocyanin in a Cystic Fibrosis Airway Epithelial Cell Line

“…As determined from measurements of ⌿ cyto , [PYO] as low as 1 M caused small oxidation of ⌿ cyto , and 10 and 100 M PYO caused large and sustained oxidation of ⌿ cyto by 20 and 40 mV. These quantitative measurements of PYO-induced oxidation of the cytosol therefore confirm previous qualitative measurements based on the use of oxidation-dependent fluorescein derivatives (21,22) or electron spin resonance (16) . at concentrations that oxidized the cytosol similar to that exhibited by 100 M PYO (i.e.…”

“…Altering the redox properties of the endoplasmic reticulum could trigger an unfolded protein response that leads to activation of NF-B (37), but PYO did not activate the unfolded protein response (analyzed by IRE1␣-dependent splicing of XBP-1), 3 indicating that this was an unlikely explanation for the mechanism by which PYO synergizes with NF-B-activating agonists in stimulating NF-B. Previous work has indicated that PYO may oxidize mitochondria selectively (22), and such oxidation could couple to cytosolic signaling (38) to activate NF-B. By using mitochondria-targeted roGFP, we found that PYO oxidized mitochondrial redox potential (⌿ mito ), although about 20% less than ⌿ cyto , 3 indicating that PYOinduced oxidation of ⌿ mito could contribute to activation of NF-B.…”

“…It is widely assumed that the effects of PYO on signaling are mediated at least in part through its ability to redox cycle with cellular NADPH and/or GSH leading to the production of ROS (9,13,20,21) and oxidation of the cytosol and/or mitochondria (21,22). Experiments using the electron spin resonance method showed that PYO caused the production of superoxide (O 2 . )…”

Interspecies competition triggers virulence and mutability in Candida albicans–Pseudomonas aeruginosa mixed biofilms

“…This protein is essential in the mitochondrial respiratory chain and is repressed under iron-deficient conditions and when there is a lack of heme groups (Dorsman and Grivell, 1990). In addition to the repression of these enzymes, it is possible that the Krebs cycles of C. albicans in mixed biofilms is affected by pyocyanin (Figure 8), which has been demonstrated to block respiration by inhibiting the activity of aconitase and affect the membrane potential of mitochondria from human cells (O'Malley et al, 2003).…”

Phenazine-1-carboxylic acid, a secondary metabolite ofPseudomonas aeruginosa, alters expression of immunomodulatory proteins by human airway epithelial cells

“…Because PCA is structurally similar to pyocyanin, we initially hypothesized that PCA may exert prooxidant effects in mammalian cells. Thus it was of interest to determine whether PCA, like pyocyanin (9,11,28), stimulates intracellular oxidant formation in human airway epithelial cells.…”