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. 2003 Nov 25;100(24):14315-20.
doi: 10.1073/pnas.2332354100. Epub 2003 Nov 6.

Human targets of Pseudomonas aeruginosa pyocyanin

Affiliations

Human targets of Pseudomonas aeruginosa pyocyanin

Huimin Ran et al. Proc Natl Acad Sci U S A. .

Abstract

Pseudomonas aeruginosa produces copious amounts of the redoxactive tricyclic compound pyocyanin that kills competing microbes and mammalian cells, especially during cystic fibrosis lung infection. Cross-phylum susceptibility to pyocyanin suggests the existence of evolutionarily conserved physiological targets. We screened a Saccharomyces cerevisiae deletion library to identify presumptive pyocyanin targets with the expectation that similar targets would be conserved in humans. Fifty S. cerevisiae targets were provisionally identified, of which 60% have orthologous human counterparts. These targets encompassed major cellular pathways involved in the cell cycle, electron transport and respiration, epidermal cell growth, protein sorting, vesicle transport, and the vacuolar ATPase. Using cultured human lung epithelial cells, we showed that pyocyanin-mediated reactive oxygen intermediates inactivate human vacuolar ATPase, supporting the validity of the yeast screen. We discuss how the inactivation of V-ATPase may negatively impact the lung function of cystic fibrosis patients.

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Figures

Fig. 1.
Fig. 1.
PCN causes oxidative stress in S. cerevisiae.(A and B) PCN increases the levels of H2O2 and formula image in yeasts. BY4741 cells were exposed to PCN at indicated concentrations. The production of H2O2 and formula image was observed by measuring fluorescence emitted by oxidation of DCFH-DA and dihydroethidine, respectively. (C and D) Increasing amounts of PCN-generated ROI are accompanied by higher level of catalase and SOD activities in yeasts. BY4741 cells were exposed to PCN, and total proteins were harvested at the indicated time intervals and concentrations to assay for catalase and SOD. The experiments were repeated five times with similar results. The results from one representative experiment are shown.
Fig. 2.
Fig. 2.
PCN causes oxidative stress in lung epithelial cells. (A and B) PCN increases the levels of H2O2 and formula image in A549 cells. A549 cells were exposed to PCN or BA1 at indicated concentrations. The production of H2O2 and formula image was observed by measuring fluorescence emitted by oxidation of DCFH-DA and dihydroethidine, respectively. (C and D) Increasing amounts of PCN-generated ROI are accompanied by higher level of catalase and SOD activities in the lung epithelial cells. A549 cells were exposed to PCN, and total proteins were harvested at the indicated time intervals and concentrations to assay for catalase and SOD. The experiments were repeated five times with similar results. The results from one representative experiment are shown.
Fig. 3.
Fig. 3.
PCN alters the growth phenotype of yeast V-ATPase mutants. Yeast strains with mutations in V-ATPase were followed for 21 h while shaking at 150 rpm in yeast extract/peptone/dextrose (YPD) or YPD supplemented with 100 μg/ml PCN. (A) Cell density of parental strain BY4741 according to OD630 over time. (B) Viability of PCN-treated BY4741 and V-ATPase mutants as determined by colony-forming unit counts on YPD agar plates. Similar results were obtained from six independent experiments. Results from one representative experiment are shown.
Fig. 4.
Fig. 4.
PCN inactivates V-ATPase and prevents vacuole acidification in lung epithelial cells. The A549 cells were treated for 1 h with 0.5 and 2 μM BA1 or with 25, 50, and 100 μg/ml PCN or DMSO as control. Treated cells were stained with 5 μg/ml AO and examined with a fluorescence light microscope. (Original magnification, ×40.)
Fig. 5.
Fig. 5.
PCN inhibits ATP hydrolysis of V-ATPase from lung epithelial cells. Shown are ATP hydrolysis assays with microsomes isolated from A549 cells after1hof exposure to DMSO (control) or to indicated concentrations of H2O2, PCN, or BA1. The percent of ATP hydrolysis was calculated relative to the DMSO control. The mean of three independent experiments and standard deviation are shown. All data were statistically significant at P < 0.05.

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