Figure 1.

Killing of P. aeruginosa by reagent HOCl and loss of low-molecular-weight thiols. Bacteria were treated with increasing concentrations of HOCl in HBSS at 37 °C for 10 min with end–over–end rotation. The reaction was stopped by the addition of 1 mm methionine, and viable bacteria were determined by the CFU plating assay. A, % survival is expressed as (CFU_treated/CFU_untreated) × 100. After exposure to HOCl, NEM (20 mm) and protease inhibitors were added, and the bacteria were lysed by sonication for 5 min. The NEM adducts of reduced glutathione (GSH) (B), cysteine (Cys) (C), and coenzyme A (CoA) (D) were measured by LC-MS/MS and expressed as a percentage of untreated control. Data are presented as mean ± S.E. of at least five independent experiments. Dose-response curves with variable slope (four parameters) were fitted. The line fitted to the survival data in A is also shown as a dotted line in B–D. A significant difference when compared with untreated control was identified by ANOVA with Dunnett's multiple comparison test and is indicated by asterisk (*, p < 0.01; **, p < 0.01; and ***, p < 0.001).