ExoU-induced redox imbalance and oxidative stress in airway epithelial cells during Pseudomonas aeruginosa pneumosepsis

Abstract

ExoU is a potent proinflammatory toxin produced by Pseudomonas aeruginosa, a major agent of severe lung infection and sepsis. Because inflammation is usually associated with oxidative stress, we investigated the effect of ExoU on free radical production and antioxidant defense mechanisms during the course of P. aeruginosa infection. In an experimental model of acute pneumonia, ExoU accounted for increased lipid peroxidation in mice lungs as soon as 3 h after intratracheal instillation of PA103 P. aeruginosa strain. The contribution of airway cells to the generation of a redox imbalance was assessed by in vitro tests carried out with A549 airway epithelial cells. Cultures infected with the ExoU-producing PA103 P. aeruginosa strain produced significantly increased concentrations of lipid hydroperoxides, 8-isoprostane, reactive oxygen intermediates, peroxynitrite and nitric oxide (NO), when compared to cells infected with exoU-deficient mutants. Overproduction of NO by PA103-infected cells likely resulted from overexpression of both inducible and endothelial NO synthase isoforms. PA103 infection was also associated with a significantly increased activity of superoxide dismutase (SOD) and decreased levels of reduced glutathione (GSH), a major antioxidant compound. Our findings unveil another potential mechanism of tissue damage during infection by ExoU-producing P. aeruginosa strains.

Keywords: ExoU; Lipid peroxidation; NO synthase; Oxidative stress; Pneumosepsis; Pseudomonas aeruginosa.

Fig. 1

ExoU induces oxidative stress in lungs of P. aeruginosa-infected mice. Malondialdehyde (MDA) concentration in lungs of mice infected for 3 h with the ExoU-producing P. aeruginosa strain (n = 15) was significantly higher than that in lungs of control (n = 10) or PA103ΔexoU-infected animals (n = 15). *p < 0.05 when the values obtained in PA103-infected mice were compared with those obtained in animals from the other two groups

Fig. 2

ExoU induces oxidative stress in PA103-infected A549 cells. Oxidative stress was characterized by enhanced production of lipid hydroperoxides (a) and 8-isoprostane (b). The graphs represent the mean ± SEM from values obtained in three different assays performed in triplicate; *p < 0.05, **p < 0.01 or ***p < 0.001 when the values obtained in PA103-infected cultures were compared with those obtained in control or in PA103ΔexoU-infected cultures

Fig. 3

ExoU-induced oxidative stress results from the enhanced production of ROS, NO and peroxynitrite. At 1 h after infection, the level of ROS (a), NO (b) and peroxynitrite (c) in cells infected with the ExoU-producing PA103 strain was significantly higher than that in cells infected with the PA103ΔexoU mutant, which does not produce ExoU, or with the PA103ΔUT/S142A mutant that produces a toxin with impaired PLA₂ activity. The graphs represent the mean ± SEM of the percentual increases, considering the values obtained in control noninfected cultures as 100 %. *p < 0.05, **p < 0.01 and ***p < 0.001 when the values obtained in PA103-infected cells were compared with those obtained in cells infected with the mutants

Fig. 4

ExoU increases eNOS and iNOS expression by A549 cells. a, b Western blot detection of iNOS, eNOS and β-actin in control and infected cells. Graphs in c and d represents mean ± SEM from values obtained in three different western blot assays. *p < 0.05 and **p < 0.01 when the values obtained in PA103-infected cells were compared with those obtained in control or in PA103ΔexoU-infected cells

Fig. 5

ExoU impairs the antioxidant defense mechanisms of A549 cells. At 48 h post-infection, ExoU significantly increased SOD activity (a) and decreased GSH levels (b) in A549 cells. The graphs represent the mean ± SEM from values obtained in three different assays performed in triplicate. ***p < 0.001 when the values obtained in PA103-infected cultures were compared with those obtained in the other cultures