Central role of PAFR signalling in ExoU-induced NF-κB activation

Abstract

SummaryExoU is an important virulence factor in acute Pseudomonas aeruginosa infections. Here, we unveiled the mechanisms of ExoU-driven NF-κB activation by using human airway cells and mice infected with P. aeruginosa strains. Several approaches showed that PAFR was crucially implicated in the activation of the canonical NF-κB pathway. Confocal microscopy of lungs from infected mice revealed that PAFR-dependent NF-κB activation occurred mainly in respiratory epithelial cells, and reduced p65 nuclear translocation was detected in mice PAFR−/− or treated with the PAFR antagonist WEB 2086. Several evidences showed that ExoU-induced NF-κB activation regulated PAFR expression. First, ExoU increased p65 occupation of PAFR promoter, as assessed by ChIP. Second, luciferase assays in cultures transfected with different plasmid constructs revealed that ExoU promoted p65 binding to the three κB sites in PAFR promoter. Third, treatment of cell cultures with the NF-κB inhibitor Bay 11-7082, or transfection with IκBα negative-dominant, significantly decreased PAFR mRNA. Finally, reduction in PAFR expression was observed in mice treated with Bay 11-7082 or WEB 2086 prior to infection. Together, our data demonstrate that ExoU activates NF-κB by PAFR signalling, which in turns enhances PAFR expression, highlighting an important mechanism of amplification of response to this P. aeruginosa toxin.

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

“…Lung infection by ExoU-producing P. aeruginosa is accompanied by a robust infiltration of inflammatory cells consisting primarily of neutrophils [9,[11][12][13]. However, this highly cytotoxic toxin kills both resident alveolar macrophages and recruited neutrophils, thereby interfering with the clearance of bacteria from infected lungs [14,15].…”

“…Upon injection, ExoU is activated by host cell cofactors [5][6][7] and targeted to the plasma membranes, where it cleaves membrane phospholipids, resulting in rapid lysis of a variety of cell types [8]. ExoU PLA 2 activity also accounts for a potent stimulation of inflammatory response and increased release of free arachidonic acid, cytokines, eicosanoids and PAF from infected cells [9][10][11], as well as for the activation of the NF-κB transcription factor via a PAFR signaling pathway [12,13]. The proinflammatory effect of ExoU has been extensively confirmed in experimental models of pneumosepsis in which P. aeruginosa inoculation into mice airways resulted in a rapid influx of large numbers of inflammatory cells, primarily neutrophils, into the lungs [9,[11][12][13][14][15][16].…”

Temporal upregulation of host surface receptors provides a window of opportunity for bacterial adhesion and disease

“…Of these, WEB-2086 has recently been demonstrated to significantly inhibit both NTHi and Streptococcus pneumoniae adherence to bronchial epithelial cells in vitro [46]. WEB-2086 also caused a significant reduction in exotoxin ExoU-expressing P. aeruginosa bacterial load in both in vitro and in vivo infections of A549 human alveolar epithelial cells and mouse lungs, respectively [111]. Besides WEB-2086, CAS-99103-16-9 has been shown to inhibit P. aeruginosa infection in in vitro and in vivo experimental models [42].…”

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

“…ExoU induces NF-B activation through the canonical pathway by platelet activating factor (PAF) receptor signaling, leading to IL-8 production and neutrophil recruitment (158). The ExoU-mediated activation of NF-B in turn favors PAF receptor expression, which amplifies its effects (159). ExoU leads epithelial cells to overexpress both the inducible and endothelial nitric oxide synthases, which leads to a redox imbalance that results in increased concentrations of lipid hydroperoxides and decreased levels of reduced glutathione (155).…”