Pseudomonas aeruginosa is the major cause of chronic pulmonary disease in cystic fibrosis (CF) patients. During chronic infection, P. aeruginosa lose certain virulence factors, transform into a mucoid phenotype, and develop antibiotic resistance. We hypothesized that these genetic and phenotypic alterations of P. aeruginosa affect the airway epithelial responses. A549 cells were infected with 27 well-characterized isolates of P. aeruginosa from CF patients obtained during longitudinal observation, or with P. aeruginosa mutant strains lacking flagella, pili, lipopolysaccharide, or pyocyanin. Pseudomonas aeruginosa isolates from the early stages of the infection exhibited high adherence to A549 cells, were readily internalized, and able to induce reactive oxygen species (ROS) production, apoptosis of infected cells, and the release of granulocyte macrophage colonystimulating factor. Late P. aeruginosa isolates collected from patients with chronic lung infection were shown to have reduced adherence to and internalization into A549 cells compared with bacteria from patients with intermittent P. aeruginosa colonization, and induced lower production of ROS and apoptosis, but caused high proinflammatory cytokine and adhesion molecule expression. Our findings suggest that despite the loss of virulence factors during the adaptation process in the CF lung by late P. aeruginosa strains, they retain high proinflammatory abilities that likely contribute to the disease pathogenesis.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen, which is the major cause of severe chronic lung infection in cystic fibrosis patients. It is also responsible for systemic infections in immunocompromised individuals and those presenting with significant pulmonary conditions in intensive care units. This microorganism has the capacity to initiate severe inflammation in infected lungs resulting in detrimental tissue damage. We have hypothesized that Syk protein tyrosine kinase mediates lung epithelial cellular responses to P. aeruginosa infection, and that a naturally occurring non-toxic Syk inhibitor piceatannol can protect infected human cells against the deleterious effects associated with this infection. We infected Syk-positive H292 or Syk-negative A549 human lung epithelial cell lines with P. aeruginosa and assessed the resulting cellular responses, i.e. production of proinflammatory cytokines, adhesion molecule expression, generation of reactive oxygen species, and apoptosis of infected cells, utilizing a multiplex bead-based immunoassay and flow cytometry. We also studied the internalization of P. aeruginosa using the gentamicin exclusion assay. We found that the piceatannol treatment significantly suppressed inflammation, oxidative stress and apoptosis in H292, but not in A549 cells implicating Syk participation in the regulation of the pathological processes induced by P. aeruginosa infection. Intriguingly, piceatannol was able to down-regulate the internalization of P. aeruginosa by both Syk-positive and Syk-negative cell lines, implying that the mechanisms of action of this compound extend beyond Syk inhibition. As piceatannol can interfere with several mechanisms of bacterial pathogenesis this natural compound deserves further study as a potential therapeutic option in P. aeruginosa infection.