Pseudomonas aeruginosa (P.a.) is an opportunistic pathogen that is responsible for nocosomial infections and is the major bacterium found in the cystic fibrosis (CF) lungs. Our group has shown that host endogenous proteases such as neutrophil elastase are able to cleave and inactivate CFTR in the airways [1 Am J Respir Crit Care Med 2013 ;  187 (2) : 170-179

Cliccare qui per andare alla sezione Riferimenti]. Here we hypothesize that the P.a. virulence factor elastase LasB could modulate CFTR expression and innate immune responses in airway epithelial cells.

The effect of recombinant LasB and secretomes from P.a. strains (WT and ΔLasB PAO1, referred as WT-SEC and ΔLasB-SEC) on CFTR integrity was evaluated in the alveolar epithelial cell line A549 infected with the adenovirus Ad GFP-WT-CFTR and in the Calu-3 cell line grown on semi-permeable supports. The P.a. elastase and secretomes were used to treat the corrected CF cell line CFBE41o-WT-CFTR for ion transport experiments in Ussing chambers and the NCI-H292 and the polarized Calu-3 cell lines for the measurement of secreted cytokines and that of elafin, an important mucosal anti-microbial molecule.

SDS-PAGE analysis of the secretomes showed that LasB is the main secreted protein by WT PAO1. Recombinant LasB (10nM) and the WT-SEC (10%) but not the ΔLasB-SEC, induced a slight decrease in CFTR expression in our CFTR overexpressing model. The recombinant elastase did not show any effect on the activity of ENaC or CFTR in Ussing chamber experiments. However, WT-SEC (10%) seemed to decrease CFTR activity as reported by the measurement of forskolin/ibmx induced short-circuit current (ΔIsc) and CFTR_(Inh-172)-sensitive ΔIsc, when compared to the effect of the ΔLasB-SEC or the LB medium alone. Finally, recombinant LasB, as well as the WT-SEC but not ΔLasB-SEC decreased IL-6 and elafin secretion whereas it had no effect on IL-8 secretion.

Taken together, our results show that LasB, the major type 2 secretion system virulence factor, is able to modulate the expression of CFTR and its activity when used in the P.a. secretome. Moreover, it targets the immune response by regulating the expression of cytokines and anti-microbial peptides and we are currently investigating the molecular mechanisms and signalling pathways underlying these processes.