Identification of DksA as a novel pro-inflammatory mediator of <i>Pseudomonas aeruginosa</i> under conditions mimicking chronic cystic fibrosis lung infection

Chronic infection with <i>Pseudomonas aeruginosa</i> is a major driver of airway inflammation, which plays a central role in the progression of cystic fibrosis (CF) lung disease. During long-term colonization, <i>P. aeruginosa</i> adapts to the CF lung by downregulating virulence factors and adopting a biofilm-associated, mucoid lifestyle. Despite the expected reduction in immune activation due to these adaptations, excessive inflammation persists, a paradox that remains poorly understood. Our objective was to identify novel bacterial mediators sustaining persistent inflammation by <i>P. aeruginosa</i> in the CF lung. To this end, we analyzed clinical <i>P. aeruginosa</i> CF isolates, cultured them in synthetic CF sputum medium, and exposed 3D lung epithelial cell cultures to the resulting cell-free supernatants. There was considerable variability in pro-inflammatory activity among the isolates, with a subset of the isolates inducing strong IL-8 secretion by the 3D cells despite low production of known virulence factors. Comparative proteomics analysis of the cell-free supernatants of pro-inflammatory and immunosuppressive isolates revealed several mediators not previously linked to inflammation. Thirteen of these candidate pro-inflammatory mediators were selected for further analysis. Using <i>P. aeruginosa</i> transposon mutants lacking the respective mediators, DksA (a transcription factor) was confirmed as an immunomodulatory mediator in the 3D lung model. Finally, analysis of existing transcriptomes of <i>P. aeruginosa</i> in CF sputum revealed that <i>dksA</i> was found to be one of the most strongly expressed genes in this patient population, highlighting the relevance of our findings. In conclusion, we identified a novel <i>P. aeruginosa</i> mediator that may contribute to CF airway inflammation.