Establishment and characterization of persistent <i>Pseudomonas aeruginosa</i> infections in air–liquid interface cultures of human airway epithelial cells

Bacteria exhibit distinct behaviors in laboratory settings compared to infection environments. The presence of host cells induces changes in bacterial activity, while pathogens trigger immune responses that shape the microenvironment. Studying infection dynamics by microscopy, cytokine screening, and dual RNA sequencing in an air-liquid interface model, we found that prolonged <i>Pseudomonas aeruginosa</i> colonization of airway epithelium led to a pro-inflammatory response, consistent across <i>P. aeruginosa</i> strains, despite differences in the dynamics of this response. Concurrently, <i>P. aeruginosa</i> formed non-attached aggregates on the apical side of the cell layer and upregulated genes involved in biofilm formation and virulence. Notably, there was remarkable resemblance between the <i>P. aeruginosa</i> transcriptional profile in our model and that previously reported upon host cell contact. Developing a platform that replicates host microenvironments is vital not only for gaining deeper insights into the interplay between host and pathogen but also for evaluating therapeutic strategies in conditions that closely mirror clinical environments.