From Functional Genomics to Tissue Models: Investigating Pseudomonas aeruginosa-Macrophage Interactions

During infection, Pseudomonas aeruginosa faces diverse immune defences that restrict colonization and dissemination across host tissues. Among the first responders are innate immune cells, including both tissue resident and recruited populations. P. aeruginosa infections are most prevalent in the lung, primarily affecting ventilated patients in intensive care and individuals with underlying disease. Airway macrophages are central to host responses against respiratory pathogens, contributing to microbial clearance, inflammatory signalling, and infection resolution. However, their response to bacteria during chronic infection remains poorly understood. To model bacterial diversity arising from mutagenesis and host adaptation, I employed a transposon insertion sequencing (Tn-seq) screen to dissect determinants of phagocytic uptake and global fitness during P. aeruginosa competition with macrophages. This approach identified genes regulating swimming and twitching motility as key factors in phagocytic evasion. Live-cell imaging of alveolar macrophage like cells further demonstrated that impaired surface colonization by swimming mutants accounted for their reduced uptake. I also uncovered a previously unrecognized role of type IV pili in promoting phagocytosis, suggesting that pili retraction may actively trigger engulfment. To better mimic physiological conditions during infection, I evaluated two different lung tissue models for co-culture with macrophages. I adapted Transwells for immune cell migration assays across infected lung epithelia; however, macrophages rarely traversed into epithelia. Therefore, I shifted to studying infections in the presence of macrophages at the mucosal surface using a lung-on-chip platform, AirGels, developed in our laboratory. High-resolution timelapse imaging in this system revealed P. aeruginosa biofilm formation around macrophages associated with mucus. These findings raise new questions about the role of type IV pili in phagocytosis and the mechanisms by which macrophages become engulfed by bacterial biofilms at mucosal surfaces. Overall, this thesis establishes novel approaches to study bacterial interactions with macrophages, combining functional genomics with live-cell imaging. It also underscores the importance of physiologically relevant tissue models to capture immune-pathogen dynamics during infection.