Swimming motility and type IV pili modulate phagocytosis by shaping physical interactions between Pseudomonas aeruginosa and macrophages

During chronic lung infections, Pseudomonas aeruginosa diversifies under selection from antibiotics, metabolic constraints, and host defenses. Macrophages are key sentinels of the innate immune system and play a central role in clearing airway pathogens. Yet, how they process heterogeneous bacterial populations remains poorly understood. Here, we investigate how P. aeruginosa evades phagocytosis under conditions that resemble chronic infection. We use an attenuated mutant lacking a functional type III secretion system (T3SS), which reduces macrophage killing, allowing us to isolate determinants of bacterial susceptibility to phagocytosis. Using transposon insertion sequencing (Tn-seq), we identify bacterial fitness factors under phagocytic selection. Our screen reveals that disruption of genes involved in swimming and twitching motility reduces uptake by macrophages. We find that defects in flagellar and type IV pili (T4P) function interfere with the physical interactions between bacteria and macrophages. Live-cell imaging shows that motility-deficient bacteria exhibit reduced surface accumulation and unstable attachment to macrophages, limiting their internalization. Clinical isolates with reduced swimming or twitching motility display similarly impaired uptake. We also test the influence of bacterial motility on macrophage survival by restoring T3SS activity in flagellar and T4P mutants. In the PAO1 background, most motility mutants retained the ability to kill macrophages indicating that cytotoxicity dominates bacteria-macrophage competition in the presence of a functional T3SS. However, the non-flagellated, hyper-piliated Δ fliC Δ pilT mutant showed a surprisingly attenuated killing, which may reflect reduced bacterial encounters with macrophages, impaired engagement of cytotoxic effectors, or both. Together, our results show that swimming motility and type IV pili shape physical interactions between P. aeruginosa and macrophages, thereby influencing phagocytic uptake.