Pseudomonas aeruginosa balances cytotoxicity and motility to counter phagocytosis by 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 mimic 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 motility defects interfere with the physical interactions between bacteria and macrophages. Live-cell imaging shows that motility-deficient bacteria exhibit reduced surface exploration and unstable attachment to macrophages, limiting their internalization. Clinical isolates with reduced swimming or twitching motility display similarly impaired uptake. Restoring T3SS activity in these motility mutants rescues cytotoxicity toward macrophages, with one notable exception: flagellum-less, hyper piliated P. aeruginosa remains avirulent and resistant to phagocytosis due to their lack of engagement with macrophages. Together, these results support two distinct immune evasion strategies: during chronic infection, reduced motility promotes a ”freeze”-like state that limits detection and engulfment, whereas during acute infection, P. aeruginosa adopts a ”fight”-like strategy by activating its T3SS to eliminate macrophages.