Gene expression data underlying S1A and S1B Fig.

The opportunistic pathogen Pseudomonas aeruginosa causes antibiotic–recalcitrant pneumonia by forming biofilms in the respiratory tract. Despite extensive in vitro experimentation, how P. aeruginosa forms biofilms at the airway mucosa is unresolved. To investigate the process of biofilm formation in realistic conditions, we developed AirGels: 3D, optically accessible tissue–engineered human lung models that emulate the airway mucosal environment. AirGels recapitulate important factors that mediate host–pathogen interactions including mucus secretion, flow and air–liquid interface (ALI), while accommodating high–resolution live microscopy. With AirGels, we investigated the contributions of mucus to P. aeruginosa biofilm biogenesis in in vivo–like conditions. We found that P. aeruginosa forms mucus–associated biofilms within hours by contracting luminal mucus early during colonization. Mucus contractions facilitate aggregation, thereby nucleating biofilms. We show that P. aeruginosa actively contracts mucus using retractile filaments called type IV pili. Our results therefore suggest that, while protecting epithelia, mucus constitutes a breeding ground for biofilms.