Modeling airway persistent infection of and nontypeable by using human models.

Non-typeable (NTHi) and (Mcat) are two common respiratory tract pathogens often associated with acute exacerbations in Chronic Obstructive Pulmonary Disease (COPD) as well as with otitis media (OM) in children. Although there is evidence that these pathogens can adopt persistence mechanisms such as biofilm formation, the precise means through which they contribute to disease severity and chronicity remains incompletely understood, posing challenges for their effective eradication. The identification of potential vaccine candidates frequently entails the characterization of the host-pathogen interplay even though this approach is limited by the fact that conventional models do not permit long term bacterial infections. In the present work, by using air-liquid-interface (ALI) human airway models, we aimed to recreate COPD-related persistent bacterial infections. In particular, we explored an alternative use of the ALI system consisting in the assembly of an inverted epithelium grown on the basal part of a transwell membrane with the aim to enable the functionality of natural defense mechanisms such as mucociliary clearance and cellular extrusion that are usually hampered during conventional ALI infection experiments. The inversion of the epithelium did not affect tissue differentiation and considerably delayed NTHi or Mcat infection progression, allowing one to monitor host-pathogen interactions for up to three weeks. Notably, the use of these models, coupled with confocal and transmission electron microscopy, revealed unique features associated with NTHi and Mcat infection, highlighting persistence strategies including the formation of intracellular bacterial communities (IBCs) and surface-associated biofilm-like structures. Overall, this study demonstrates the possibility to perform long term host-pathogen investigations with the aim to define persistence mechanisms adopted by respiratory pathogens and individuate potential new vaccine targets.