Modeling chronic infection with <i>Mycoplasma pneumoniae</i> at an air-liquid interface: global transcriptional response of HBEC3-KT respiratory epithelial cells to biofilm towers

ABSTRACT Mycoplasma pneumoniae , a bacterial pathogen that causes chronic respiratory infections, grows as biofilm towers in both axenic culture and submerged tissue culture model systems. In these towers, virulence factor production is reduced. The clinical relevance of these towers is unclear because biofilms have not been examined in air-liquid interface (ALI) models of M. pneumoniae infection. We used differentiated HBEC3-KT human bronchial epithelial cells at an ALI to understand how M. pneumoniae grows on and interacts with airway cells in a more physiologically relevant setting than submerged systems, and to characterize the global transcriptional response of host cells in this context. We used scanning electron microscopy to examine the growth of M. pneumoniae on HBEC3-KT cells over time, employing a modified protocol to preserve mucus. This protocol revealed abundant biofilm towers associated with both ciliary tips and the mucus layer. RNA-seq analysis of HBEC3-KT cells at days 1 and 7 after infection indicated an early tempered cytokine response, followed later by induction of type III interferon, which is unexpected not only because that response is more typical of viruses, but also because M. pneumoniae is not known to enter host cells at an ALI. We assessed barrier function using transepithelial electrical resistance and culture of medium from the basal side of the host cells, revealing that disruption occurred, but only after prolonged infection. These results collectively suggest that M. pneumoniae limits damage to host cells when growing as biofilm towers by provoking only a selective inflammatory response, promoting chronic infection.