CONICET | Buscador de Institutos y Recursos Humanos

Whooping cough is a reemerging disease caused by Bordetella pertussis (Bp). Current vaccines are protective against symptoms but fail to prevent colonization and transmission, which resulted in pathogen persistence. We have previously found that Bp survives inside phagocytic and non-phagocytic cells, suggesting that an intracellular stage might be important for persistence. The aim of this study was to evaluate whether Bp compromises the epithelial barrier and uses it as an intracellular niche. To this end, human 16HBE14o- cells were used as polarized in vitro model. After 7 days of differentiation, functional tight junctions were formed, as verified by means of specific antibodies and fluorescence microscopy. We developed a subconfluent model in which cells exposed apical and basolateral membranes, allowing the study of Bp interaction with both of them. Bacterial attachment and internalization were quantified by fluorescence microscopy at 5h post-infection. The results showed that Bp has a tropism for the basolateral membrane, in which most of the attachment and internalization was observed. Confocal microscopy analysis showed that a high percentage of internalized bacteria avoided phagolysosomal fusion. Antibiotic protection assays confirmed a high survival inside these cells at late time points (48h) post-infection, suggesting that epithelial cells might constitute a niche of persistence for this pathogen. Adenylate cyclase toxin (CyaA), a virulence factor released by Bp both in its soluble form and inside outer membrane vesicles (OMV), was found involved in tight junction disruption. We recently found that Bp OMVs preferentially attach to polarized respiratory cells near tight junctions. This might facilitate CyaA delivery to these sites, eventually granting Bp access to the basolateral membrane during infection. Furthermore, these results also suggest that the respiratory epithelium might have been underestimated as a persistence intracellular niche.