Respiratory epithelial cells: a transient niche for B. pertussis?

RODRIGUEZ, MARIA EUGENIA; GORGOJO, JUAN PABLO; LAMBERTI, YANINA

Simposio; Imaging Techniques for Biotechnological and Biomedical Applications Workshop; 2016

Secretaria de Ciencia y Técnica, Facultad de Ciencias Exactas, UNLP

Bordetella pertussis is a strictly human pathogen and the main causative agent of whooping cough, aka pertussis. Despite a high vaccination cover pertussis remains endemic within the world population. The persistence of pertussis in countries with highly vaccinated populations has been attributed to various causes including suboptimal vaccines, a waning immunity, and pathogen adaptation. B. pertussis colonizes the mucosa of the respiratory tract, where the bacterium interacts with epithelial cells and local immune-surveillance cells. Although some potential contributors to colonization have been described, the mechanisms that allow this pathogen to evade immune clearance, causing a highly contagious and prolonged respiratory disease, are still under investigation. Although B. pertussis is usually regarded as a noninvasive pathogen, a number of studies suggests that this bacterium is able to enter into and eventually survive inside host cells. We previously found that B. pertussis survives inside human macrophages in non-acidic compartments having the characteristics of early endosomes. In the present study, we investigated whether B. pertussis is also able to hide inside epithelial respiratory cells and proliferate from there. Previous in vitro studies revealed the presence of B. pertussis inside epithelial cells, however, little is known regarding the internalization and the fate of B. pertussis inside the epithelia. By mean of microscopy and biochemical techniques, we have devealed everal aspects of this interaction. Scanning electron microscopy was used to evaluate the interaction between B. pertussis and the human alveolar epithelial cell line A549 at the ultrastructural level. Microvillus-like extensions emanating from the surface of the cells, closely associated with adhering bacteria were observed. In some adhering bacteria, the presence of membrane protrusions engulfing the bacteria resembled a zipper-like entrance mechanism. The contribution of microfilaments during B. pertussis invasion was further evaluated by the analysis of the spatial association of B. pertussis with the actin of the host cell cytoskeleton by confocal microscopy. Intracellular bacteria were found not associated with the host filamentous suggesting that actin mobilization of F-actin is not implicated in the bacterial entry into A549 cells. Accordingly, bacterial internalization by respiratory epithelial cells was not inhibited by agents that inhibit actin polymerization. Further studies showed that the entry of B. pertussis into respiratory-epithelial cells requires microtubule assembly, lipid raft integrity, and the activation of tyrosine kinases. To investigate the fate of B. pertussis inside respiratory cells, CFU counts and trafficking studies by confocal microscopy were ran in parallel. The association of B. pertussis with acidic (LysoTracker?-positive) vacuoles and LAMP1 (a late endosomal and lysosomal marker)- and EEA-1 (an early endosomal marker)-containing vacuoles were evaluated. Our overall data suggest that part of the internalized bacteria manage to avoid the lysosomal-degradation pathway. Accordingly, a correlation between the number of viable intracellular bacteria and the number of either LAMP1-negative, LysoTracker?-negative, or EEA-1 positive bacteria per cell was observed. These results suggest that those bacteria had survived by avoiding the lysosomal pathway and remaining in EEA-1 positive compartments. These results suggest that B. pertussis is able to enter and survive within respiratory epithelial cells potentially contributing to host immune evasion and persistence.