Although Bordetella pertussis has been observed to survive inside macrophages, its ability to resist or evade degradation in phagolysosomes has not been defined. We here investigated the trafficking of B. pertussis upon entry into human macrophages. During the first hours following phagocytosis, a high percentage of bacteria were destroyed within acidic compartments positive for the lysosome-associated membrane proteins (LAMP). However, roughly one-fourth of the bacteria taken up evade this initial killing event, remaining in nonacidic compartments. Forty-eight hours after infection, the number of intracellular bacteria per cell increased, suggesting that B. pertussis is capable of replicating in this type of compartment. Viable bacteria accumulated within phagosomal compartments positive for the early endosomal marker Rab5 but not the late endosomal marker LAMP. Moreover, B. pertussis-containing phagosomes acquired exogenously added transferrin, indicating that intracellular bacteria have access to extracellular components and essential nutrients via the host cell recycling pathway. Overall, these results suggest that B. pertussis survives and eventually replicates in compartments with characteristics of early endosomes, potentially contributing to its extraordinary ability to persist within hosts and populations.

Bordetella pertussis is a re-emerging human respiratory pathogen whose infectious process is not fully understood, hampering the design of effective vaccines. The nature of bacterial attachment to host cells is a key event in the outcome of the infection. However, host cell receptors involved in B. pertussis colonization of the respiratory tract are still under investigation. Here, we report that cholesterol-rich domains are involved in B. pertussis adhesion to epithelial cells. Treatment of A549 cells with cholesterol-sequestering drugs such as methyl-beta-cyclodextrin, nystatin, or filipin resulted in a significant decrease of B. pertussis attachment. Confocal laser microscopy studies showed B. pertussis associated with cholesterol-rich domains. Accordingly, B. pertussis was found in detergent-resistant membrane domain fractions isolated from bacterial-infected A549 cells. Our results indicate a main role of filamentous hemagglutinin, an environmentally regulated virulence factor, in this interaction, and a specific affinity for cholesterol, one of the major components of tracheal secretions, which might additionally contribute to the effective colonization of the respiratory tract.

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