Intracellular Trafficking of Bordetella pertussis in Human Macrophages

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 colonizes the human respiratory tract, causing a disease known as whooping cough or pertussis, which affects around 4 million people worldwide and causes more than 300,000 deaths each year. Despite high vaccination rates, whooping cough remains a serious threat to human health and its incidence has been increasing in recent years in vaccinated populations. Although some potential contributors to initial colonization have been described, the mechanisms that allow this pathogen to evade immune clearance and to cause the extraordinarily prolonged disease known in China as Bai Ri Ke (100-day cough) are not known.B. pertussis expresses a number of potent virulence factors, adhesins, and toxins (23) with known or predicted roles during infection. Although B. pertussis is described as an extracellular pathogen, several studies indicate that the immunomodulatory properties of several of these virulence factors enable the bacterium to persist within epithelial cells and leukocytes (1, 3, 22, 24), leading to speculation that the infection might also comprise an intracellular stage. The dual extra- and intracellular locations of B. pertussis are also consistent with the reported need for both cellular and humoral immune responses for bacterial elimination from the respiratory tract (12, 17, 33, 38).It is presumed that macrophages play an important role in the clearance of B. pertussis (21). However, in vitro studies indicated that B. pertussis is capable of surviving intracellularly in human macrophages for several days in the absence of opsonins (11). Moreover, B. pertussis was found viable in alveolar macrophage cells of mice for more than 21 days after infection (16). These observations have led to speculation that alveolar macrophages might represent an intracellular niche for B. pertussis (16, 41). The recovery of viable B. pertussis from human hosts several weeks after infection (19, 30) and the observation of B. pertussis within pulmonary alveolar macrophages of HIV-infected children (7) and in infants with confirmed B. pertussis pneumonia (28) provide support for this theory.Efforts to characterize the interaction between B. pertussis and human macrophages have been mainly focused on B. pertussis adherence. Several B. pertussis virulence factors facilitate interaction with phagocytes. B. pertussis fimbriae mediate the binding to the very late antigen 5 receptor on monocytes and macrophages, inducing the upregulation of complement receptor 3 (CR3: CD11b/CD18) (14). CR3 expression is further upregulated by pertussis toxin and filamentous hemagglutinin (FHA) (18, 42). It has been demonstrated that CR3 serves as a docking molecule for B. pertussis binding by FHA (18, 31), which eventually leads to B. pertussis uptake in a nonbactericidal way (15). However, little is known about the fate of B. pertussis inside macrophages. Recent studies by our group showed that neutrophil uptake of B. pertussis in the absence of specific antibodies leads to the failure of lysosomal maturation and bacterial clearance (20). These observations are intriguing evidence that B. pertussis has mechanisms that can allow for evasion of phagolysosome biogenesis. However, short-lived neutrophils are unlikely to provide a prolonged reservoir of bacteria, and evasion of phagolysosome biogenesis in macrophages appears to be an important aspect of the persistence of many other pathogens (9, 10, 29, 40).The aim of this study was to determine the fate of B. pertussis following phagocytosis by macrophages. Although many ingested bacteria were rapidly killed by macrophages, a significant fraction of internalized B. pertussis was capable of evading phagosome-lysosome fusion, surviving for days and eventually replicating in nonacidic compartments with characteristics of early endosomes. These results reveal a pathway that may contribute to both the extraordinarily long persistence of the coughing illness caused by B. pertussis and its ability to persist within largely immune populations.