Intracellular trafficking of Bordetella pertussis in human macrophages

Abstract

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.

FIG. 1.

Increase in the number of B. pertussis cells inside macrophages over time. GFP-expressing B. pertussis cells were incubated with human macrophages (MOI, 50) for 20 min at 37°C. After being washed, the macrophages were incubated for 2 h (panel a) or 48 h (panel b) in DMEM-10% NS and further stained with the acidotropic dye Lysotracker. Results representative of one out of three independent experiments are shown.

FIG. 2.

Phagocytosis and survival of B. pertussis in human macrophages. (A) GFP-expressing B. pertussis cells were incubated with human macrophages (MOI, 20) for 20 min at 37°C. After being washed, the cells were fixed and extracellular and intracellular bacteria were quantified by double immunofluorescence staining. The data represent the mean ± SD of three independent experiments. The number of intracellular IgG-opsonized B. pertussis bacteria was significantly different from the number of intracellular nonopsonized B. pertussis bacteria (*, P < 0.05). (B) GFP-expressing B. pertussis was incubated with human macrophages (MOI, 20) for 20 min at 37°C. After three washing steps, the cells were incubated with polymyxin B to kill extracellular bacteria and the number of CFU of B. pertussis per cell was determined at different time points postinfection. The data represent the mean ± SD of three independent experiments. The number of viable intracellular nonopsonized bacteria per cell at 48 h postinfection was significantly different from the number of viable intracellular bacteria per cell at both 2 and 24 h postinfection. (*, P < 0.05).

FIG. 3.

Time course of B. pertussis colocalization with the acidotropic dye Lysotracker. GFP-expressing B. pertussis bacteria were incubated with human macrophages (MOI, 20) for 20 min at 37°C. After being washed, B. pertussis-infected macrophages were incubated with Lysotracker (20 min postinfection) or treated with polymyxin B before Lysotracker staining at 2 or 48 h after infection. The bars indicate percentages of Lysotracker-positive phagosomes. The data represent the mean ± SD of three independent experiments. Confocal microscopy images representative of one out of three independent experiments are shown.

FIG. 4.

Quantification of bacterial loads in macrophages over time. Macrophages incubated with GFP-expressing bacteria at 37°C for 20 min were washed and further incubated with polymyxin B to kill the extracellular bacteria. At 2, 24, or 48 h after infection, cells were fixed and the number of bacteria per macrophage (b/c) was analyzed by fluorescence microscopy. The number of intracellular bacteria was determined by analyzing 100 macrophages in each sample. The data represent the mean ± SD of three independent experiments.

FIG. 5.

Characterization of B. pertussis-containing phagosome. Macrophages incubated with GFP-expressing B. pertussis at 37°C for 20 min were washed and further incubated with polymyxin B. At 2 or 48 h after infection, macrophages were analyzed by confocal microscopy for Alexa transferrin-594 uptake or LAMP-1 and Rab5 staining. The bars indicate percentages of LAMP-, Rab5-, and transferrin-positive phagosomes. The data represent the mean ± SD of three independent experiments. Representative confocal microscopy images of the colocalization of B. pertussis with LAMP, Rab5, or transferrin in macrophages at 48 h postinfection are shown.