Intracellular trafficking of Brucella abortus in J774 macrophages

Abstract

Brucella abortus is a facultative intracellular bacterium capable of surviving inside professional and nonprofessional phagocytes. The microorganism remains in membrane-bound compartments that in several cell types resemble modified endoplasmic reticulum structures. To monitor the intracellular transport of B. abortus in macrophages, the kinetics of fusion of phagosomes with preformed lysosomes labeled with colloidal gold particles was observed by electron microscopy. The results indicated that phagosomes containing live B. abortus were reluctant to fuse with lysosomes. Furthermore, newly endocytosed material was not incorporated into these phagosomes. These observations indicate that the bacteria strongly affect the normal maturation process of macrophage phagosomes. However, after overnight incubation, a significant percentage of the microorganisms were found in large phagosomes containing gold particles, resembling phagolysosomes. Most of the Brucella bacteria present in phagolysosomes were not morphologically altered, suggesting that they can also resist the harsh conditions prevalent in this compartment. About 50% colocalization of B. abortus with LysoSensor, a weak base that accumulates in acidic compartments, was observed, indicating that the B. abortus bacteria do not prevent phagosome acidification. In contrast to what has been described for HeLa cells, only a minor percentage of the microorganisms were found in compartments labeled with monodansylcadaverine, a marker for autophagosomes, and with DiOC6 (3,3'-dihexyloxacarbocyanine iodide), a marker for the endoplasmic reticulum. These results indicate that B. abortus bacteria alter phagosome maturation in macrophages. However, acidification does occur in these phagosomes, and some of them can eventually mature to phagolysosomes.

FIG. 1

Fusion of Brucella-containing phagosomes with preformed lysosomes. Colloidal gold particles (20 nm) coated with mannosylated BSA were internalized in J774 macrophages for 15 min at 37°C and chased into lysosomes by an additional 60-min incubation. B. abortus (opsonized [squares] or not opsonized [circles]) was incubated with the macrophages (100 Brucella bacteria/macrophage) for 5 min at 37°C. Cells were then washed five times with BME to remove nonadherent bacteria. Macrophages were incubated at 37°C for different times, fixed, and processed for transmission electron microscopy. (A) The percentage of phagosomes containing gold particles was calculated by counting at least 100 phagosomes for each condition. (B) The percentage of digested Brucella was estimated by counting at least 150 intraphagosomal bacteria. The results are from one of three independent experiments performed.

FIG. 2

Images of the fusion of Brucella-containing phagosomes with preformed lysosomes. Lysosomes were loaded with 20-nm colloidal gold particles as described in the Fig. 1 legend. Live (a, c, and e) and heat-killed (b, d, and f) Brucella bacteria were internalized for 5 min and chased for 45 min (top panels), 2 h (middle panels), and 24 h (bottom panels). Gold-containing compartments are abundant in these cells. Live Brucella bacteria are located in small, gold-free phagosomes, except in the 24-h phagosome. The Brucella shown at this time point (e) is not digested in spite of being located in a phagolysosome containing gold particles. Two partially digested Brucella bacteria are shown in panel c (arrows). Colloidal gold particles are present in all phagosomes containing heat-killed Brucella (b, d, and f). The phagosome in panel f is especially large and presents several membranous bodies that may represent highly digested Brucella. For morphological comparison, extracellular live and dead Brucella bacteria are shown as inserts in panels a and b, respectively. Bars, 1 μm.

FIG. 3

Gallery of phagosomes containing live (a to c) and heat-killed (d to f) Brucella bacteria. Panels a and d correspond to 45-min phagosomes, panels b and e correspond to 2-h phagosomes, and panels c and f correspond to 24-h phagosomes. Small phagosomes were prevalent with live Brucella (a1, a3, a4, b2, b4, and c2). However, large phagosomes were also observed. A large phagosome with a cytoplasm-like content and double membrane (arrows) resembling an autophagosome is shown in panel a2. The phagosomes in panels b1, b3, and c1 present abundant internal membranes and resemble phagolysosomes. The phagosome in panel c4 is spacious but contains little intravesicular content. Phagosomes containing heat-killed Brucella generally present characteristics of phagolysosomes, i.e., they present abundant intravesicular membranes and gold particles (d to f). Bar, 1 μm.

FIG. 4

Accessibility of internalized gold particles to preformed phagosomes containing live and heat-killed Brucella bacteria. Live and heat-killed Brucella bacteria were internalized by J774 macrophages for 5 min and chased for 45 min (A) or 120 min (B). Colloidal gold particles were then internalized for 15 min and chased for 0 to 60 min. Colocalization of gold particles with Brucella was quantified for all conditions in at least 100 phagosomes. The results are from one of the two independent experiments performed.

FIG. 5

Hydrolysis of a radiolabeled antibody attached to live or heat-killed Brucella. Brucella bacteria opsonized with a mouse anti-Brucella antibody were incubated with a rabbit anti-mouse IgG antibody labeled with ¹²⁵I. The Brucella bacteria were then bound to J774 macrophages, and the release of TCA-soluble radioactivity into the medium was assessed after different times of internalization. The values are expressed as percentages of the total radioactivity bound to the cells, and they are the means of duplicate samples. Four independent experiments were performed with similar results.

FIG. 6

Colocalization of live Brucella with probes that label acidic compartments, autophagosomes, and the ER. Live Brucella bacteria were labeled with TAMRA (a, c, and d, left and middle panels) or BCECF (b, left and middle panels) and incubated with J774 macrophages for 20 h. Cells were labeled with LysoSensor, a green fluorescent weak base that accumulates in acidic compartments (a, right and middle panels); LysoTracker, a red fluorescent weak base that accumulates in acidic compartments (b, right panel); MDC, a marker for autophagosomal compartments (c, right and middle panels); and DiOC6, a marker for the ER (d, right and middle panels). Arrows, Brucella bacteria present in acidic compartments. Bar, 15 μm.