INTRODUCTION

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The facultative intracellular parasite Brucella abortus causes abortion and infertility in cattle and undulant fever in humans. The bacterium is endemic in many underdeveloped countries and responsible for large economic losses and chronic infections in human beings (30). Brucella infects its hosts through mucosae and wounds and initially is incorporated into professional phagocytes where it survives and reproduces (14). Afterwards, the bacterium infects several types of nonprofessional phagocytic cells including those of endocardium, brain, joints, and bones. Brucella has a special tropism for reproductive organs, causing a high rate of abortion in pregnant animals (28).

The intracellular survival of Brucella has been documented for several cell types. According to multiple observations, B. abortus is incorporated into phagosomes and remains in membrane-bound compartments until the host cell dies. In nonprofessional phagocytes, Brucella is located in structures that resemble the endoplasmic reticulum (ER) (6). Recent evidence indicates that Brucella is transported through the autophagic pathway before accumulating in the ER (22, 23).

Macrophages are particularly important for the survival and spreading of Brucella during infection (14). The intracellular transport of Brucella in these cells has not been thoroughly characterized. To study the maturation process of Brucella-containing phagosomes in phagocytes, we have monitored the intracellular transport of a virulent strain of B. abortus in J774 macrophages, a well-characterized murine cell line. The normal maturation process of phagosomes has been extensively studied with these macrophages (2). As soon as new phagosomes are formed, they exchange material with early endosomes. This active process permits the recycling of membrane-associated proteins and soluble proteins to the cell surface. As the composition of the phagosomal membrane changes, it becomes fusogenic with late endocytic compartments and the phagosome interacts with lysosomes, acquiring a complex cocktail of hydrolytic enzymes (4, 21, 25).

The aim of the present work was to monitor the interaction of phagosomes containing dead and live B. abortus bacteria with different endocytic compartments in macrophages. The results indicate that, soon after internalization, Brucella alters the transport to hydrolytic compartments and prevents fusion with newly formed endosomes. However, the bacterium does not prevent phagosome acidification and survives in vesicles that do not resemble ER structures.

	MATERIALS AND METHODS

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Reagents, materials, and solutions. LysoSensor (L7535), LysoTracker (L-7528), BCECF AM [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein; acetoxymethyl ester; B1170], TAMRA [5-(and-6)-carboxytetramethylrhodamine; succinimidyl ester; C1171], and DiOC6 (3,3'-dihexyloxacarbocyanine iodide; D273) were from Molecular Probes, Eugene, Oreg. Unless specified, all other reagents were from Sigma Chemical Co., St. Louis, Mo. A polyclonal mouse anti-Brucella antibody was generated in our laboratory, and an immunoglobulin G (IgG) fraction was purified from ascites fluid. Rabbit anti-mouse IgG was obtained from Cappel Organon Teknika Corp., Malvern, Pa., and labeled with ¹²⁵I using chloramine T (final activity, 3 × 10⁶ cpm/µg) (29). Bovine serum albumin (BSA) was mannosylated as previously described (7). Colloidal gold particles were obtained using the citrate reducing method and coated with mannosylated BSA as described previously (17). Eagle basic medium containing 20 mM HEPES-NaOH, pH 7, and supplemented with 5 mg of BSA per ml or 5% fetal calf serum (FCS) was used for short incubations of macrophages (BME).

Bacteria. B. abortus 2308, a virulent smooth strain, was grown at 37°C in Brucella agar (Merck Diagnostica for Microbiology) with 10% CO₂ for 48 h to stationary phase, resuspended in phosphate-buffered saline (PBS), washed, and resuspended in the same buffer (approximately 10¹⁰ CFU/ml) and used immediately. Bacterial numbers were determined by comparing the optical density at 600 nm with a standard curve. Direct bacterial counts (CFU) were determined by plating a serial dilution on Brucella agar and incubating the plate at 37°C for 3 days. When required, the microbes were killed by heating them to 60°C for 60 min. No bacterial growth was observed during 10 days after plating these preparations at 37°C. For some experiments, Brucella was opsonized with a polyclonal mouse anti-Brucella antibody (8 × 10⁷ bacteria were incubated with 2 µg of the antibody in 40 µl of BME for 1 h at 20°C and washed three times with BME). A radiolabeled rabbit anti-mouse IgG antibody was used as a secondary antibody to assess hydrolysis (8 × 10⁷ opsonized bacteria were incubated with 0.3 µg of ¹²⁵I-labeled rabbit anti-mouse antibody in 40 µl of BME for 1 h at 20°C and washed three times with BME). For light microscopy, Brucella was labeled with tetramethylrhodamine (8 × 10⁷ Brucella bacteria were incubated with 5 µg of TAMRA in 50 µl of PBS [pH 8] for 1 h at 20°C and washed five times with BME). To label only live bacteria, Brucella was loaded with BCECF (8 × 10⁷ Brucella bacteria were incubated with 10 µM BCECF AM in 200 µl of BME for 1 h at 25°C and washed five times with BME). Labeling the bacteria with antibodies, TAMRA, or BCECF did not affect the CFU of the preparation.

Bacterium uptake by macrophages. J-774-E clone cells, a murine macrophage cell line, were grown in minimum essential medium containing Earle's salts supplemented with 10% FCS in a 5% CO₂ atmosphere. To label endocytic compartments with colloidal gold particles, the cells were washed with BME and resuspended in the same medium containing 20-nm colloidal gold particles coated with mannosylated BSA. After a 15-min uptake at 37°C, the cells were washed to eliminate noninternalized ligand and incubated at 37°C for 60 min to chase the gold particles into lysosomes. B. abortus (dead or alive, opsonized or not opsonized) bacteria were 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 then incubated at 37°C for 0, 15, and 45 min and 2 and 24 h; fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7); and processed for transmission electron microscopy. For the 24-h time point, 5% FCS replaced BSA in the BME.

Bacterium digestion. Macrophages were grown in six-well plates for 24 to 48 h. The medium was then removed, and cells were inoculated with 1 ml of BME containing opsonized Brucella labeled with radioactive rabbit anti-mouse antibody (200 bacteria/cell, 0.1 cpm/bacterium). Culture plates were centrifuged for 10 min at 170 × g at 20°C and washed three times with BME to remove nonadherent bacteria. Monolayers were incubated with 1 ml of BME. The medium was replaced at 0, 15, 30, and 45 min and 1.5, 2, 3, and 20 h. After the first 15 min, the medium was supplemented with gentamicin (40 µg/ml) in order to kill extracellular Brucella. For the 20-h time point, 5% FCS replaced BSA in the BME. The conditioned media were precipitated with 5% trichloroacetic acid (TCA), and the radioactivity in the pellets and supernatants was measured. At the end of the experiment, the cells were solubilized in 0.5% Triton X-100 and the radioactivity was counted. The percentage of total TCA-soluble radioactivity released into the medium at each time point was calculated. The total amount of counts was obtained by adding the radioactivity of pellets and supernatants and the cell-associated radioactivity at the end of the experiment.

Phagosome acidification. Macrophages were plated for 24 h on coverslips and incubated with opsonized Brucella labeled with TAMRA or BCECF for 1 h at 20°C (100 Brucella bacteria/cell). Cells were then washed with BME and chased for different periods of time at 37°C. The coverslips were mounted in BME containing 40 µg of gentamicin per ml and 5 µM LysoSensor or 1 µM LysoTracker for experiments carried out with TAMRA- or BCECF-labeled Brucella, respectively. Each slide was finally analyzed for up to 30 min in an Eclipse TE300 Nikon microscope equipped with a Hamamatsu Orca 100 camera operated with the Metaview software (Universal Imaging Corp., West Chester, Pa.). Images were taken with two sets of filters (excitation, 510 to 560, and barrier, 590, for TAMRA; and excitation, 450 to 490, and barrier, 520, for LysoSensor) and processed with the Paint Shop Pro program (Jasc Software, Inc., Eden Prairie, Mn.).

Autophagosome and ER labeling. Macrophages were plated for 24 h on coverslips and incubated with opsonized Brucella labeled with TAMRA for 1 h at 20°C (100 Brucella bacteria/cell). Cells were then washed with BME and chased for different periods of time at 37°C. To label autophagosomes, the coverslips were mounted in BME containing 40 µg of gentamicin per ml and 50 µM monodansylcadaverine (MDC). Slides were analyzed for up to 15 min as described above using a set of filters for MDC (excitation, 330 to 380; barrier, 420). To label the ER, the coverslips were fixed for 5 min in 0.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, containing 0.1 M sucrose. After several washes with the sucrose-phosphate buffer, the coverslips were incubated for 10 s with 2.5 µg of DiOC6 per ml in the same buffer. The coverslips were then washed in PBS-sucrose and analyzed as described above using a set of filters for fluorescein (450 to 490; barrier, 520).

	RESULTS

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Colocalization of Brucella and colloidal gold particles loaded in lysosomes. To monitor by electron microscopy the intracellular transport of B. abortus to preformed lysosomes, late endocytic compartments from J774 macrophages were labeled with colloidal gold particles (15-min internalization, 60-min chase). Heat-killed and live bacteria were then internalized for 5 min and chased for up to 24 h. At different time points, the cells were fixed and the colocalization of bacteria and gold particles was quantified by electron microscopy. The results showed that dead bacteria accumulated in gold-containing phagosomes soon after internalization, whereas most live Brucella bacteria remained in gold-free phagosomes for more than 2 h (Fig. 1A). However, after 24 h of uptake about 60% of the phagosomes formed with live Brucella contained colloidal gold particles.

View larger version (17K): [in this window] [in a new window]   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.

View larger version (144K): [in this window] [in a new window]   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.

View larger version (161K): [in this window] [in a new window]   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.

Accessibility of Brucella-containing phagosomes to newly internalized gold particles. The above results indicate that phagosomes containing live Brucella mature with different kinetics than those of normal phagosomes. The question remains whether such phagosomes stay as early phagosomes or whether they represent a different compartment. It is well known that phagosomes are accessible to newly endocytosed markers and that early phagosomes receive newly internalized markers faster than do late phagosomes. In order to assess the accessibility of endocytic markers to phagosomes containing Brucella, heat-killed and live bacteria were internalized for 45 min (early phagosomes) or 120 min (late phagosomes). Afterwards, the cells were incubated with colloidal gold particles and the kinetics of the arrival of gold in the phagosomes was monitored for 60 min. Colloidal gold particles reached early phagosomes loaded with heat-killed bacteria after 15 min of uptake, whereas a 60-min uptake was necessary to reach late phagosomes (Fig. 4). Conversely, phagosomes formed by the internalization of live Brucella were reluctant to incorporate gold particles at either of the time points assessed (Fig. 4).

View larger version (18K): [in this window] [in a new window]   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.

Digestion of Brucella-associated proteins. The above results imply that live Brucella hampers transport to lysosomes whereas the heat-killed bacterium is readily transported to these hydrolytic organelles. To study the arrival of Brucella in proteolytic compartments, opsonized live and heat-killed Brucella bacteria were coated with a radiolabeled antibody. The bacteria were bound to the macrophages and internalized for different periods of time. The release of TCA-soluble radioactivity into the medium was used as an indication of the arrival of the bacteria in a protease-rich compartment. Proteolysis of heat-inactivated Brucella was evident after a few minutes of internalization. The kinetics of digestion of live Brucella was not very different during the first minutes of uptake, but afterwards the rate of release of TCA-soluble radioactivity into the medium was much lower than that with the heat-inactivated bacterium. These results indicate that there was a significant delay in the transport of live bacteria to proteolytic compartments (Fig. 5).

View larger version (18K): [in this window] [in a new window]   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.

Acidification of Brucella-containing phagosomes. As phagosomes mature, the intravesicular pH decreases from 6.5 in newly formed phagosomes to about 4 in mature phagolysosomes. It was important to assess whether the presence of live Brucella could alter the acidification of phagosomes. The pH of phagosomes containing heat-killed and live Brucella was monitored by colocalization with LysoSensor, a weak base probe that accumulates in acidic compartments and fluoresces at acidic pH (pK_a = 5.2). About 40% of the live bacteria colocalize with LysoSensor after 60 min of uptake (Table 1). This percentage had not decreased even after 20 h, indicating that the initial acidification of Brucella-containing phagosomes was not due to the presence of altered bacteria facing digestion (Table 1 and Fig. 6a). In agreement with this observation, 50% of live Brucella bacteria labeled with BCECFa fluorescent marker that accumulates in and is retained exclusively by live bacteriawere present in acidic compartments (Table 1 and Fig. 6b). The percentage of colocalization of heat-killed Brucella was similar or lower than that observed for live Brucella. After 20 h of uptake, heat-killed Brucella was difficult to recognize inside macrophages. The results indicate that Brucella does not abrogate phagosome acidification and that it can survive under low-pH conditions inside macrophages.

View larger version (39K): [in this window] [in a new window]   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.

Limited colocalization of B. abortus with MDC, an autophagosomal marker, and DiOC6, an ER marker. In HeLa cells, Brucella is found transiently in autophagosomes before localizing to the ER (22, 23). According to the electron microscopy observations, a small percentage of phagosomes containing live Brucella present morphological characteristics of autophagosomes in J774 macrophages. To assess whether live and heat-killed Brucella bacteria were transported to autophagosomal compartments in macrophages, the transport of TAMRA-labeled Brucella was monitored in cells stained with MDC, a fluorescent marker for autophagosomes. The results indicate that most of the Brucella-containing phagosomes did not colocalize with MDC as they matured (Table 2 and Fig. 6c). During the first hour of internalization, the percentage of colocalization was similar to that observed for heat-killed bacteria. At later internalization times, the colocalization with MDC decreased for live Brucella and did not change for dead bacteria. To label the ER, the cells were fixed and incubated with DiOC6. Colocalization with the ER marker was very rare for live and heat-killed Brucella bacteria after 2 or 20 h of internalization. According to these results, although a small percentage of B. abortus transiently travel through autophagosomal compartments, most of them survive inside structures not related to autophagosomes or the ER.

	DISCUSSION

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Professional phagocytes are central effector cells in defense against microbial pathogens that kill a variety of microorganisms by ingesting them in phagosomes where they are exposed to a harmful environment. The bactericidal condition inside the vacuole is related to the presence of reactive oxygen species, hydrolytic enzymes, and specialized antimicrobial proteins and peptides. Low pH and depletion of nutrients and cations have also been proposed as part of the toxic conditions in phagosomes (20). Not all microbes that enter into a professional phagocyte are exposed to the same toxic environment. For example, the oxidative burst is triggered by a complex mechanism that depends in part on the receptor engaged in the phagocytic process (19). Also, fusion with specific granules in neutrophils is determined by the receptor involved in the uptake (13). Moreover, some microbes are not really phagocytosed by phagocytes. They invade cells and form a specialized vacuole with characteristics that are controlled by the microbe and not by the phagocyte (18).

It is now known that intracellular microbes have developed a series of strategies to survive inside cells (27). Alteration of the normal process of phagosome maturation has been described for several microorganisms such as Mycobacterium, Legionella, Chlamydia, and Listeria spp. (1, 27). In the case of Brucella, inhibition of phagosome-lysosome fusion has been reported by several authors (9, 22, 23). However, phagosome maturation is a complex process that involves a series of fusions with different endocytic compartments and recycling of membranes and proteins by means of tubular connections and budding of transport vesicles (2). We have monitored by electron microscopy the fusion of newly formed phagosomes with preexisting late endocytic compartments labeled with colloidal gold particles. Additionally, the entrance of newly endocytosed colloidal gold particles into preformed phagosomes containing Brucella was assessed. The results show that Brucella significantly delays fusion with preformed lysosomes and prevents the interaction with newly formed endosomes. Alteration in the intracellular transport of Brucella is also supported by the observation that the arrival of the bacterium in proteolytic compartments was very slow. However, at late time points, a significant percentage of Brucella were found in gold-containing phagosomes with morphological characteristics of phagolysosomes. The presence of B. abortus in phagolysosomes of professional phagocytes has been reported by other authors (5, 11). In contrast to what has been described for other cell types, we observed a very limited colocalization of B. abortus with markers of the autophagosomal pathway and the ER. Moreover, there was no preferential colocalization of the live versus the heat-killed bacterium with these markers.

In other cell types, Brucella also hampers fusion with lysosomes, but is found first in autophagosomal vacuoles and later in vesicles that correspond to specialized regions of the ER (22, 23). According to what is presently known, the differences observed between the intracellular transport of B. abortus in professional phagocytes and that in nonprofessional phagocytes may be a consequence of the same survival strategy. Brucella canby means of a still-unknown mechanismdelay the fusion of newly formed phagosomes with late endocytic compartments. In HeLa cells, the lack of fusion may allow the interaction of the phagosome with early autophagic vesicles that normally fuse with endosomes (15) and presumably with other related endosomal structures such as newly formed phagosomes. The presence of the microbe in the autophagic vesicle would render this vesicle less fusogenic with late endocytic compartments and hamper the maturation of the autophagosome to an autophagolysosome. The fact that autophagosomes have an ER origin (8) may allow the interaction of the Brucella-containing autophagosome with some regions of the ER. In macrophages, which have a more active endocytic route, the mechanism employed by Brucella to delay fusion may not be sufficient to permanently prevent fusion with a late compartment. Hence, phagosomes will eventually mature to phagolysosomes. Autophagosomes interacting with Brucella-containing phagosomes will also mature to autophagolysosomes and not to ER-derived vacuoles. It has been shown that B. abortus expresses specific proteins after phagocytosis, oxidative stress, and acidic pH (26). It would be interesting to know whether the differences between the intracellular destinations of B. abortus in HeLa cells and that in macrophages are reflected in differential patterns of protein expression.

Opsonization of B. abortus bacteria did not affect their ability to prevent fusion with other endocytic compartments. Also, survival inside the macrophage was not significantly affected by entering through the Fc receptor. Similar observations have been made for Brucella suis (24). However, opsonization may affect survival in interferon-treated J774 macrophages (10). The concentration and type of antibody used may also modify the effect of opsonization on Brucella survival (12). Although antibodies may have a role in the relationship between Brucella and the cell host, our results indicate that B. abortus can alter intracellular transport independently of opsonization with specific antibodies.

The delayed fusion with late endocytic compartments does not prevent acidification of the phagosomes. A large percentage of live B. abortus bacteria were present in acidic vacuoles after an overnight incubation. Porte et al. (24) have reported that acidification of phagosomes may favor the survival of B. suis inside J774 macrophages. Acidic intravesicular pH may trigger the expression of several proteins necessary for intracellular survival of the microbe (26). Acidification is important for some transport steps in the endocytic pathway, and alkalinization of vesicles may prevent fusion with late compartments (3). However, a low pH seems to be necessary but not sufficient for fusion, and acidic phagosomes containing Brucella evinced attenuated fusion with late compartments.

Localization of B. abortus to vesicles resembling phagolysosomes was frequent after 24 h of internalization. However, most of the Brucella bacteria within these phagosomes presented an intact morphology, suggesting that they were resistant to the lysosomal environment. It has been reported that the outer membrane of B. abortus is resistant to bactericidal cationic peptides (16) and that phagocytosis induces the production of specific proteins (26). Hence, it is possible that Brucella can resist digestion inside phagolysosomes. Normally, maturation of newly formed phagosomes to phagolysosomes is a fast process. The delayed maturation observed for Brucella phagosomes may be very important to prevent early digestion and to allow the bacteria to express new genes necessary for intracellular survival.

In the future, it will be important to understand at the molecular level the alteration in the mechanism of intracellular transport caused by Brucella and the genes in the bacteria responsible for this remarkable effect.