INTRODUCTION

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As a response to bacterial attack, phagocytic cells have developed various instruments to kill pathogens. These antimicrobial defense mechanisms comprise the generation of oxygen radicals during the oxidative burst, the acidification of pathogen-containing phagosomes to a harmful, low pH, and the fusion of phagosomes with lysosomes, allowing pathogen killing by defensins or degradation by lysosomal enzymes. Facultatively intracellular bacteria, in return, have developed various strategies to counteract these host cell assaults, leading to survival and multiplication in macrophages (14): (i) escape from the phagosome into the cytoplasm, as reported for Listeria monocytogenes and Shigella spp. (11), (ii) inhibition of phagosome acidification, associated with (iii) the absence of phagosome-lysosome fusion, as described for mycobacteria, Legionella pneumophila, and Chlamydia trachomatis (10, 18, 19, 20, 32, 40, 42), and (iv) adaptation to acidic phagolysosomes, achieved by Coxiella burnetii and Francisella tularensis (4, 12, 18, 28). Salmonella typhimurium, another facultative intracellular bacterium, survives and replicates in an acidic phagosome as well (38). Pathogens have adapted to these hostile growth conditions, imposing numerous stresses on the bacteria by synthesizing a complex set of specific proteins within the intracellular environment (1, 2). In the case of S. typhimurium, phagosome acidification is essential for the activation of virulence gene transcription via a two-component system (3, 15).

Brucella spp. are gram-negative, facultatively intracellular bacteria divided into six species, which infect humans and animals. These organisms belong to the group that can survive and replicate within host phagocytic cells, and multiplication in phagocytes is crucial to the pathogenesis of Brucella infections (25). It has been shown for Brucella abortus that the bacteria multiply within bovine macrophages (35) and remain enclosed in intracellular compartments during phagocyte infection (17). Similar results have been obtained for B. abortus and B. suis in studies using murine and human monocytes or macrophage-like cell lines (8, 16, 22).

However, very little is known about the intracellular compartment containing Brucella spp., and the environmental conditions that the bacteria encounter. It has not been established whether Brucella spp. inhibit phagosome maturation or whether they have developed mechanisms allowing resistance to bactericidal factors encountered along the normal endocytic pathway of the phagocyte. It has been shown that the chaperones GroEL and DnaK are induced during heat shock, reduced pH, and intracellular survival of B. abortus (26, 36), suggesting harsh conditions inside the phagosome. Earlier, we have demonstrated that DnaK is induced under the same conditions in B. suis and that dnaK inactivation abolishes bacterial multiplication in U937-derived phagocytes (23).

As a consequence of these results and earlier work on other intracellular bacteria suggesting a coordinated gene activation in response to the microenvironment encountered inside the phagosome (29), we contributed in this study to the characterization of B. suis-containing vacuoles. We measured by videofluorescence microscopy the phagosomal pH in murine J774 macrophages infected by B. suis and analyzed the influence of vacuolar pH on survival and replication of the pathogen within the macrophages. Our results demonstrate that Brucella-containing phagosomes acidify rapidly and that an acidic environment during the early phase of infection is necessary for the survival and multiplication of B. suis in macrophages.

	MATERIALS AND METHODS

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Reagents. N-Hydroxysuccinimidyl ester 5- (and -6)-carboxyfluorescein (NHS-CF), N-hydroxysuccinimidyl ester 5- (and -6)-carboxytetramethylrhodamine (NHS-Rho), and Oregon Green 488 carboxylic acid succinimidyl ester (Oregon Green 488) were purchased from Molecular Probes (Eugene, Oreg.); bafilomycin A₁ and monensin were purchased from Sigma.

Bacterial strains and growth conditions. The strain used throughout the experiments was B. suis 1330 (ATCC 23444). Bacteria were grown in tryptic soy (TS) broth (Difco Laboratories) at 37°C for 24 h to stationary phase. The bacteria were harvested by centrifugation, washed twice in phosphate-buffered saline (PBS), and used immediately. Killed organisms were obtained either by incubation at 65°C for 30 min or by incubation at 37°C for 1 h with 300 µg of gentamicin per ml.

Preparation of opsonized bacteria. B. suis were opsonized with polyclonal murine anti-Brucella antibodies for 30 min at 37°C and washed once in PBS.

Labeling of bacteria with fluorescent probes. Log-phase cultures (10⁹ bacteria/ml) were washed twice in PBS and resuspended in 1 ml of PBS containing 0.05% Tween 80. After addition of the mix of fluorescent probes NHS-CF or Oregon Green 488 and NHS-Rho (10 µl of each at 10 mg/ml), the suspension was vortexed. The bacteria were incubated at 4°C for 30 min in the dark. The labeled bacteria were centrifuged, and the reaction was stopped by addition of Tris-HCl (pH 8.3) at a final concentration of 100 mM. The bacteria were again incubated at 4°C for 15 min in the dark, washed twice in PBS, and used immediately for infection. Under these conditions 100% of the bacteria were labeled, as controlled by fluorescence microscopy.

Cell culture. J774.A1 cells (ATCC TIB 67), a murine macrophage-like cell line, were grown in RPMI 1640 medium (Gibco/BRL) containing 10% heat-inactivated fetal calf serum and 5 mM glutamine (complete medium) at 37°C and 5% CO₂. Cells were resuspended at 10⁵ cells/ml and cultured for 1 day.

Infection and intracellular viability assay of B. suis in J774 cells. Experiments were performed as described previously (8). Briefly, J774 cells were infected at a multiplicity of infection (MOI) of 20 bacteria per cell with stationary-phase B. suis 1330 in RPMI 1640 for 30 min. Cells were washed three times with PBS and reincubated in RPMI 1640 with 10% fetal calf serum and gentamicin at 30 µg/ml for at least 1 h (1 h postinfection). At different time points, cells were washed once with PBS and lysed in 0.2% Triton X-100. Bacterial counts (CFU) were determined by plating serial dilutions on TS agar and incubation at 37°C for 3 days. The relative intracellular survival has been defined as 100% at 1 h postinfection.

Addition of vacuolar-pH-neutralizing reagents to infected cells. At 1 h postinfection, one of the following reagents was added to J774 cells: 30 mM NH₄Cl, 100 nM bafilomycin, or 50 µM monensin. The effect of these substances was studied by determining the number of viable bacteria 3 and 5 h after addition of the reagents. Infected but untreated cells were analyzed in parallel. All experiments were performed in quadruplicate. A possible toxic effect of the substances on B. suis was tested by exposure in TS broth for 5 h followed by viability assays. Macrophage viability was verified by trypan blue dye exclusion assays.

Measurement of phagosomal pH by fluorescence microscopy. J774 cells were cultured in Lab-Tek chambered coverglass (Nunc) with 400 µl per well of a suspension at 10⁵ cells/ml. After 1 day of incubation, cells were infected for 45 min by adding carboxyfluorescein (CF)-Rho or Oregon Green 488-rhodamine (Rho)-labeled bacteria in serum-free RPMI 1640 medium (200 µl per well) at a ratio of 100 bacteria per cell. Cells were then washed twice in PBS and incubated in complete medium containing gentamicin at 30 µg/ml to eliminate extracellular bacteria. For pH neutralization, bafilomycin A₁ was added at a concentration of 100 nM together with gentamicin at the end of the infection period. At various time points postinfection, the medium was removed and replaced by PBS for observation. For each point, several pairs of images (generally four) were acquired at CF- and Rho-specific wavelengths. Acquisitions were made by videofluorescence microscopy with a Cool View camera (Photonic Science, East Sussex, England) and an image processor linked to an inverted Leica DM IRB microscope (Leica, Rueil-Malmaison, France). Filters used to detect CF or Oregon Green 488 fluorescence consisted of an excitation bandpass filter (450 to 490 nm), a dichroic mirror (510 nm), and an emission bandpass filter (515 to 560 nm). Filters used to detect Rho fluorescence consisted of an excitation bandpass filter (515 to 560 nm), a dichroic mirror (580 nm), and a longpass emission filter (>590 nm). Microscope settings were performed under Rho fluorescence to avoid extensive CF photobleaching, and CF or Oregon Green 488 fluorescence was acquired rapidly. After acquisition, images were analyzed by the imaging system VISIOLAB 1000 (Biocom, Les Ulis, France). For each image, background was defined as the grey-level value corresponding to the majority of pixels. Fluorescent objects were selected by the software on the Rho image (the number of objects per image generally varied from 20 to 50), and the selection was reported on the CF or Oregon Green 488 image. The average grey-level value was calculated for each selection, the background was subtracted from the corresponding value, and the CF/Rho or Oregon Green 488/Rho ratio was calculated for each pair of image. Standard deviations were obtained from the mean of four CF/Rho or Oregon Green 488/Rho ratio values.

	RESULTS

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Labeling of B. suis with fluorescent probes does not affect bacterial viability and intracellular replication of the bacteria. The method that we used to determine the pH in Brucella-containing vacuoles was first described by Oh and Straubinger (32) and is based on the fact that CF emission intensity varies with pH whereas Rho fluorescence intensity is independent of pH and is used as a reference signal. The ratio of CF and Rho fluorescence therefore allows calculation of the pH. The fluorescent products NHS-CF and NHS-Rho had to be covalently bound to the surface of the bacteria prior to infection. Compared to control labelings performed with Escherichia coli in preliminary experiments, the efficiency of CF and Rho labeling of B. suis was always lower, regardless of the labeling conditions with respect to concentrations of probes, temperature, or time of incubation (not shown). With heat-killed B. suis, the labeling efficiency was significantly higher, possibly due to alterations of the bacterial cell surface structure. However, the method was validated for the labeling of live B. suis as well, allowing measurement of the pH of phagosomes containing live or nonviable B. suis.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Intracellular survival of B. suis 1330 in murine J774 cells. Macrophages were infected with unlabeled () and with CF-Rho-labeled (), opsonized bacteria as described in the text. Each point represents the mean ± standard deviation of three experiments.

Quantitation of pH in individual phagosomes containing B. suis. For in situ calibration allowing the calculation of pH from the CF/Rho emission ratio, the ionophore nigericin was used to artificially adjust the intracellular pH to the values defined for the incubation buffers, i.e., pH 3.6, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0. A representative calibration curve is shown in Fig. 2A. For each experiment, an independent calibration curve was established because of variability in labeling intensity and in data acquisition parameters.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Acidification of phagosomes containing live B. suis labeled with CF and Rho. Bacteria were labeled with fluorescent probes and then opsonized with specific antibodies. (A) In situ calibration curve for the calculation of phagosomal pH from the CF/Rho emission ratio. Cells infected with live B. suis were incubated in buffers of defined pH containing the ionophore nigericin. The plotted emission ratios represent the means ± standard deviations calculated from the acquisition of four pairs of images for each pH value. (B) Phagosomal pH after infection with live bacteria. At the times indicated, ranging from 1 to 5 h postinfection, four pairs of CF and Rho images were acquired from every observation, and pH was calculated from the calibration curve. Experiments were done in quadruplicate.

Quantitation of pH in individual phagosomes containing live B. suis, using Oregon Green 488 as reporter of pH. Fluorescein derivatives are the most widely used pH-sensitive probes, with a pK_a of around 6.4, which therefore allows accurate pH measurements in the range 5 to 8. Nevertheless, by using NHS-CF in our experiments, we could determine phagosomal pH values in the range 4 to 8 (Fig. 2A), as also reported previously by Oh and Straubinger (32) and Montcourrier et al. (30). Recently, a new probe, Oregon Green 488, was described as a more appropriate reporter of acid pH, since its pK_a of around 4.7 allows a more accurate measurement of pH in the range 3.5 to 6.0 (41) than CF. Oregon Green 488 has the same maximum excitation and emission wavelengths as CF. Using the method described above, we performed experiments with this probe. A representative calibration curve, obtained with Oregon Green 488-Rho-double-labeled B. suis, is shown in Fig. 3A. The pH dependence was optimal between pH 3.6 and 5.0. Our results revealed that the pH inside phagosomes containing live bacteria varied between 4.2 and 4.5 and remained at this level until at least 5 h postinfection (Fig. 3B). These results confirmed those obtained with CF.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Acidification of phagosomes containing live B. suis labeled with Oregon Green 488 and Rho. Bacteria were labeled with fluorescent probes and then opsonized with specific antibodies. (A) In situ calibration curve for the calculation of phagosomal pH from the Oregon Green/Rho emission ratio. Cells infected with live B. suis were incubated in buffers of defined pH containing the ionophore nigericin. The plotted emission ratios, represent the means ± standard deviations calculated from the acquisition of four pairs of images for each pH value. (B) Phagosomal pH after infection with live bacteria. At the times indicated, ranging from 2 to 5 h postinfection, four pairs of Oregon green 488 and Rho images were acquired from every observation, and pH was calculated from the calibration curve.

Effect of the specific vacuolar proton-ATPases inhibitor bafilomycin A₁ on phagosome acidification. Vacuolar proton-ATPases participate in the variety of mechanisms that have been found to be implicated in the acidification of vacuoles (27). To investigate the nature of the mechanism responsible for acidification of the compartment containing B. suis, we studied the effect of the macrolide antibiotic bafilomycin A₁ on phagosome acidification. BafilomycinA₁ has been described as a specific inhibitor of vacuolar ATPases (38), but it has no toxic effect on B. suis (our control experiments [data not shown]) or on other gram-negative bacteria (6). Acidification of the compartment containing live B. suis was inhibited by bafilomycin A₁ compared to the vacuoles in untreated cells (Fig. 4A). One hour of treatment with the inhibitor was sufficient to obtain a shift of the pH to about 6. 

View larger version (27K): [in this window] [in a new window]   FIG. 4.   Effect of bafilomycin A1 on acidification of phagosomes containing live or gentamicin-killed B. suis. (A) Phagosomal pH after infection with live bacteria, in the absence (open bars) or presence (grey bars) of 100 nM bafilomycin A1, added at the end of the infection period. Live bacteria were labeled with fluorescent probes (CF and Rho) and then opsonized with specific antibodies. (B) Phagosomal pH after infection with killed bacteria, in the absence (open bars) or presence (grey bars) of 100 nM bafilomycin A1. Bacteria were first gentamicin killed, labeled with fluorescent probes (CF and Rho), and then opsonized with specific antibodies. Calibration curves were made for each experiment. Values represent means ± standard deviations.

Effect of bafilomycin A₁ and other vacuolar-pH-neutralizing reagents on short-term survival of B. suis in macrophages. The results described above led us to investigate the importance of low phagosomal pH in the survival and replication of B. suis within J774 cells. In addition to bafilomycin, two other reagents raising the pH of acidic compartments by different mechanisms were used: NH₄Cl as a lysosomotrophic substance, and monensin as a cationic ionophore. One hour postinfection, macrophages were treated with the vacuolar-pH-neutralizing reagents, and viable intracellular bacteria were enumerated following recovery 3 and 5 h later, i.e., 4 and 6 h postinfection. Infected but untreated cells were analyzed in parallel.

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Effects of vacuolar-pH-neutralizing reagents on survival of opsonized (A) or nonopsonized (B) B. suis in macrophages. Bafilomycin (BAF; 100 nM), NH4Cl (30 mM), or monensin (MON; 50 µM) was added to J774 cells 1 h postinfection. The number of surviving bacteria was determined 3 and 5 h later (4 and 6 h postinfection [p.i.]). Percentages are indicated with respect to the number of viable bacteria at 1 h, prior to addition of the reagents, considered to be 100%. NT, no treatment (control). All experiments were performed in quadruplicate. Values represent means ± standard deviations.

Early but not late neutralization of vacuolar pH by ammonium chloride inhibits survival of B. suis in J774 macrophages. The strong reduction of early intracellular survival of opsonized and nonopsonized B. suis observed in the presence of the three vacuolar-pH-neutralizing agents described above led us to investigate the fate of the intracellular bacteria over a longer period of infection, until 48 h. Our study had to be limited to the effects of NH₄Cl, as monensin and bafilomycin were cytotoxic to J774 cells starting at 12 and 24 h postinfection, respectively, whereas 30 mM NH₄Cl had no toxic effect until the end of the experiments (not shown). Early neutralization of intracellular compartments at 90 min after the beginning of infection resulted in a constant decline of the bacteria until their complete eradication at 48 h postinfection, as opposed to infection of untreated cells (Fig. 6). In contrast, the addition of NH₄Cl to J774 cells at 7 h had no effect on normal intracellular growth of B. suis: after an initial reduction in intracellular viability of 30 to 50%, the bacteria multiplied 800- to 1,000-fold (Fig. 6). These results indicated that for intracellular multiplication of B. suis, an acidic intraphagosomal pH is essential only in the early phase of infection. In our system, neutralization at 6 to 7 h after the first contact of B. suis with the macrophage, i.e., when bacteria began to multiply, did not alter the outcome of infection.

View larger version (14K): [in this window] [in a new window]   FIG. 6.   Time-dependent effect of the neutralization of cellular compartments by 30 mM NH4Cl on intracellular survival of B. suis 1330 in murine J774 cells. During the course of infection, macrophages remained untreated () or were treated with NH4Cl at 90 min () or 7 h () after the beginning of infection. Experiments were performed in triplicate, and the values represent means ± standard deviations.

	DISCUSSION

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This work has provided for the first time evidence that in murine macrophages, vacuoles containing live B. suis rapidly acidified to a pH of around 4.0 to 4.5. The mechanism responsible for this acidification was linked to the activity of host cell vacuolar proton-ATPases, which can be specifically inhibited by bafilomycin A₁. In our hands, phagosomes containing killed B. suis acidified to pH values of about 4.5 to 4.7, slightly higher than the values obtained for live brucellae. It is interesting to speculate that live B. suis may enhance phagosome acidification by a yet unknown, active mechanism. In analogy to certain results obtained with S. typhimurium (38), acidification was an early and fast event in the process of macrophage infection: the low pH values were measured 1 h after the infection period and remained at that level for at least 5 h, the duration of the experiment. Analysis at time points beyond 5 h was difficult and unreliable with our approach, due to the division of labeled bacteria and hence dilution of the fluorescent markers within the intracellular bacterial population. Therefore, it cannot be excluded that the pH of the phagosomes varies at later stages of infection.

Quantitation of pH has been feasible only with opsonized bacteria, as the rate of phagocytosis is very poor for nonopsonized brucellae, independently of the bacterial species and the origin of the macrophages used (9, 17). As a consequence, we have no direct experimental data proving that the pH is as acidic in phagosomes containing nonopsonized B. suis. Nevertheless, survival and replication of opsonized and nonopsonized bacteria within macrophages were inhibited by bafilomycin A₁, suggesting vacuolar acidification in both cases. Recently, Oh and Straubinger (32) found little difference in the environmental pH encountered by opsonized and nonopsonized Mycobacterium avium in the intracellular compartments of J774 cells. Other studies, in contrast, have reported the important role of phagocytosis receptors in the intracellular fate of pathogens (5, 21). In the work described here, phagosome acidification by a vacuolar proton-ATPase(s) was undoubtedly a phenomenon associated with B. suis in murine macrophages, independently of the mechanism of bacterial entry. This can be either Fc receptor-mediated phagocytosis involved in uptake of opsonized brucellae or uptake of nonopsonized bacteria that could be mediated by integrins or mannose-binding receptors (7).

Our results furthermore described the survival of B. suis in these acidic compartments. Strong reduction of intracellular bacterial viability by addition of vacuolar-pH-neutralizing reagents may have several reasons: the inhibitors may have side effects on a variety of cellular processes, compromising cell functions necessary for B. suis survival. These effects include inhibition of the interactions between endocytic compartments, dissipation of membrane potentials, and effect on the host cell exocytic pathway. An alternate hypothesis is that the bacterium requires a low-pH environment for survival and multiplication inside the macrophage, at least in the early phase of infection. One explanation for the necessity of an acidic pH in the vacuoles has been given for the intracellular bacterium F. tularensis, where it was found that endosome acidification favors dissociation of iron from transferrin, making it available for the bacteria (12).

On the assumption that B. suis has to be located in an acidic phagosome for survival in macrophages, we defend the hypothesis that low pH acts as an intracellular signal on the regulation of genes involved in survival and multiplication within the phagocytic cell. This has been shown to be true for virulence gene transcription in S. typhimurium, where the acidified phagosome has been previously described as the trigger for PhoP-dependent gene expression (3). Recent studies indicated, however, that changes in pH are not sensed by PhoQ or transmitted to PhoP by a sensor that might respond to pH (15). Two-dimensional gel electrophoretical analysis of protein profiles of the same pathogen revealed that certain subsets of stress-induced proteins are also induced during macrophage infection (1). Studies on proteins of B. abortus induced under stress conditions and during macrophage infection describe the existence of several proteins with increased expression at low pH and inside bovine or murine macrophages (26, 36). We have demonstrated that the molecular chaperone DnaK is induced at low pH in B. suis in vitro and is required for bacterial growth in U937 macrophages (23). Moreover, B. suis maintains its capacity to grow, though slowly, at acid pH as low as pH 4.6 and to resist well pH 3.2 for several hours (24). Thus, acid pH rapidly encountered by B. suis inside the phagosome could be a signal for the induction of a specific set of genes essential for subsequent multiplication within the host cell. The results presented here on the effects of early or late neutralization of Brucella-containing phagosomes on intracellular survival favored this hypothesis. Early neutralization at 1 h postinfection by substances that differ in their mechanisms of action always led to a strong decrease in live intramacrophagic brucellae. Infection assays over 48 h resulted in complete eradication of the bacteria. In contrast, later neutralization, at 7 h, did not affect intracellular multiplication, suggesting that the pH of the phagosome containing B. suis either was still low but not essential for the outcome of infection or had changed to less acidic values. Additional work has to be done on the later stages of brucellae-macrophage interactions to address this question. The characteristic decrease of 30 to 50% in viable intracellular brucellae always observed during the first 7 h of infection might be explained by a lag phase during which part of the bacteria were eliminated before having adapted to this potentially hostile environment. This period coincided with the phase of infection where low pH was essential to allow subsequent multiplication of B. suis. These observations further support the idea of a specific adaptation, possibly by differential gene activation in B. suis during the early phase of infection.

The question is raised whether acidification of phagosomes containing B. suis is linked to phagolysosome fusion. Several reports described a decrease in the fusion of Brucella spp.-containing phagosomes with lysosomes within macrophages (13, 17, 31). Recently, Pizarro-Cerda et al. (33) reported that virulent B. abortus avoid lysosome fusion in HeLa cells. Experiments studying phagolysosome fusion by confocal microscopy revealed that phagosomes containing live B. suis never fused with lysosomes and that in contrast, phagosomes containing killed B. suis fused with these compartments, to be processed along the degradative route of the host cell (34). The process of fusion appeared to be dissociated from the acquisition of a vacuolar proton-ATPase, since phagosome acidification takes place with killed and live bacteria. Fusion and phagosome acidification are clearly regulated by different mechanisms. Live B. suis can avoid killing in the phagosome, either by actively inducing a mechanism which prevents fusion with the lysosome or by following a different, nondegradative route which is still unknown for brucellae but may be similar to that described for S. typhimurium (37). Moreover, our experiments (data not shown) revealed that opsonization of B. suis did not affect intracellular traffic and allowed us to use opsonized bacteria when necessary, for example, for the determination of intraphagosomal pH.

In this study, we reported that phagosomes containing live B. suis are rapidly acidified by a vacuolar proton-ATPase in murine macrophages. The acidic environment is necessary for survival and replication of bacteria within cells. Further investigations on the bacterial factors and the host cell processes involved will contribute to our understanding of the endocytic traffic of B. suis within the macrophage.