Opsonized Virulent Brucella abortus Replicates within Nonacidic, Endoplasmic Reticulum-Negative, LAMP-1-Positive Phagosomes in Human Monocyt
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感染与免疫杂志 2005年第6期
Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932
Depatment of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27834
ABSTRACT
Cells in the Brucella spp. are intracellular pathogens that survive and replicate within host monocytes. Brucella maintains persistent infections in animals despite the production of high levels of anti-Brucella-specific antibodies. To determine the effect of antibody opsonization on the ability of Brucella to establish itself within monocytes, the intracellular trafficking of virulent Brucella abortus 2308 and attenuated hfq and bacA mutants was followed in the human monocytic cell line THP-1. Early trafficking events of B. abortus 2308-containing phagosomes (BCP) were indistinguishable from those seen for control particles (heat-killed B. abortus 2308, live Escherichia coli HB101, or latex beads). All phagosomes transiently communicated the early-endosomal compartment and rapidly matured into LAMP-1+, cathepsin D+, and acidic phagosomes. By 2 h postinfection, however, the number of cathepsin D+ BCP was significantly lower for live B. abortus 2308-infected cells than for either Brucella mutant strains or control particles. B. abortus 2308 persisted within these cathepsin D–, LAMP-1+, and acidic vesicles; however, at the onset of intracellular replication, the numbers of acidic B. abortus 2308 BCP decreased while remaining cathepsin D– and LAMP-1+. In contrast to B. abortus 2308, the isogenic hfq and bacA mutants remained in acidic, LAMP-1+ phagosomes and failed to initiate intracellular replication. Notably, markers specific for the host endoplasmic reticulum were absent from the BCPs throughout the course of the infection. Thus, opsonized B. abortus in human monocytes survives within phagosomes that remain in the endosomal pathway and replication of virulent B. abortus 2308 within these vesicles corresponds with an increase in intraphagosomal pH.
INTRODUCTION
Brucella species are bacterial intracellular pathogens of mammals that maintain chronic infections by surviving and replicating within host monocytes and macrophages (34). This intracellular niche is critical for Brucella pathogenesis, as demonstrated by the attenuated nature of mutants defective in in vitro intracellular survival assays and the importance of cell-mediated immunity in controlling Brucella infections (2, 3, 10). In general, successful intracellular pathogens either escape from the phagosome soon after internalization or remain in the phagosome and disrupt the complex host cell trafficking machinery to avoid delivery to the phagosome/lysosomal compartment (21). The maturation of phagosomes into the destructive lysosomal compartment is a highly regulated process that involves the endosomal, phagosomal, and lysosomal trafficking pathways (36). Brucella spp. do not escape from phagosomes, and intracellular survival is achieved by inhibiting phagosome-lysosome fusion (9, 25, 27).
In the absence of opsonizing antibody, internalization of the brucellae into host macrophages is facilitated by the binding of the bacteria to lipid rafts on the host cell plasma membrane (23, 29, 39, 40). There is a considerable amount of experimental evidence, however, that opsonic entry plays an important role in the early stages of Brucella infections (12, 14, 16, 42). Although opsonization of the brucellae with specific immunoglobulin G (IgG) enhances the brucellacidal activity of cultured macrophages, virulent strains of Brucella can still resist killing by these phagocytes and eventually demonstrate net intracellular replication (1, 14, 15, 42). This ability to replicate after opsonin-mediated phagocytosis likely plays an important role in the ability of the brucellae to persist for prolonged periods in the host. It is well established that Brucella-specific IgG levels become elevated during the early stages of infection in both natural hosts and humans, but there is no correlation between the appearance of these serologic responses and resolution of the infection (12, 15, 32). In fact, elevated IgG levels are considered an indicator of active Brucella infections in humans and animals (37).
Studies with cultured murine macrophages have shown that following nonopsonic entry, virulent Brucella strains are trafficked to intracellular compartments that are favorable for intracellular survival and replication and that these intracellular compartments are enriched in membrane components originating from the endoplasmic reticulum (ER) (6, 17). Studies examining the interactions of Brucella melitensis and B. suis strains with cultured human peripheral blood monocytes, on the other hand, demonstrated that the Brucella-containing phagosomes avoid fusion with lysosomes in these host cells, but no association was detected between the Brucella-containing phagosomes and the ER of the human peripheral blood monocytes (30). The experiments described in this report were performed to gain a better understanding of the trafficking patterns of virulent B. abortus 2308 in the human monocytic cell line THP-1 after IgG-mediated phagocytosis and to assess the interaction of the B. abortus 2308-containing phagosomes (BCPs) with components of the host cell ER after entry of the brucellae into these phagocytes via this route. B. abortus hfq and bacA mutants derived from virulent strain 2308 were also included in this study because experimental evidence obtained in the mouse model suggests that the inability of the hfq and bacA mutants to resist killing by macrophages following IgG-mediated entry plays an important role in their inability to maintain chronic infection in experimental hosts (20, 31).
MATERIALS AND METHODS
Bacterial culture. All chemicals were obtained from Sigma-Aldrich unless otherwise stated. Virulent B. abortus laboratory strain 2308 was cultured on trypticase soy agar (TSA; Difco) supplemented with 5% bovine blood (BA) at 37°C under 5% CO2. Attenuated B. abortus mutants Hfq3 (2308 hfq) (31) and KL7 (2308 bacA) (20) were grown on BA supplemented with 50 μg/ml of kanamycin. Escherichia coli HB101 was grown on TSA at 37°C under 5% CO2. A green fluorescent protein (GFP)-expressing derivative of the broad-host-range plasmid pBBR1MCS (designated pBBR1MCS6-Y) (22) was introduced into the B. abortus strains and E. coli HB101 by electroporation, and the bacterial strains carrying this plasmid were maintained on BA or TSA supplemented with 6 μg/ml of chloramphenicol. Heat-killed B. abortus cells were prepared by incubating cell suspensions at 70°C in water for 30 min. Loss of viability was confirmed by plating portions of the heated cell suspension on BA and subsequent incubation at 37°C for 4 days.
THP-1 culture and differentiation into adherent monocytes. The human monocytic cell line THP-1 was cultured in RPMI 1640 medium with 2 mM glutamine supplemented with 1.5 g/ml sodium bicarbonate (Mediatech) and 10% fetal bovine serum (Gemini Bioproducts). Suspension cultures of THP-1 cells were maintained at a cell density between 5 x 105 and 2 x 106 cells/ml of cell culture medium and split two to three times per week. Cell culture viability was monitored by hemocytometer and trypan blue dye exclusion. THP-1 cells growing in suspension were harvested at a density of 1 x 106 cells/ml, resuspended in fresh medium supplemented with 5 nM phorbol myristic acid (PMA) to terminally differentiate the cells into adherent monocytes (35), and placed into 12- or 24-well tissue (Costar, NY) culture plates containing sterile #1 glass coverslips in each well. After overnight culture in the presence of PMA, adherent cells were washed three times gently with phosphate-buffered saline (PBS; pH 7.4) and incubated for an additional 24 h in complete RPMI 1640 medium with no PMA. The additional 24-h incubation following PMA differentiation allowed the cells to increase attachment to the glass surface and develop morphology characteristics similar to those of monocytes.
Intracellular survival and replication of the B. abortus strains in THP-1 cells. THP-1 cells used for the evaluation of the intracellular survival and replication profiles of the B. abortus strains were treated in a similar fashion but plated in 96-well flat-bottom tissue culture plates. Bacterial suspensions were prepared and generated by scraping 48-h cultures of the B. abortus strains grown on BA into screw-cap microfuge tubes containing PBS. E. coli cultures were grown on TSA medium for 24 h prior to harvesting. Pellets of bacteria were resuspended by vigorous vortexing, and numbers of bacteria present in the suspensions were determined by optical density at 600 nm measurements. These suspensions were used to generate dilute Brucella and E. coli preparations using complete RPMI 1640 medium whereby the bacterial density was adjusted to desired levels to account for variations in the numbers of target monocytes. Opsonization took place within these dilute suspensions containing either rabbit anti-Brucella (Difco) or anti-E. coli (Molecular Probes) IgG. Concentrations of antibody necessary to mediate opsonization without agglutinating the bacterial suspensions were achieved using antibody dilutions ranging from 1/2,000 to 1/5,000. Bacterial cell suspensions and antisera were incubated together either at 37°C in a shaking water bath for 20 min or at room temperature for 30 min followed by brief vortexing. Suspensions of opsonized bacteria were added to monocyte monolayers at a multiplicity of infection (bacteria/monocyte ratio) of 10:1 for B. abortus KL7 and E. coli HB101 and 20:1 for B. abortus 2308 and Hfq3. Tissue culture plates were gently agitated by hand and then centrifuged at 4°C for 10 min at 270 x g. Monolayers were washed gently with cold PBS to remove nonadherent bacteria and then incubated in fresh medium for 20 min at 37°C with 5% CO2 to allow for phagocytosis of adherent bacteria. Monolayers were washed three times with PBS to remove any remaining nonadherent bacteria. Fresh media containing 100 μg/ml gentamicin was added following the last washing step to kill adherent, extracellular bacteria. For experiments lasting longer than 2 h, the 100 μg/ml-gentamicin-supplemented medium was replaced with medium containing 10 μg/ml gentamicin after 1 h and the bacteria remained in this medium for the duration of the experiment. Viability of intracellular Brucella was determined by lysing monocytes with 0.1% deoxycholate, diluting suspensions in PBS, and plating aliquots in triplicate on BA medium (31). Percentages of bacterial survival at 24 and 48 h were calculated based on the number of internalized bacteria detected at 1 h postinfection which represents 100% of internalized bacteria. Statistical comparisons were made using Student's t test.
Antibodies and reagents for fluorescence microscopy. Primary antibodies used for immunofluorescence microscopy were as follows: mouse anti-LAMP-1 monoclonal, anti-mannose-6-phosphate receptor (Iowa State Hybridoma), mouse anti-EEA1, anti-calnexin, anti-BiP/GRP74, anti-p115, anti-p230, anti-SRP54, anti-Rab5 (Transduction Laboratories), mouse anti-transferrin receptor (Molecular Probes), rabbit anti-Brucella antibody (Difco), rabbit anti-E. coli antibody (Molecular Probes), and mouse and rabbit anti-cathepsin D (Oncogene). Primary antibodies were used routinely at the concentration of 1/100 except for the following: mouse anti-LAMP-1, 1/20; mouse anti-mannose-6-phosphate receptor, 1/5. Slow-fade and Pro-long antifade mounting solution, DiOC6, and Lysotracker Red DND-99 were also purchased from Molecular Probes.
Immunofluorescence microscopy. Coverslips harboring the adherent and infected monocytes were fixed with freshly prepared 4% paraformaldehyde in PBS (pH 7.4) at room temperature for 20 min. Following fixation, adherent and extracellular bacteria were differentiated from internalized bacteria by differential antibody staining performed prior to permeabilizaing monocytes for antibody staining of cellular antigens for time course experiments under 2 h. Differential staining was initiated by washing monolayers with cold PBS, followed by incubating coverslips in PBS at 1 h at 4°C successively with rabbit anti-Brucella antibody (1/500), followed with IgG-specific anti-rabbit secondary antibodies conjugated with either AMCA or Alexa 350 fluorochromes. Monolayers were routinely permeabilized with bovine serum albumin-PBS solution containing a final concentration of 0.1% saponin (BSP). However, the preferred permeabilization method for monolayers to be incubated either with anti-cathepsin D or with anti-LAMP-1 was treatment with ice-cold methanol for 1 min, followed by multiple washes with ice-cold PBS. Incubations with primary and secondary antibodies (at concentrations indicated above) were at room temperature on a shaking platform for 1 h, at which time coverslips were washed three times with cold BSP and affixed to a glass slide with Pro-long or Slow-fade mounting solutions. Immunofluorescence microscopy was performed with either an Olympus BTX upright microscope equipped with DAPI (4',6'-diamidino-2-phenylindole), eGFP, and Texas Red filter sets, with a cooled charge-coupled device image sensor and MetaView software (Fig. 2 through 5) or a Bio-Rad Radiance 2000 inverted scanning laser confocal microscope equipped with Ar/HeNe/Red Diode lasers and AGR-3 filter configuration (Bio-Rad Lasersharp software) (Fig. 6 through 11). Postacquisition image processing was performed with ImageJ v1.33g (http://rsb.info.nih.gov/ij/index.html).
RESULTS
Human THP-1 monocytes accurately model chronic B. abortus intracellular infection. The infection protocol employed for these experiments was designed to follow the maturation of newly formed Brucella containing phagosomes into vesicles harboring replicating bacteria. Through opsonization, a relatively low multiplicity of infection of 20 bacteria per monocyte can be used, which allows for a sufficient number of monocytes to be infected with a single bacterium, though conditions are needed to study the trafficking of phagosomes containing individual bacteria. Synchronization of bacterial uptake was achieved by low-speed centrifugation at 4°C, followed by rapid warming to 37°C for 20 min to allow for phagocytosis to be completed. Using this protocol with an multiplicity of infection of 20:1, approximately 10% of the monocytes were infected, of which there were typically between 1 and 10 bacteria per macrophage (data not shown).
The intracellular survival and replication profile exhibited by B. abortus 2308 in THP-1 cells under these experimental conditions resembles that displayed by this strain when it is introduced into cultured murine macrophages after opsonization with IgG (3, 12, 16). Specifically, the number of viable intracellular B. abortus 2308 cells decreased between 1 and 24 h postinfection in the THP-1 cells, but net intracelluar replication of strain 2308 was observed in these phagocytes between 24 and 48 h postinfection (Fig. 1A). Likewise, B. abortus Hfq3 (2308 hfq) and KL7 (2308 bacA) displayed significant attenuation in the THP-1 cultures compared to the parental 2308 strain (Fig. 1A), which is consistent with the attenuation displayed by the B. abortus hfq and bacA mutants in cultured murine macrophages and experimentally infected mice (20, 31). Genetic complementation of the hfq mutation in B. abortus Hfq3 with a plasmid-borne copy of hfq (31) and directed reversion of the bacA mutation in B. abortus KL7 through reconstruction of the bacA locus (20) restored virulence of these mutants in THP-1 cells (data not shown). Similar viability experiments performed with the parental B. abortus 2308 strain compared the intracellular survival of Ig opsonized or nonopsonized in THP-1 monocytes. The multiplicity of infection of 20:1 was used for both opsonized and nonopsonized and the same method described above for infection monocytes was employed for these experiments. As expected, the number of bacteria recovered at 1 h postinfection was a log higher for the opsonized bacteria than that for nonopsonized (nonopsonized, 2.8 ± 0.10 log10 CFU; opsonized, 4.2 ± 0.17 log10 CFU). Percent survival calculations account for the differences in internalization between these two groups, and the results show that both opsonized and nonopsonized bacteria are equally capable of replicating within differentiated THP-1 monocytes (Fig. 1B).
Nascent Brucella phagosomes communicate with early and late endosomes. Previous reports have shown that murine monocytes internalize nonopsonized Brucella into phagosomes that communicate with the early endosomes, but not late endosomes/lysosomes (1, 5, 30). To determine if this is also the case for opsonized Brucella internalized by human monocytes, fluorescence microscopy was used to examine the acquisition of various endosomal and phagosomal markers by phagosomes containing either live B. abortus 2308, Hfq3, or KL7; heat-killed B. abortus 2308; live E. coli HB101; or 0.8-μm latex beads. Phagosomes were scored for the presence of these vesicular markers on a per-phagosome basis; thus, a vesicle containing several bacteria would count as one phagosome. For early time points at >1 h, adherent extracellular bacteria were excluded from analysis by performing differential staining that fluorescently labels extracellular bacteria (see Materials and Methods).
(i) Interactions of Brucella containing phagosomes with the early-endosomal compartment. Newly formed phagosomes containing live B. abortus or E. coli strains, heat-killed B. abortus 2308 (Fig. 2), or latex beads (data not shown) rapidly acquired the early-endocytic marker EEA1. EEA1 colocalization was transient and levels dropped rapidly after peaking at 20 min. Additional early-endosomal markers, Rab5 and TfR also transiently associated with phagosomes containing live B. abortus or E. coli strains, heat-killed B. abortus 2308 or latex beads at similar rates to that observed for EEA1 (data not shown). These experimental findings are consistent with earlier reports stating that nascent Brucella phagosomes communicate with early endosomes (1, 5, 30).
(ii) Interactions of Brucella containing phagosomes with the late-endosomal compartment. Maturing phagosomes eventually replace early-endocytic components on the vesicle membrane with those associated with late endosomes and lysosomes such as the mannose-6-phosphate receptor, LAMP-1, the vacuolar ATPase complex, and Rab7 (reviewed in reference 38). This transition is critical not only for general phagosome trafficking but it also represents a key juncture in the development of the replicative niche of the intracellular brucellae since acidification of this compartment during the early stages of infection appears to be necessary for the intracellular replication of these bacteria (18, 28). At 40 min postinfection, phagosomes containing either live B. abortus 2308 or E. coli HB101 or heat-killed B. abortus 2308 were found to be heavily enriched with LAMP-1 (Fig. 3). Maximal levels of LAMP-1 colocalization for all of these phagosomes were observed by 60 min postinfection. In addition to the arrival of LAMP-1, components of the proton pump ATPase were also delivered to these phagosomes, causing the rapid acidification of the lumen of these vesicles. Vesicle acidification was detected using the acidotropic dye Lysotracker (Molecular Probes), which is retained in vesicles with a pH of 5.5 (Fig. 4). Addition of the proton-pump-specific inhibitor bafilomycin A abolished all Lysotracker staining of the B. abortus- and E. coli-containing phagosomes, demonstrating that the observed vesicle acidity was the direct result of an active proton pump complex present in the phagosomal membrane (data not shown). These experimental findings indicate that phagosomes containing B. abortus 2308 do not deviate from the normal early-phagosomal trafficking pathway in THP-1 cells.
Interactions of Brucella-containing phagosomes with lysosomes. Fusion of phagosomes with lysosomes represents the end point of phagosome maturation. Experiments performed with nonopsonized Brucella in murine monocytes demonstrated that phagosomes containing virulent Brucella do not fuse with lysosomes. This altered trafficking is detected as early as 1 h postinfection for live and heat-killed B. suis in the murine macrophage-like cell line J774.A1 (29), while Celli et al. reported cathepsin D levels below 40% throughout the first 24 h postinfection for phagosomes containing B. abortus 2308 in cultured bone marrow-derived macrophages from C57BL6 mice (5). In contrast, we did not see a statistically significant difference between the number of phagosomes containing live or heat-killed B. abortus 2308 or live E. coli HB101 that stained cathepsin D positive in THP-1 cells until 60 min postinfection, when these bacteria were introduced by the opsonic route (Fig. 5). The kinetics of cathepsin D delivery to these phagosomes coincided with the arrival of LAMP-1 (Fig. 3) and acidification of the lumen of the phagosome (Fig. 4). In noninfected cells, cathepsin D-positive lysosomes were always found to be acidic and LAMP-1 positive (data not shown). Thus, a significant number of phagosomes containing live B. abortus 2308 matured into phagolysosomes in the THP-1 cells that were indistinguishable from those containing heat-killed B. abortus 2308 or live E. coli HB101 phagosomes at 20 and 40 min postinfection. As time progressed, however, the percentage of cathepsin D-positive phagosomes containing live B. abortus 2308 phagosomes began to decrease, although the levels of LAMP-1-positive and acidic vesicles containing live B. abortus 2308 remained elevated (Fig. 3 and 4).
B. abortus 2308 replicates in LAMP-1-positive, deacidified vesicles in THP-1 cells. As shown in Fig. 1, the majority of the brucellacidal activity occurs within the initial 24 h postinfection and net intracellular replication of the brucellae is observed between 24 and 48 h postinfection. Accordingly, microscopic analysis of infected THP-1 cells revealed that after 24 h, the number of phagosomes containing multiple brucellae increases, as does the total number of bacteria observed within individual phagosomes as the infection progresses. The increase in the number of bacteria within these vesicles was attributed to bacterial replication, and vesicles harboring these replicating bacteria were deemed replicative vesicles. Interestingly, not all of the intracellular brucellae observed in the THP-1 cells at 24 or 48 h postinfection were found in these replicative vesicles. In fact, many phagosomes containing a single bacterium were seen late in infection in THP-1 cells that also harbored large replicative vesicles containing many brucellae (Fig. 6, 7, and 9).
We suspected that the transition of brucellae from a nonreplicating physiological state into a replication competent state corresponded with a change in the nature of the intraphagosomal environment. To examine this possibility, phagosomes containing live B. abortus 2308 were characterized at various intervals between 2 and 48 h postinfection in the THP-1 cells. During this period, the phagosomes containing B. abortus 2308 displayed the same colocalization patterns as BCPs observed at 2 h postinfection (LAMP-1+ and cathepsin D–). For example, the B. abortus 2308-containing phagosomes were largely LAMP-1 positive (80.0% ± 3.6% positive) and cathepsin D negative (46.3% ± 16%) at 24 h postinfection in these phagocytes. This pattern of high LAMP-1 (Fig. 7 and 9) and low cathepsin D on Brucella phagosomes remained constant for the remainder of the experiment and was also detected surrounding large vesicles that contained numerous densely packed, replicating brucellae.
Despite the constant colocalization of LAMP-1 on phagosomes harboring B. abortus 2308, microscopic analysis of these vesicles at 24 (Fig. 6) and 48 (Fig. 7) h postinfection indicated that these replicative vesicles were not highly acidic, as they did not stain with the fluorescent acidotropic marker. A more detailed analysis focused on phagosomes that contained only single bacteria in the THP-1 cells, since those that harbor multiple bacteria were assumed to have already undergone the transition into a replication-permissive vesicle and to have become nonacidic. Between 2 and 24 h postinfection, the number of single B. abortus 2308 cells residing in acidified (e.g., Lysotracker-positive) phagosomes decreased from 83.1% ± 2% at 2 h to 51% ± 17% by 24 h (Fig. 6). These results demonstrate a positive correlation between the deacidification of single Brucella-containing phagosomes with the ability of Brucella to form large replicative phagosomes.
Attenuated B. abortus mutants Hfq3 (2308 hfq) and KL7 (2308 bacA) remain confined to acidic phagosomes. Unlike B. abortus 2308, the isogenic hfq mutant Hfq3 and bacA mutant KL7 do not exhibit net intracellular replication in THP-1 cells (Fig. 1). Microscopic analysis of infected THP-1 monolayers also showed that in contrast to their parent strain, B. abortus Hfq3 and KL7 remain largely confined to acidic phagosomes for the duration of their intracellular residence in these phagocytes (Fig. 6, graph). These experimental findings are particularly interesting given the fact that the colocalization patterns of Hfq3- and KL7-containing phagosomes with EEA1-, LAMP-1-, and cathepsin D-specific markers were nearly identical to those displayed by B. abortus 2308-containing phagosomes in THP-1 cells at 20 min, 1, 2, 24, 48, and 72 h postinfection. Also, Hfq3- and KL7-containing phagosomes acidified at the same rate in these phagocytes as those containing B. abortus 2308 (data not shown). As shown in Fig. 3 and 5, methanol treatment of infected monolayers for fixation decreases the GFP signal detected for heat-killed B. abortus 2308 cells due to the delivery of these BCP to the degradative phagolysosomal compartment. The persistence of fluorescent B. abortus Hfq and KL7 in acidic phagosomes in THP-1 cells (Fig. 6) strongly suggests that the B. abortus hfq and bacA mutants are able to maintain viability, but are unable to initiate replication, in these intracellular compartments.
Ig-opsonized B. abortus 2308 cells do not colocalize with the ER in THP-1 cells. Markers specific for the membrane of the ER (calnexin, Sec61, and SRP54) and the ER lumen (Bip/GRP74), as well as the ER-tropic fluorescent dye DIOC6, were used in microscopic analysis to examine the B. abortus 2308-containing phagosomes for evidence of interaction with the ER in THP-1 cells. Throughout the course of infection of these phagocytes, the B. abortus 2308-containing phagosomes (including the large replicative vesicles containing multiple bacteria) either were found to be devoid of ER markers entirely or appeared to be located in closed proximity to, but physically separated from, ER-positive structures (Fig. 8 and 9). Specifically, close microscopic inspection of these areas of apparent ER association revealed that the ER-specific markers were more distant from the intracellular brucellae than LAMP-1-specific markers; moreover, the ER markers were contiguous with ER structures in the THP-1 cells and not wholly integrated with the B. abortus 2308-containing phagosomes. It is also noted that latex beads and the attenuated B. abortus hfq and bacA mutants exhibited this same pattern of limited association with the ER in THP-1 cells at 48 h postinfection (Fig. 10). In contrast, THP-1 cells infected with nonopsonized bacteria were frequently found to harbor replicating Brucella within ER-positive compartments (Fig. 11). The distinct ER-positive Brucella-containing vesicles were found only within the monolayers infected with nonopsonized bacteria, although some nonopsonized Brucella cells were also negative for ER markers.
DISCUSSION
These experiments were undertaken to the effect that antibody opsonization had on the ability of Brucella abortus 2308. Intramacrophage survival is the cornerstone of Brucella pathogenesis and affords the brucellae the ability to cause chronic disease in a wide range of host animals (32). Brucellosis in humans presents a chronic debilitating disease known as undulant or Malta fever (41). Brucella-specific immunoglobulin is produced during infection; however, these antibodies appear to promote infection by giving the noninvasive bacteria a rapid means to enter new monocytes and macrophages (1, 14, 15, 42) Experimental evidence suggests that survival of the brucellae within the host cell is achieved by bacterial interference of intracellular trafficking events whereby phagosomes containing Brucella avoid fusion with lysosomes (9, 25, 27). In doing so, this highly adapted intracellular pathogen is not exposed to toxic hydrolases, lipases, and defensins and is allowed to survive and replicate within phagocytes. The virB operon encoding the type IV secretion machinery is necessary for the development of the replicative phagosome within which the brucellae reside in cultured murine macrophages (5), HeLa cells (7), and the human monocytic cell line THP-1 (4) after nonopsonic entry into these cells. The effector molecule or molecules transported by the type IV secretion apparatus and how they influence the trafficking patterns of Brucella-containing vacuoles in these host cells, however, is presently unknown.
The binding of immunoglobulin-opsonized bacteria to the Fc receptor complex on host monocytes and macrophages results in rapid phagocytosis (8). Once the bacteria are engulfed via this route, plasma membrane components are rapidly removed from the nascent phagosomes and these vesicles acquire membrane components associated with early endosomes (24). Thus, it was not surprising to find that virulent B. abortus 2308 cells internalized through IgG-mediated phagocytosis were found within phagosomes that communicated with the early-endocytic compartment in THP-1 cells (Fig. 12). Newly formed B. abortus 2308-containing phagosomes transiently associated with the early-endosomal marker EEA1 soon after internalization by the THP-1 cells. The rates of association and intensity of phagosome staining for EEA1 were equivalent for live or heat-killed B. abortus 2308 and live E. coli HB101 cells. The association of the B. abortus 2308- and E. coli HB101-containing phagosomes with the early-endosomal compartment in the THP-1 cells was further supported by the observation that these phagosomes also exhibit transient colocalization with the transferrin receptor and the endosomal membrane-trafficking protein Rab5 during analysis by immunofluorescence microscopy (data not shown).
Following the loss of endosomal components, phagosomes containing either live or dead B. abortus 2308 or live E. coli HB101 simultaneously acquired the late-endosomal/lysosomal marker LAMP-1 and were acidified by the delivery of the proton pump (V+ATPase), marking the successful transition of these vesicles into the late-endosomal compartment. The majority of these phagosomes also appeared to be destined to form phagolysosomes, as indicated by their early acquisition of the lysosomal hydrolase cathepsin D. As late as 40 min postinfection, most of the phagosomes containing live virulent B. abortus 2308 had fused with lysosomes. Following the 40-min time point, however, the percentage of cathepsin D-positive phagosomes containing B. abortus 2308 decreased significantly at 60 min and was further reduced by 120 min postinfection. No such reduction in cathepsin D levels was observed for phagosomes containing either heat-killed B. abortus 2308 or live E. coli HB101. There are two possible explanations for these results. Either the cathepsin D is lost from the phagosomes containing the live B. abortus 2308 during the period between 40 and 60 min postinfection in the THP-1 cells or a significant number of the B. abortus 2308 cells residing in the cathepsin D-positive phagosomes were killed and subsequently degraded, resulting in an enrichment for the B. abortus 2308-containing phagosomes that were cathepsin D negative at earlier times. The second scenario appears more plausible since the selective removal or destruction of cathepsin D within these vesicles would appear mechanically difficult to perform while retaining other late-endosomal/lysosomal characteristics (e.g., remaining LAMP-1 positive).
Another interesting and potentially informative observation was that there appears to be a distinct temporal correlation between the deacidification of LAMP-1-positive, cathepsin D-negative phagosomes containing single bacteria and the onset of net intracellular Brucella replication. Others have proposed that a rise in intraphagosomal pH beyond a threshold level is necessary before the brucellae can initiate replication in these intracellular compartments (17). Such a requirement is also consistent with the fact that although Brucella strains are generally quite resistant to in vitro exposure to acidic conditions down to pH values of 4 and below (11, 19, 31), these strains are capable of replicating when cultured at pH 5 and above. The mechanism underlying the rise in intraphagosomal pH in the B. abortus 2308-containing phagosomes is unknown, but it is apparently dependent upon the viability of the intracellular brucellae, since a corresponding rise in intravesicular pH was not observed for the phagosomes containing individual heat-killed B. abortus 2308 cells.
Although the brucellae end up residing in an intracellular compartment in host macrophages in which they can survive and replicate, there is considerable evidence that they encounter a variety of environmental stresses during their intracellular residence, including exposure to reactive oxygen intermediates, acidic pH, and nutrient deprivation (17, 32). Genetic evidence suggests that the product of the hfq gene (an RNA binding protein known as host factor I, or HF-I) is required for efficient stationary-phase gene expression in B. abortus 2308. Moreover, the generalized increase in resistance to environmental stresses that accompanies the transition into stationary phase appears to be critical for allowing the brucellae to appropriately adapt to the environmental conditions encountered in the phagosomal compartments of host macrophages. The phenotype displayed by the B. abortus hfq mutant Hfq3 in THP-1 cells is consistent with the HF-I-dependent stationary-phase gene expression playing a role in successful adaptation to the environmental conditions encountered in the replicative phagosome, since this mutant is trafficked to the same acidic, LAMP-1-positive, cathepsin D-negative intracellular compartment in these phagocytes as the parental 2308 strain but does not replicate in these phagosomes.
Although the precise function of the bacA gene product in B. abortus 2308 remains unresolved, genetic evidence suggests that it plays a role in modifying the fatty acid composition of the lipid A moiety of the lipopolysaccharide in this bacterium (13). The B. abortus bacA mutant KL7 also ends up in acidic, LAMP-1-positive, cathepsin D-negative phagosomes in THP-1 cells when it enters these cells via the opsonic route, but like the hfq mutant, KL7 does not appear to be able to replicate within this intracellular compartment. The altered cell envelope of the B. abortus bacA mutant makes this strain more susceptible than its parental strain to a number of environmental stresses, including exposure to acidic pH and membrane-damaging agents such as deoxycholate and sodium dodecyl sulfate (33). Consequently, the intracellular behavior of the B. abortus bacA mutant in THP-1 cells suggests that the lipid A modifications made by BacA play a critical role in allowing the brucellae to resist the environmental conditions encountered in the replicative phagosome.
Studies with cultured murine macrophages and HeLa cells have shown that there is a clear correlation between entry into a replicative phagosome that is enriched in membrane components arising from the host cell ER and intracellular replication of virulent Brucella strains in these host cells (5-7, 26). Thus, it was interesting to find that B. abortus 2308 demonstrated extensive intracellular replication in phagosomes that showed no clear association with ER components when introduced into THP-1 cells via IgG opsonization. It is quite possible that the route of entry into these host cells plays a major role in the divergent nature of these experimental findings. In the earlier studies showing the association between intracellular replication of the brucellae and entrance into the ER-derived replicative phagosome in murine macrophages and HeLa cells, the brucellae were not opsonized and likely entered these cells via their interactions with lipid rafts on the host cell surface (23, 29, 39, 40). Indeed, we observed a significant portion of nonopsonized Brucella in replicative compartments that were positive for ER constituents. It is also important to note that Rittig et al. reported that B. suis- and B. melitensis-containing phagosomes in cultured human peripheral blood monocytes were lacking ER components for both opsonized and nonopsonized brucellae (30). Nevertheless, the studies described here demonstrate that the primary intracellular niche for B. abortus internalized by opsonin-mediated phagocytosis is in phagosomes that are devoid of ER components. Although we predict that internalization by this method is the key contributing factor to the differences between our observations and those in other published reports, there is also considerable likelihood that other variables in the host-pathogen interaction contribute to the outcome of Brucella infection. These contributing factors would include the type and activation status of the monocyte/macrophage population, differences in Brucella spp. pathogenesis, and types of bacterial internalization, as well as possible variation among host species. Further experiments are needed to determine if this intracellular niche reached by opsonized Brucella in the human THP-1 cell line is also observed within human peripheral blood monocytes or within murine monocyte/macrophage cell lines and primary monocytes. Preliminary results with human peripheral blood monocytes support our observations within THP-1 cells whereby opsonized B. abortus cells were found viable in single LAMP-1-positive, ER-negative vesicles at 36 h postinfection.
In summary, the results of the studies described in this report indicate that the membrane composition of the replicative phagosome within which the brucellae survive and replicate within host macrophages differs based upon their route of entry into these phagocytes. Moreover, they show that entrance into an ER-enriched intracellular compartment in these host cells is not an absolute requirement for intracellular survival and replication of the brucellae. These experimental findings have the most relevance with regard to the interactions of the brucellae with host macrophages during the period after the onset of a specific humoral immune response. Although strong IgG responses are induced during Brucella infections both in natural hosts and in humans, there does not appear to be a strong correlation between the responses and resolution of the infection (12, 15, 32). In fact, it has been postulated that Brucella-specific antibodies may actually help the brucellae gain entry into their intracellular niche in host macrophages (12, 14, 16, 42). The capacity of the brucellae to resist killing by host macrophages when taken up by IgG-mediated phagocytosis would certainly be predicted to help these bacteria maintain chronic infections in their hosts. The fact that the brucellae appear to be able to replicate within phagosomes that have progressed to different stages along the endocytic pathway provides further evidence of how well adapted the brucellae are for their intracellular lifestyle in the host.
ACKNOWLEDGMENTS
This work was supported by an NRSA fellowship from the National Institute of Allergy and Infectious Disease (F32-AI056965-01) and by a contract from the United States Army Medical Research and Material Command (DAMD-98-C-4054).
We also thank Kathleen Llorens and Shane Smith for their technical assistance and the Research Core Facility for the use of the confocal microscopy suite.
REFERENCES
1. Arenas, G. N., A. S. Staskevich, A. Aballay, and L. S. Mayorga. 2000. Intracellular trafficking of Brucella abortus in J774 macrophages. Infect. Immun. 68:4255-4263.
2. Baldwin, C. L., and M. Parent. 2002. Fundamentals of host immune response against Brucella abortus: what the mouse model has revealed about control of infection. Vet. Microbiol. 90:367-382.
3. Baldwin, C. L., and A. J. Winter. 1994. Macrophages and Brucella. Immunol. Ser. 60:363-380.
4. Boschiroli, M. L., S. Ouahrani-Bettache, V. Foulongne, S. Michaux-Charachon, G. Bourg, A. Allardet-Servent, C. Cazevieille, J. P. Liautard, M. Ramuz, and D. O'Callaghan. 2002. The Brucella suis virB operon is induced intracellularly in macrophages. Proc. Natl. Acad. Sci. USA 99:1544-1549.
5. Celli, J., C. de Chastellier, D. M. Franchini, J. Pizarro-Cerda, E. Moreno, and J. P. Gorvel. 2003. Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum. J. Exp. Med. 198:545-556.
6. Celli, J., and J. P. Gorvel. 2004. Organelle robbery: Brucella interactions with the endoplasmic reticulum. Curr. Opin. Microbiol. 7:93-97.
7. Comerci, D. J., M. J. Martinez-Lorenzo, R. Sieira, J. P. Gorvel, and R. A. Ugalde. 2001. Essential role of the VirB machinery in the maturation of the Brucella abortus-containing vacuole. Cell. Microbiol. 3:159-168.
8. Coppolino, M. G., M. Krause, P. Hagendorff, D. A. Monner, W. Trimble, S. Grinstein, J. Wehland, and A. S. Sechi. 2001. Evidence for a molecular complex consisting of Fyb/SLAP, SLP-76, Nck, VASP and WASP that links the actin cytoskeleton to Fcgamma receptor signalling during phagocytosis. J. Cell Sci. 114:4307-4318.
9. Detilleux, P. G., B. L. Deyoe, and N. F. Cheville. 1990. Entry and intracellular localization of Brucella spp. in Vero cells: fluorescence and electron microscopy. Vet. Pathol. 27:317-328.
10. Dornand, J., A. Gross, V. Lafont, J. Liautard, J. Oliaro, and J. P. Liautard. 2002. The innate immune response against Brucella in humans. Vet. Microbiol. 90:383-394.
11. Endley, S., D. McMurray, and T. A. Ficht. 2001. Interruption of the cydB locus in Brucella abortus attenuates intracellular survival and virulence in the mouse model of infection. J. Bacteriol. 183:2454-2462.
12. Eze, M. O., L. Yuan, R. M. Crawford, C. M. Paranavitana, T. L. Hadfield, A. K. Bhattacharjee, R. L. Warren, and D. L. Hoover. 2000. Effects of opsonization and gamma interferon on growth of Brucella melitensis 16M in mouse peritoneal macrophages in vitro. Infect. Immun. 68:257-263.
13. Ferguson, G. P., A. Datta, J. Baumgartner, R. M. Roop II, R. W. Carlson, and G. C. Walker. 2004. Similarity to peroxisomal-membrane protein family reveals that Sinorhizobium and Brucella BacA affect lipid-A fatty acids. Proc. Natl. Acad. Sci. USA 101:5012-5017.
14. Gross, A., S. Spiesser, A. Terraza, B. Rouot, E. Caron, and J. Dornand. 1998. Expression and bactericidal activity of nitric oxide synthase in Brucella suis-infected murine macrophages. Infect. Immun. 66:1309-1316.
15. Hoffmann, E. M., and J. J. Houle. 1995. Contradictory roles for antibody and complement in the interaction of Brucella abortus with its host. Crit. Rev. Microbiol. 21:153-163.
16. Jones, S. M., and A. J. Winter. 1992. Survival of virulent and attenuated strains of Brucella abortus in normal and gamma interferon-activated murine peritoneal macrophages. Infect. Immun. 60:3011-3014.
17. Kohler, S., S. Michaux-Charachon, F. Porte, M. Ramuz, and J. P. Liautard. 2003. What is the nature of the replicative niche of a stealthy bug named Brucella Trends Microbiol. 11:215-219.
18. Kohler, S., F. Porte, V. Jubier-Maurin, S. Ouahrani-Bettache, J. Teyssier, and J. P. Liautard. 2002. The intramacrophagic environment of Brucella suis and bacterial response. Vet. Microbiol. 90:299-309.
19. Kulakov, Y. K., P. G. Guigue-Talet, M. R. Ramuz, and D. O'Callaghan. 1997. Response of Brucella suis 1330 and B. canis RM6/66 to growth at acid pH and induction of an adaptive acid tolerance response. Res. Microbiol. 148:145-151.
20. LeVier, K., R. W. Phillips, V. K. Grippe, R. M. Roop II, and G. C. Walker. 2000. Similar requirements of a plant symbiont and a mammalian pathogen for prolonged intracellular survival. Science 287:2492-2493.
21. Meresse, S., O. Steele-Mortimer, E. Moreno, M. Desjardins, B. Finlay, and J. P. Gorvel. 1999. Controlling the maturation of pathogen-containing vacuoles: a matter of life and death. Nat. Cell. Biol. 1:E183-E188.
22. Murphy, E., G. T. Robertson, M. Parent, S. D. Hagius, R. M. Roop II, P. H. Elzer, and C. L. Baldwin. 2002. Major histocompatibility complex class I and II expression on macrophages containing a virulent strain of Brucella abortus measured using green fluorescent protein-expressing brucellae and flow cytometry. FEMS Immunol. Med. Microbiol. 33:191-200.
23. Naroeni, A., and F. Porte. 2002. Role of cholesterol and the ganglioside GM1 in entry and short-term survival of Brucella suis in murine macrophages. Infect. Immun. 70:1640-1644.
24. Pitt, A., L. S. Mayorga, P. D. Stahl, and A. L. Schwartz. 1992. Alterations in the protein composition of maturing phagosomes. J. Clin. Investig. 90:1978-1983.
25. Pizarro-Cerdá, J., S. Meresse, R. G. Parton, G. van der Goot, A. Sola-Landa, I. Lopez-Goi, E. Moreno, and J.-P. Gorvel. 1998. Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes. Infect. Immun. 66:5711-5724.
26. Pizarro-Cerda, J., E. Moreno, and J. P. Gorvel. 2000. Invasion and intracellular trafficking of Brucella abortus in nonphagocytic cells. Microbes Infect. 2:829-835.
27. Pizarro-Cerdá, J., E. Moreno, V. Sanguedolce, J. L. Mege, and J. P. Gorvel. 1998. Virulent Brucella abortus prevents lysosome fusion and is distributed within autophagosome-like compartments. Infect. Immun. 66:2387-2392.
28. Porte, F., J. P. Liautard, and S. Kohler. 1999. Early acidification of phagosomes containing Brucella suis is essential for intracellular survival in murine macrophages. Infect. Immun. 67:4041-4047.
29. Porte, F., A. Naroeni, S. Ouahrani-Bettache, and J. P. Liautard. 2003. Role of the Brucella suis lipopolysaccharide O antigen in phagosomal genesis and in inhibition of phagosome-lysosome fusion in murine macrophages. Infect. Immun. 71:1481-1490.
30. Rittig, M. G., M. T. Alvarez-Martinez, F. Porte, J. P. Liautard, and B. Rouot. 2001. Intracellular survival of Brucella spp. in human monocytes involves conventional uptake but special phagosomes. Infect. Immun. 69:3995-4006.
31. Robertson, G. T., and R. M. Roop II. 1999. The Brucella abortus host factor I (HF-I) protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice. Mol. Microbiol. 34:690-700.
32. Roop, R. M., II, B. H. Bellaire, M. W. Valderas, and J. A. Cardelli. 2004. Adaptation of the brucellae to their intracellular niche. Mol. Microbiol. 52:621-630.
33. Roop, R. M., II, G. T. Robertson, G. P. Ferguson, L. E. Milford, M. E. Winkler, and G. C. Walker. 2002. Seeking a niche: putative contributions of the hfq and bacA gene products to the successful adaptation of the brucellae to their intracellular home. Vet. Microbiol. 90:349-363.
34. Smith, L. D., and T. A. Ficht. 1990. Pathogenesis of Brucella. Crit. Rev. Microbiol. 17:209-230.
35. Takashiba, S., T. E. Van Dyke, S. Amar, Y. Murayama, A. W. Soskolne, and L. Shapira. 1999. Differentiation of monocytes to macrophages primes cells for lipopolysaccharide stimulation via accumulation of cytoplasmic nuclear factor B. Infect. Immun. 67:5573-5578.
36. Tuvim, M. J., R. Adachi, S. Hoffenberg, and B. F. Dickey. 2001. Traffic control: Rab GTPases and the regulation of interorganellar transport. News Physiol. Sci. 16:56-61.
37. Vendrell, J. P., A. M. Conge, M. Segondy, S. Lombroso, M. F. Huguet, A. Bertrand, F. Janbon, and A. Serre. 1992. In vitro antibody secretion by peripheral blood mononuclear cells as an expression of the immune response to Brucella spp. in humans. J. Clin. Microbiol. 30:2200-2203.
38. Vieira, O. V., R. J. Botelho, and S. Grinstein. 2002. Phagosome maturation: aging gracefully. Biochem. J. 366:689-704.
39. Watarai, M., S. Makino, Y. Fujii, K. Okamoto, and T. Shirahata. 2002. Modulation of Brucella-induced macropinocytosis by lipid rafts mediates intracellular replication. Cell. Microbiol. 4:341-355.
40. Watarai, M., S. Makino, M. Michikawa, K. Yanagisawa, S. Murakami, and T. Shirahata. 2002. Macrophage plasma membrane cholesterol contributes to Brucella abortus infection of mice. Infect. Immun. 70:4818-4825.
41. Young, E. J. 1995. An overview of human brucellosis. Clin. Infect. Dis. 21:283-289.
42. Young, E. J., M. Borchert, F. L. Kretzer, and D. M. Musher. 1985. Phagocytosis and killing of Brucella by human polymorphonuclear leukocytes. J. Infect. Dis. 151:682-690.(Bryan H Bellaire, R. Mart)
Depatment of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27834
ABSTRACT
Cells in the Brucella spp. are intracellular pathogens that survive and replicate within host monocytes. Brucella maintains persistent infections in animals despite the production of high levels of anti-Brucella-specific antibodies. To determine the effect of antibody opsonization on the ability of Brucella to establish itself within monocytes, the intracellular trafficking of virulent Brucella abortus 2308 and attenuated hfq and bacA mutants was followed in the human monocytic cell line THP-1. Early trafficking events of B. abortus 2308-containing phagosomes (BCP) were indistinguishable from those seen for control particles (heat-killed B. abortus 2308, live Escherichia coli HB101, or latex beads). All phagosomes transiently communicated the early-endosomal compartment and rapidly matured into LAMP-1+, cathepsin D+, and acidic phagosomes. By 2 h postinfection, however, the number of cathepsin D+ BCP was significantly lower for live B. abortus 2308-infected cells than for either Brucella mutant strains or control particles. B. abortus 2308 persisted within these cathepsin D–, LAMP-1+, and acidic vesicles; however, at the onset of intracellular replication, the numbers of acidic B. abortus 2308 BCP decreased while remaining cathepsin D– and LAMP-1+. In contrast to B. abortus 2308, the isogenic hfq and bacA mutants remained in acidic, LAMP-1+ phagosomes and failed to initiate intracellular replication. Notably, markers specific for the host endoplasmic reticulum were absent from the BCPs throughout the course of the infection. Thus, opsonized B. abortus in human monocytes survives within phagosomes that remain in the endosomal pathway and replication of virulent B. abortus 2308 within these vesicles corresponds with an increase in intraphagosomal pH.
INTRODUCTION
Brucella species are bacterial intracellular pathogens of mammals that maintain chronic infections by surviving and replicating within host monocytes and macrophages (34). This intracellular niche is critical for Brucella pathogenesis, as demonstrated by the attenuated nature of mutants defective in in vitro intracellular survival assays and the importance of cell-mediated immunity in controlling Brucella infections (2, 3, 10). In general, successful intracellular pathogens either escape from the phagosome soon after internalization or remain in the phagosome and disrupt the complex host cell trafficking machinery to avoid delivery to the phagosome/lysosomal compartment (21). The maturation of phagosomes into the destructive lysosomal compartment is a highly regulated process that involves the endosomal, phagosomal, and lysosomal trafficking pathways (36). Brucella spp. do not escape from phagosomes, and intracellular survival is achieved by inhibiting phagosome-lysosome fusion (9, 25, 27).
In the absence of opsonizing antibody, internalization of the brucellae into host macrophages is facilitated by the binding of the bacteria to lipid rafts on the host cell plasma membrane (23, 29, 39, 40). There is a considerable amount of experimental evidence, however, that opsonic entry plays an important role in the early stages of Brucella infections (12, 14, 16, 42). Although opsonization of the brucellae with specific immunoglobulin G (IgG) enhances the brucellacidal activity of cultured macrophages, virulent strains of Brucella can still resist killing by these phagocytes and eventually demonstrate net intracellular replication (1, 14, 15, 42). This ability to replicate after opsonin-mediated phagocytosis likely plays an important role in the ability of the brucellae to persist for prolonged periods in the host. It is well established that Brucella-specific IgG levels become elevated during the early stages of infection in both natural hosts and humans, but there is no correlation between the appearance of these serologic responses and resolution of the infection (12, 15, 32). In fact, elevated IgG levels are considered an indicator of active Brucella infections in humans and animals (37).
Studies with cultured murine macrophages have shown that following nonopsonic entry, virulent Brucella strains are trafficked to intracellular compartments that are favorable for intracellular survival and replication and that these intracellular compartments are enriched in membrane components originating from the endoplasmic reticulum (ER) (6, 17). Studies examining the interactions of Brucella melitensis and B. suis strains with cultured human peripheral blood monocytes, on the other hand, demonstrated that the Brucella-containing phagosomes avoid fusion with lysosomes in these host cells, but no association was detected between the Brucella-containing phagosomes and the ER of the human peripheral blood monocytes (30). The experiments described in this report were performed to gain a better understanding of the trafficking patterns of virulent B. abortus 2308 in the human monocytic cell line THP-1 after IgG-mediated phagocytosis and to assess the interaction of the B. abortus 2308-containing phagosomes (BCPs) with components of the host cell ER after entry of the brucellae into these phagocytes via this route. B. abortus hfq and bacA mutants derived from virulent strain 2308 were also included in this study because experimental evidence obtained in the mouse model suggests that the inability of the hfq and bacA mutants to resist killing by macrophages following IgG-mediated entry plays an important role in their inability to maintain chronic infection in experimental hosts (20, 31).
MATERIALS AND METHODS
Bacterial culture. All chemicals were obtained from Sigma-Aldrich unless otherwise stated. Virulent B. abortus laboratory strain 2308 was cultured on trypticase soy agar (TSA; Difco) supplemented with 5% bovine blood (BA) at 37°C under 5% CO2. Attenuated B. abortus mutants Hfq3 (2308 hfq) (31) and KL7 (2308 bacA) (20) were grown on BA supplemented with 50 μg/ml of kanamycin. Escherichia coli HB101 was grown on TSA at 37°C under 5% CO2. A green fluorescent protein (GFP)-expressing derivative of the broad-host-range plasmid pBBR1MCS (designated pBBR1MCS6-Y) (22) was introduced into the B. abortus strains and E. coli HB101 by electroporation, and the bacterial strains carrying this plasmid were maintained on BA or TSA supplemented with 6 μg/ml of chloramphenicol. Heat-killed B. abortus cells were prepared by incubating cell suspensions at 70°C in water for 30 min. Loss of viability was confirmed by plating portions of the heated cell suspension on BA and subsequent incubation at 37°C for 4 days.
THP-1 culture and differentiation into adherent monocytes. The human monocytic cell line THP-1 was cultured in RPMI 1640 medium with 2 mM glutamine supplemented with 1.5 g/ml sodium bicarbonate (Mediatech) and 10% fetal bovine serum (Gemini Bioproducts). Suspension cultures of THP-1 cells were maintained at a cell density between 5 x 105 and 2 x 106 cells/ml of cell culture medium and split two to three times per week. Cell culture viability was monitored by hemocytometer and trypan blue dye exclusion. THP-1 cells growing in suspension were harvested at a density of 1 x 106 cells/ml, resuspended in fresh medium supplemented with 5 nM phorbol myristic acid (PMA) to terminally differentiate the cells into adherent monocytes (35), and placed into 12- or 24-well tissue (Costar, NY) culture plates containing sterile #1 glass coverslips in each well. After overnight culture in the presence of PMA, adherent cells were washed three times gently with phosphate-buffered saline (PBS; pH 7.4) and incubated for an additional 24 h in complete RPMI 1640 medium with no PMA. The additional 24-h incubation following PMA differentiation allowed the cells to increase attachment to the glass surface and develop morphology characteristics similar to those of monocytes.
Intracellular survival and replication of the B. abortus strains in THP-1 cells. THP-1 cells used for the evaluation of the intracellular survival and replication profiles of the B. abortus strains were treated in a similar fashion but plated in 96-well flat-bottom tissue culture plates. Bacterial suspensions were prepared and generated by scraping 48-h cultures of the B. abortus strains grown on BA into screw-cap microfuge tubes containing PBS. E. coli cultures were grown on TSA medium for 24 h prior to harvesting. Pellets of bacteria were resuspended by vigorous vortexing, and numbers of bacteria present in the suspensions were determined by optical density at 600 nm measurements. These suspensions were used to generate dilute Brucella and E. coli preparations using complete RPMI 1640 medium whereby the bacterial density was adjusted to desired levels to account for variations in the numbers of target monocytes. Opsonization took place within these dilute suspensions containing either rabbit anti-Brucella (Difco) or anti-E. coli (Molecular Probes) IgG. Concentrations of antibody necessary to mediate opsonization without agglutinating the bacterial suspensions were achieved using antibody dilutions ranging from 1/2,000 to 1/5,000. Bacterial cell suspensions and antisera were incubated together either at 37°C in a shaking water bath for 20 min or at room temperature for 30 min followed by brief vortexing. Suspensions of opsonized bacteria were added to monocyte monolayers at a multiplicity of infection (bacteria/monocyte ratio) of 10:1 for B. abortus KL7 and E. coli HB101 and 20:1 for B. abortus 2308 and Hfq3. Tissue culture plates were gently agitated by hand and then centrifuged at 4°C for 10 min at 270 x g. Monolayers were washed gently with cold PBS to remove nonadherent bacteria and then incubated in fresh medium for 20 min at 37°C with 5% CO2 to allow for phagocytosis of adherent bacteria. Monolayers were washed three times with PBS to remove any remaining nonadherent bacteria. Fresh media containing 100 μg/ml gentamicin was added following the last washing step to kill adherent, extracellular bacteria. For experiments lasting longer than 2 h, the 100 μg/ml-gentamicin-supplemented medium was replaced with medium containing 10 μg/ml gentamicin after 1 h and the bacteria remained in this medium for the duration of the experiment. Viability of intracellular Brucella was determined by lysing monocytes with 0.1% deoxycholate, diluting suspensions in PBS, and plating aliquots in triplicate on BA medium (31). Percentages of bacterial survival at 24 and 48 h were calculated based on the number of internalized bacteria detected at 1 h postinfection which represents 100% of internalized bacteria. Statistical comparisons were made using Student's t test.
Antibodies and reagents for fluorescence microscopy. Primary antibodies used for immunofluorescence microscopy were as follows: mouse anti-LAMP-1 monoclonal, anti-mannose-6-phosphate receptor (Iowa State Hybridoma), mouse anti-EEA1, anti-calnexin, anti-BiP/GRP74, anti-p115, anti-p230, anti-SRP54, anti-Rab5 (Transduction Laboratories), mouse anti-transferrin receptor (Molecular Probes), rabbit anti-Brucella antibody (Difco), rabbit anti-E. coli antibody (Molecular Probes), and mouse and rabbit anti-cathepsin D (Oncogene). Primary antibodies were used routinely at the concentration of 1/100 except for the following: mouse anti-LAMP-1, 1/20; mouse anti-mannose-6-phosphate receptor, 1/5. Slow-fade and Pro-long antifade mounting solution, DiOC6, and Lysotracker Red DND-99 were also purchased from Molecular Probes.
Immunofluorescence microscopy. Coverslips harboring the adherent and infected monocytes were fixed with freshly prepared 4% paraformaldehyde in PBS (pH 7.4) at room temperature for 20 min. Following fixation, adherent and extracellular bacteria were differentiated from internalized bacteria by differential antibody staining performed prior to permeabilizaing monocytes for antibody staining of cellular antigens for time course experiments under 2 h. Differential staining was initiated by washing monolayers with cold PBS, followed by incubating coverslips in PBS at 1 h at 4°C successively with rabbit anti-Brucella antibody (1/500), followed with IgG-specific anti-rabbit secondary antibodies conjugated with either AMCA or Alexa 350 fluorochromes. Monolayers were routinely permeabilized with bovine serum albumin-PBS solution containing a final concentration of 0.1% saponin (BSP). However, the preferred permeabilization method for monolayers to be incubated either with anti-cathepsin D or with anti-LAMP-1 was treatment with ice-cold methanol for 1 min, followed by multiple washes with ice-cold PBS. Incubations with primary and secondary antibodies (at concentrations indicated above) were at room temperature on a shaking platform for 1 h, at which time coverslips were washed three times with cold BSP and affixed to a glass slide with Pro-long or Slow-fade mounting solutions. Immunofluorescence microscopy was performed with either an Olympus BTX upright microscope equipped with DAPI (4',6'-diamidino-2-phenylindole), eGFP, and Texas Red filter sets, with a cooled charge-coupled device image sensor and MetaView software (Fig. 2 through 5) or a Bio-Rad Radiance 2000 inverted scanning laser confocal microscope equipped with Ar/HeNe/Red Diode lasers and AGR-3 filter configuration (Bio-Rad Lasersharp software) (Fig. 6 through 11). Postacquisition image processing was performed with ImageJ v1.33g (http://rsb.info.nih.gov/ij/index.html).
RESULTS
Human THP-1 monocytes accurately model chronic B. abortus intracellular infection. The infection protocol employed for these experiments was designed to follow the maturation of newly formed Brucella containing phagosomes into vesicles harboring replicating bacteria. Through opsonization, a relatively low multiplicity of infection of 20 bacteria per monocyte can be used, which allows for a sufficient number of monocytes to be infected with a single bacterium, though conditions are needed to study the trafficking of phagosomes containing individual bacteria. Synchronization of bacterial uptake was achieved by low-speed centrifugation at 4°C, followed by rapid warming to 37°C for 20 min to allow for phagocytosis to be completed. Using this protocol with an multiplicity of infection of 20:1, approximately 10% of the monocytes were infected, of which there were typically between 1 and 10 bacteria per macrophage (data not shown).
The intracellular survival and replication profile exhibited by B. abortus 2308 in THP-1 cells under these experimental conditions resembles that displayed by this strain when it is introduced into cultured murine macrophages after opsonization with IgG (3, 12, 16). Specifically, the number of viable intracellular B. abortus 2308 cells decreased between 1 and 24 h postinfection in the THP-1 cells, but net intracelluar replication of strain 2308 was observed in these phagocytes between 24 and 48 h postinfection (Fig. 1A). Likewise, B. abortus Hfq3 (2308 hfq) and KL7 (2308 bacA) displayed significant attenuation in the THP-1 cultures compared to the parental 2308 strain (Fig. 1A), which is consistent with the attenuation displayed by the B. abortus hfq and bacA mutants in cultured murine macrophages and experimentally infected mice (20, 31). Genetic complementation of the hfq mutation in B. abortus Hfq3 with a plasmid-borne copy of hfq (31) and directed reversion of the bacA mutation in B. abortus KL7 through reconstruction of the bacA locus (20) restored virulence of these mutants in THP-1 cells (data not shown). Similar viability experiments performed with the parental B. abortus 2308 strain compared the intracellular survival of Ig opsonized or nonopsonized in THP-1 monocytes. The multiplicity of infection of 20:1 was used for both opsonized and nonopsonized and the same method described above for infection monocytes was employed for these experiments. As expected, the number of bacteria recovered at 1 h postinfection was a log higher for the opsonized bacteria than that for nonopsonized (nonopsonized, 2.8 ± 0.10 log10 CFU; opsonized, 4.2 ± 0.17 log10 CFU). Percent survival calculations account for the differences in internalization between these two groups, and the results show that both opsonized and nonopsonized bacteria are equally capable of replicating within differentiated THP-1 monocytes (Fig. 1B).
Nascent Brucella phagosomes communicate with early and late endosomes. Previous reports have shown that murine monocytes internalize nonopsonized Brucella into phagosomes that communicate with the early endosomes, but not late endosomes/lysosomes (1, 5, 30). To determine if this is also the case for opsonized Brucella internalized by human monocytes, fluorescence microscopy was used to examine the acquisition of various endosomal and phagosomal markers by phagosomes containing either live B. abortus 2308, Hfq3, or KL7; heat-killed B. abortus 2308; live E. coli HB101; or 0.8-μm latex beads. Phagosomes were scored for the presence of these vesicular markers on a per-phagosome basis; thus, a vesicle containing several bacteria would count as one phagosome. For early time points at >1 h, adherent extracellular bacteria were excluded from analysis by performing differential staining that fluorescently labels extracellular bacteria (see Materials and Methods).
(i) Interactions of Brucella containing phagosomes with the early-endosomal compartment. Newly formed phagosomes containing live B. abortus or E. coli strains, heat-killed B. abortus 2308 (Fig. 2), or latex beads (data not shown) rapidly acquired the early-endocytic marker EEA1. EEA1 colocalization was transient and levels dropped rapidly after peaking at 20 min. Additional early-endosomal markers, Rab5 and TfR also transiently associated with phagosomes containing live B. abortus or E. coli strains, heat-killed B. abortus 2308 or latex beads at similar rates to that observed for EEA1 (data not shown). These experimental findings are consistent with earlier reports stating that nascent Brucella phagosomes communicate with early endosomes (1, 5, 30).
(ii) Interactions of Brucella containing phagosomes with the late-endosomal compartment. Maturing phagosomes eventually replace early-endocytic components on the vesicle membrane with those associated with late endosomes and lysosomes such as the mannose-6-phosphate receptor, LAMP-1, the vacuolar ATPase complex, and Rab7 (reviewed in reference 38). This transition is critical not only for general phagosome trafficking but it also represents a key juncture in the development of the replicative niche of the intracellular brucellae since acidification of this compartment during the early stages of infection appears to be necessary for the intracellular replication of these bacteria (18, 28). At 40 min postinfection, phagosomes containing either live B. abortus 2308 or E. coli HB101 or heat-killed B. abortus 2308 were found to be heavily enriched with LAMP-1 (Fig. 3). Maximal levels of LAMP-1 colocalization for all of these phagosomes were observed by 60 min postinfection. In addition to the arrival of LAMP-1, components of the proton pump ATPase were also delivered to these phagosomes, causing the rapid acidification of the lumen of these vesicles. Vesicle acidification was detected using the acidotropic dye Lysotracker (Molecular Probes), which is retained in vesicles with a pH of 5.5 (Fig. 4). Addition of the proton-pump-specific inhibitor bafilomycin A abolished all Lysotracker staining of the B. abortus- and E. coli-containing phagosomes, demonstrating that the observed vesicle acidity was the direct result of an active proton pump complex present in the phagosomal membrane (data not shown). These experimental findings indicate that phagosomes containing B. abortus 2308 do not deviate from the normal early-phagosomal trafficking pathway in THP-1 cells.
Interactions of Brucella-containing phagosomes with lysosomes. Fusion of phagosomes with lysosomes represents the end point of phagosome maturation. Experiments performed with nonopsonized Brucella in murine monocytes demonstrated that phagosomes containing virulent Brucella do not fuse with lysosomes. This altered trafficking is detected as early as 1 h postinfection for live and heat-killed B. suis in the murine macrophage-like cell line J774.A1 (29), while Celli et al. reported cathepsin D levels below 40% throughout the first 24 h postinfection for phagosomes containing B. abortus 2308 in cultured bone marrow-derived macrophages from C57BL6 mice (5). In contrast, we did not see a statistically significant difference between the number of phagosomes containing live or heat-killed B. abortus 2308 or live E. coli HB101 that stained cathepsin D positive in THP-1 cells until 60 min postinfection, when these bacteria were introduced by the opsonic route (Fig. 5). The kinetics of cathepsin D delivery to these phagosomes coincided with the arrival of LAMP-1 (Fig. 3) and acidification of the lumen of the phagosome (Fig. 4). In noninfected cells, cathepsin D-positive lysosomes were always found to be acidic and LAMP-1 positive (data not shown). Thus, a significant number of phagosomes containing live B. abortus 2308 matured into phagolysosomes in the THP-1 cells that were indistinguishable from those containing heat-killed B. abortus 2308 or live E. coli HB101 phagosomes at 20 and 40 min postinfection. As time progressed, however, the percentage of cathepsin D-positive phagosomes containing live B. abortus 2308 phagosomes began to decrease, although the levels of LAMP-1-positive and acidic vesicles containing live B. abortus 2308 remained elevated (Fig. 3 and 4).
B. abortus 2308 replicates in LAMP-1-positive, deacidified vesicles in THP-1 cells. As shown in Fig. 1, the majority of the brucellacidal activity occurs within the initial 24 h postinfection and net intracellular replication of the brucellae is observed between 24 and 48 h postinfection. Accordingly, microscopic analysis of infected THP-1 cells revealed that after 24 h, the number of phagosomes containing multiple brucellae increases, as does the total number of bacteria observed within individual phagosomes as the infection progresses. The increase in the number of bacteria within these vesicles was attributed to bacterial replication, and vesicles harboring these replicating bacteria were deemed replicative vesicles. Interestingly, not all of the intracellular brucellae observed in the THP-1 cells at 24 or 48 h postinfection were found in these replicative vesicles. In fact, many phagosomes containing a single bacterium were seen late in infection in THP-1 cells that also harbored large replicative vesicles containing many brucellae (Fig. 6, 7, and 9).
We suspected that the transition of brucellae from a nonreplicating physiological state into a replication competent state corresponded with a change in the nature of the intraphagosomal environment. To examine this possibility, phagosomes containing live B. abortus 2308 were characterized at various intervals between 2 and 48 h postinfection in the THP-1 cells. During this period, the phagosomes containing B. abortus 2308 displayed the same colocalization patterns as BCPs observed at 2 h postinfection (LAMP-1+ and cathepsin D–). For example, the B. abortus 2308-containing phagosomes were largely LAMP-1 positive (80.0% ± 3.6% positive) and cathepsin D negative (46.3% ± 16%) at 24 h postinfection in these phagocytes. This pattern of high LAMP-1 (Fig. 7 and 9) and low cathepsin D on Brucella phagosomes remained constant for the remainder of the experiment and was also detected surrounding large vesicles that contained numerous densely packed, replicating brucellae.
Despite the constant colocalization of LAMP-1 on phagosomes harboring B. abortus 2308, microscopic analysis of these vesicles at 24 (Fig. 6) and 48 (Fig. 7) h postinfection indicated that these replicative vesicles were not highly acidic, as they did not stain with the fluorescent acidotropic marker. A more detailed analysis focused on phagosomes that contained only single bacteria in the THP-1 cells, since those that harbor multiple bacteria were assumed to have already undergone the transition into a replication-permissive vesicle and to have become nonacidic. Between 2 and 24 h postinfection, the number of single B. abortus 2308 cells residing in acidified (e.g., Lysotracker-positive) phagosomes decreased from 83.1% ± 2% at 2 h to 51% ± 17% by 24 h (Fig. 6). These results demonstrate a positive correlation between the deacidification of single Brucella-containing phagosomes with the ability of Brucella to form large replicative phagosomes.
Attenuated B. abortus mutants Hfq3 (2308 hfq) and KL7 (2308 bacA) remain confined to acidic phagosomes. Unlike B. abortus 2308, the isogenic hfq mutant Hfq3 and bacA mutant KL7 do not exhibit net intracellular replication in THP-1 cells (Fig. 1). Microscopic analysis of infected THP-1 monolayers also showed that in contrast to their parent strain, B. abortus Hfq3 and KL7 remain largely confined to acidic phagosomes for the duration of their intracellular residence in these phagocytes (Fig. 6, graph). These experimental findings are particularly interesting given the fact that the colocalization patterns of Hfq3- and KL7-containing phagosomes with EEA1-, LAMP-1-, and cathepsin D-specific markers were nearly identical to those displayed by B. abortus 2308-containing phagosomes in THP-1 cells at 20 min, 1, 2, 24, 48, and 72 h postinfection. Also, Hfq3- and KL7-containing phagosomes acidified at the same rate in these phagocytes as those containing B. abortus 2308 (data not shown). As shown in Fig. 3 and 5, methanol treatment of infected monolayers for fixation decreases the GFP signal detected for heat-killed B. abortus 2308 cells due to the delivery of these BCP to the degradative phagolysosomal compartment. The persistence of fluorescent B. abortus Hfq and KL7 in acidic phagosomes in THP-1 cells (Fig. 6) strongly suggests that the B. abortus hfq and bacA mutants are able to maintain viability, but are unable to initiate replication, in these intracellular compartments.
Ig-opsonized B. abortus 2308 cells do not colocalize with the ER in THP-1 cells. Markers specific for the membrane of the ER (calnexin, Sec61, and SRP54) and the ER lumen (Bip/GRP74), as well as the ER-tropic fluorescent dye DIOC6, were used in microscopic analysis to examine the B. abortus 2308-containing phagosomes for evidence of interaction with the ER in THP-1 cells. Throughout the course of infection of these phagocytes, the B. abortus 2308-containing phagosomes (including the large replicative vesicles containing multiple bacteria) either were found to be devoid of ER markers entirely or appeared to be located in closed proximity to, but physically separated from, ER-positive structures (Fig. 8 and 9). Specifically, close microscopic inspection of these areas of apparent ER association revealed that the ER-specific markers were more distant from the intracellular brucellae than LAMP-1-specific markers; moreover, the ER markers were contiguous with ER structures in the THP-1 cells and not wholly integrated with the B. abortus 2308-containing phagosomes. It is also noted that latex beads and the attenuated B. abortus hfq and bacA mutants exhibited this same pattern of limited association with the ER in THP-1 cells at 48 h postinfection (Fig. 10). In contrast, THP-1 cells infected with nonopsonized bacteria were frequently found to harbor replicating Brucella within ER-positive compartments (Fig. 11). The distinct ER-positive Brucella-containing vesicles were found only within the monolayers infected with nonopsonized bacteria, although some nonopsonized Brucella cells were also negative for ER markers.
DISCUSSION
These experiments were undertaken to the effect that antibody opsonization had on the ability of Brucella abortus 2308. Intramacrophage survival is the cornerstone of Brucella pathogenesis and affords the brucellae the ability to cause chronic disease in a wide range of host animals (32). Brucellosis in humans presents a chronic debilitating disease known as undulant or Malta fever (41). Brucella-specific immunoglobulin is produced during infection; however, these antibodies appear to promote infection by giving the noninvasive bacteria a rapid means to enter new monocytes and macrophages (1, 14, 15, 42) Experimental evidence suggests that survival of the brucellae within the host cell is achieved by bacterial interference of intracellular trafficking events whereby phagosomes containing Brucella avoid fusion with lysosomes (9, 25, 27). In doing so, this highly adapted intracellular pathogen is not exposed to toxic hydrolases, lipases, and defensins and is allowed to survive and replicate within phagocytes. The virB operon encoding the type IV secretion machinery is necessary for the development of the replicative phagosome within which the brucellae reside in cultured murine macrophages (5), HeLa cells (7), and the human monocytic cell line THP-1 (4) after nonopsonic entry into these cells. The effector molecule or molecules transported by the type IV secretion apparatus and how they influence the trafficking patterns of Brucella-containing vacuoles in these host cells, however, is presently unknown.
The binding of immunoglobulin-opsonized bacteria to the Fc receptor complex on host monocytes and macrophages results in rapid phagocytosis (8). Once the bacteria are engulfed via this route, plasma membrane components are rapidly removed from the nascent phagosomes and these vesicles acquire membrane components associated with early endosomes (24). Thus, it was not surprising to find that virulent B. abortus 2308 cells internalized through IgG-mediated phagocytosis were found within phagosomes that communicated with the early-endocytic compartment in THP-1 cells (Fig. 12). Newly formed B. abortus 2308-containing phagosomes transiently associated with the early-endosomal marker EEA1 soon after internalization by the THP-1 cells. The rates of association and intensity of phagosome staining for EEA1 were equivalent for live or heat-killed B. abortus 2308 and live E. coli HB101 cells. The association of the B. abortus 2308- and E. coli HB101-containing phagosomes with the early-endosomal compartment in the THP-1 cells was further supported by the observation that these phagosomes also exhibit transient colocalization with the transferrin receptor and the endosomal membrane-trafficking protein Rab5 during analysis by immunofluorescence microscopy (data not shown).
Following the loss of endosomal components, phagosomes containing either live or dead B. abortus 2308 or live E. coli HB101 simultaneously acquired the late-endosomal/lysosomal marker LAMP-1 and were acidified by the delivery of the proton pump (V+ATPase), marking the successful transition of these vesicles into the late-endosomal compartment. The majority of these phagosomes also appeared to be destined to form phagolysosomes, as indicated by their early acquisition of the lysosomal hydrolase cathepsin D. As late as 40 min postinfection, most of the phagosomes containing live virulent B. abortus 2308 had fused with lysosomes. Following the 40-min time point, however, the percentage of cathepsin D-positive phagosomes containing B. abortus 2308 decreased significantly at 60 min and was further reduced by 120 min postinfection. No such reduction in cathepsin D levels was observed for phagosomes containing either heat-killed B. abortus 2308 or live E. coli HB101. There are two possible explanations for these results. Either the cathepsin D is lost from the phagosomes containing the live B. abortus 2308 during the period between 40 and 60 min postinfection in the THP-1 cells or a significant number of the B. abortus 2308 cells residing in the cathepsin D-positive phagosomes were killed and subsequently degraded, resulting in an enrichment for the B. abortus 2308-containing phagosomes that were cathepsin D negative at earlier times. The second scenario appears more plausible since the selective removal or destruction of cathepsin D within these vesicles would appear mechanically difficult to perform while retaining other late-endosomal/lysosomal characteristics (e.g., remaining LAMP-1 positive).
Another interesting and potentially informative observation was that there appears to be a distinct temporal correlation between the deacidification of LAMP-1-positive, cathepsin D-negative phagosomes containing single bacteria and the onset of net intracellular Brucella replication. Others have proposed that a rise in intraphagosomal pH beyond a threshold level is necessary before the brucellae can initiate replication in these intracellular compartments (17). Such a requirement is also consistent with the fact that although Brucella strains are generally quite resistant to in vitro exposure to acidic conditions down to pH values of 4 and below (11, 19, 31), these strains are capable of replicating when cultured at pH 5 and above. The mechanism underlying the rise in intraphagosomal pH in the B. abortus 2308-containing phagosomes is unknown, but it is apparently dependent upon the viability of the intracellular brucellae, since a corresponding rise in intravesicular pH was not observed for the phagosomes containing individual heat-killed B. abortus 2308 cells.
Although the brucellae end up residing in an intracellular compartment in host macrophages in which they can survive and replicate, there is considerable evidence that they encounter a variety of environmental stresses during their intracellular residence, including exposure to reactive oxygen intermediates, acidic pH, and nutrient deprivation (17, 32). Genetic evidence suggests that the product of the hfq gene (an RNA binding protein known as host factor I, or HF-I) is required for efficient stationary-phase gene expression in B. abortus 2308. Moreover, the generalized increase in resistance to environmental stresses that accompanies the transition into stationary phase appears to be critical for allowing the brucellae to appropriately adapt to the environmental conditions encountered in the phagosomal compartments of host macrophages. The phenotype displayed by the B. abortus hfq mutant Hfq3 in THP-1 cells is consistent with the HF-I-dependent stationary-phase gene expression playing a role in successful adaptation to the environmental conditions encountered in the replicative phagosome, since this mutant is trafficked to the same acidic, LAMP-1-positive, cathepsin D-negative intracellular compartment in these phagocytes as the parental 2308 strain but does not replicate in these phagosomes.
Although the precise function of the bacA gene product in B. abortus 2308 remains unresolved, genetic evidence suggests that it plays a role in modifying the fatty acid composition of the lipid A moiety of the lipopolysaccharide in this bacterium (13). The B. abortus bacA mutant KL7 also ends up in acidic, LAMP-1-positive, cathepsin D-negative phagosomes in THP-1 cells when it enters these cells via the opsonic route, but like the hfq mutant, KL7 does not appear to be able to replicate within this intracellular compartment. The altered cell envelope of the B. abortus bacA mutant makes this strain more susceptible than its parental strain to a number of environmental stresses, including exposure to acidic pH and membrane-damaging agents such as deoxycholate and sodium dodecyl sulfate (33). Consequently, the intracellular behavior of the B. abortus bacA mutant in THP-1 cells suggests that the lipid A modifications made by BacA play a critical role in allowing the brucellae to resist the environmental conditions encountered in the replicative phagosome.
Studies with cultured murine macrophages and HeLa cells have shown that there is a clear correlation between entry into a replicative phagosome that is enriched in membrane components arising from the host cell ER and intracellular replication of virulent Brucella strains in these host cells (5-7, 26). Thus, it was interesting to find that B. abortus 2308 demonstrated extensive intracellular replication in phagosomes that showed no clear association with ER components when introduced into THP-1 cells via IgG opsonization. It is quite possible that the route of entry into these host cells plays a major role in the divergent nature of these experimental findings. In the earlier studies showing the association between intracellular replication of the brucellae and entrance into the ER-derived replicative phagosome in murine macrophages and HeLa cells, the brucellae were not opsonized and likely entered these cells via their interactions with lipid rafts on the host cell surface (23, 29, 39, 40). Indeed, we observed a significant portion of nonopsonized Brucella in replicative compartments that were positive for ER constituents. It is also important to note that Rittig et al. reported that B. suis- and B. melitensis-containing phagosomes in cultured human peripheral blood monocytes were lacking ER components for both opsonized and nonopsonized brucellae (30). Nevertheless, the studies described here demonstrate that the primary intracellular niche for B. abortus internalized by opsonin-mediated phagocytosis is in phagosomes that are devoid of ER components. Although we predict that internalization by this method is the key contributing factor to the differences between our observations and those in other published reports, there is also considerable likelihood that other variables in the host-pathogen interaction contribute to the outcome of Brucella infection. These contributing factors would include the type and activation status of the monocyte/macrophage population, differences in Brucella spp. pathogenesis, and types of bacterial internalization, as well as possible variation among host species. Further experiments are needed to determine if this intracellular niche reached by opsonized Brucella in the human THP-1 cell line is also observed within human peripheral blood monocytes or within murine monocyte/macrophage cell lines and primary monocytes. Preliminary results with human peripheral blood monocytes support our observations within THP-1 cells whereby opsonized B. abortus cells were found viable in single LAMP-1-positive, ER-negative vesicles at 36 h postinfection.
In summary, the results of the studies described in this report indicate that the membrane composition of the replicative phagosome within which the brucellae survive and replicate within host macrophages differs based upon their route of entry into these phagocytes. Moreover, they show that entrance into an ER-enriched intracellular compartment in these host cells is not an absolute requirement for intracellular survival and replication of the brucellae. These experimental findings have the most relevance with regard to the interactions of the brucellae with host macrophages during the period after the onset of a specific humoral immune response. Although strong IgG responses are induced during Brucella infections both in natural hosts and in humans, there does not appear to be a strong correlation between the responses and resolution of the infection (12, 15, 32). In fact, it has been postulated that Brucella-specific antibodies may actually help the brucellae gain entry into their intracellular niche in host macrophages (12, 14, 16, 42). The capacity of the brucellae to resist killing by host macrophages when taken up by IgG-mediated phagocytosis would certainly be predicted to help these bacteria maintain chronic infections in their hosts. The fact that the brucellae appear to be able to replicate within phagosomes that have progressed to different stages along the endocytic pathway provides further evidence of how well adapted the brucellae are for their intracellular lifestyle in the host.
ACKNOWLEDGMENTS
This work was supported by an NRSA fellowship from the National Institute of Allergy and Infectious Disease (F32-AI056965-01) and by a contract from the United States Army Medical Research and Material Command (DAMD-98-C-4054).
We also thank Kathleen Llorens and Shane Smith for their technical assistance and the Research Core Facility for the use of the confocal microscopy suite.
REFERENCES
1. Arenas, G. N., A. S. Staskevich, A. Aballay, and L. S. Mayorga. 2000. Intracellular trafficking of Brucella abortus in J774 macrophages. Infect. Immun. 68:4255-4263.
2. Baldwin, C. L., and M. Parent. 2002. Fundamentals of host immune response against Brucella abortus: what the mouse model has revealed about control of infection. Vet. Microbiol. 90:367-382.
3. Baldwin, C. L., and A. J. Winter. 1994. Macrophages and Brucella. Immunol. Ser. 60:363-380.
4. Boschiroli, M. L., S. Ouahrani-Bettache, V. Foulongne, S. Michaux-Charachon, G. Bourg, A. Allardet-Servent, C. Cazevieille, J. P. Liautard, M. Ramuz, and D. O'Callaghan. 2002. The Brucella suis virB operon is induced intracellularly in macrophages. Proc. Natl. Acad. Sci. USA 99:1544-1549.
5. Celli, J., C. de Chastellier, D. M. Franchini, J. Pizarro-Cerda, E. Moreno, and J. P. Gorvel. 2003. Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum. J. Exp. Med. 198:545-556.
6. Celli, J., and J. P. Gorvel. 2004. Organelle robbery: Brucella interactions with the endoplasmic reticulum. Curr. Opin. Microbiol. 7:93-97.
7. Comerci, D. J., M. J. Martinez-Lorenzo, R. Sieira, J. P. Gorvel, and R. A. Ugalde. 2001. Essential role of the VirB machinery in the maturation of the Brucella abortus-containing vacuole. Cell. Microbiol. 3:159-168.
8. Coppolino, M. G., M. Krause, P. Hagendorff, D. A. Monner, W. Trimble, S. Grinstein, J. Wehland, and A. S. Sechi. 2001. Evidence for a molecular complex consisting of Fyb/SLAP, SLP-76, Nck, VASP and WASP that links the actin cytoskeleton to Fcgamma receptor signalling during phagocytosis. J. Cell Sci. 114:4307-4318.
9. Detilleux, P. G., B. L. Deyoe, and N. F. Cheville. 1990. Entry and intracellular localization of Brucella spp. in Vero cells: fluorescence and electron microscopy. Vet. Pathol. 27:317-328.
10. Dornand, J., A. Gross, V. Lafont, J. Liautard, J. Oliaro, and J. P. Liautard. 2002. The innate immune response against Brucella in humans. Vet. Microbiol. 90:383-394.
11. Endley, S., D. McMurray, and T. A. Ficht. 2001. Interruption of the cydB locus in Brucella abortus attenuates intracellular survival and virulence in the mouse model of infection. J. Bacteriol. 183:2454-2462.
12. Eze, M. O., L. Yuan, R. M. Crawford, C. M. Paranavitana, T. L. Hadfield, A. K. Bhattacharjee, R. L. Warren, and D. L. Hoover. 2000. Effects of opsonization and gamma interferon on growth of Brucella melitensis 16M in mouse peritoneal macrophages in vitro. Infect. Immun. 68:257-263.
13. Ferguson, G. P., A. Datta, J. Baumgartner, R. M. Roop II, R. W. Carlson, and G. C. Walker. 2004. Similarity to peroxisomal-membrane protein family reveals that Sinorhizobium and Brucella BacA affect lipid-A fatty acids. Proc. Natl. Acad. Sci. USA 101:5012-5017.
14. Gross, A., S. Spiesser, A. Terraza, B. Rouot, E. Caron, and J. Dornand. 1998. Expression and bactericidal activity of nitric oxide synthase in Brucella suis-infected murine macrophages. Infect. Immun. 66:1309-1316.
15. Hoffmann, E. M., and J. J. Houle. 1995. Contradictory roles for antibody and complement in the interaction of Brucella abortus with its host. Crit. Rev. Microbiol. 21:153-163.
16. Jones, S. M., and A. J. Winter. 1992. Survival of virulent and attenuated strains of Brucella abortus in normal and gamma interferon-activated murine peritoneal macrophages. Infect. Immun. 60:3011-3014.
17. Kohler, S., S. Michaux-Charachon, F. Porte, M. Ramuz, and J. P. Liautard. 2003. What is the nature of the replicative niche of a stealthy bug named Brucella Trends Microbiol. 11:215-219.
18. Kohler, S., F. Porte, V. Jubier-Maurin, S. Ouahrani-Bettache, J. Teyssier, and J. P. Liautard. 2002. The intramacrophagic environment of Brucella suis and bacterial response. Vet. Microbiol. 90:299-309.
19. Kulakov, Y. K., P. G. Guigue-Talet, M. R. Ramuz, and D. O'Callaghan. 1997. Response of Brucella suis 1330 and B. canis RM6/66 to growth at acid pH and induction of an adaptive acid tolerance response. Res. Microbiol. 148:145-151.
20. LeVier, K., R. W. Phillips, V. K. Grippe, R. M. Roop II, and G. C. Walker. 2000. Similar requirements of a plant symbiont and a mammalian pathogen for prolonged intracellular survival. Science 287:2492-2493.
21. Meresse, S., O. Steele-Mortimer, E. Moreno, M. Desjardins, B. Finlay, and J. P. Gorvel. 1999. Controlling the maturation of pathogen-containing vacuoles: a matter of life and death. Nat. Cell. Biol. 1:E183-E188.
22. Murphy, E., G. T. Robertson, M. Parent, S. D. Hagius, R. M. Roop II, P. H. Elzer, and C. L. Baldwin. 2002. Major histocompatibility complex class I and II expression on macrophages containing a virulent strain of Brucella abortus measured using green fluorescent protein-expressing brucellae and flow cytometry. FEMS Immunol. Med. Microbiol. 33:191-200.
23. Naroeni, A., and F. Porte. 2002. Role of cholesterol and the ganglioside GM1 in entry and short-term survival of Brucella suis in murine macrophages. Infect. Immun. 70:1640-1644.
24. Pitt, A., L. S. Mayorga, P. D. Stahl, and A. L. Schwartz. 1992. Alterations in the protein composition of maturing phagosomes. J. Clin. Investig. 90:1978-1983.
25. Pizarro-Cerdá, J., S. Meresse, R. G. Parton, G. van der Goot, A. Sola-Landa, I. Lopez-Goi, E. Moreno, and J.-P. Gorvel. 1998. Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes. Infect. Immun. 66:5711-5724.
26. Pizarro-Cerda, J., E. Moreno, and J. P. Gorvel. 2000. Invasion and intracellular trafficking of Brucella abortus in nonphagocytic cells. Microbes Infect. 2:829-835.
27. Pizarro-Cerdá, J., E. Moreno, V. Sanguedolce, J. L. Mege, and J. P. Gorvel. 1998. Virulent Brucella abortus prevents lysosome fusion and is distributed within autophagosome-like compartments. Infect. Immun. 66:2387-2392.
28. Porte, F., J. P. Liautard, and S. Kohler. 1999. Early acidification of phagosomes containing Brucella suis is essential for intracellular survival in murine macrophages. Infect. Immun. 67:4041-4047.
29. Porte, F., A. Naroeni, S. Ouahrani-Bettache, and J. P. Liautard. 2003. Role of the Brucella suis lipopolysaccharide O antigen in phagosomal genesis and in inhibition of phagosome-lysosome fusion in murine macrophages. Infect. Immun. 71:1481-1490.
30. Rittig, M. G., M. T. Alvarez-Martinez, F. Porte, J. P. Liautard, and B. Rouot. 2001. Intracellular survival of Brucella spp. in human monocytes involves conventional uptake but special phagosomes. Infect. Immun. 69:3995-4006.
31. Robertson, G. T., and R. M. Roop II. 1999. The Brucella abortus host factor I (HF-I) protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice. Mol. Microbiol. 34:690-700.
32. Roop, R. M., II, B. H. Bellaire, M. W. Valderas, and J. A. Cardelli. 2004. Adaptation of the brucellae to their intracellular niche. Mol. Microbiol. 52:621-630.
33. Roop, R. M., II, G. T. Robertson, G. P. Ferguson, L. E. Milford, M. E. Winkler, and G. C. Walker. 2002. Seeking a niche: putative contributions of the hfq and bacA gene products to the successful adaptation of the brucellae to their intracellular home. Vet. Microbiol. 90:349-363.
34. Smith, L. D., and T. A. Ficht. 1990. Pathogenesis of Brucella. Crit. Rev. Microbiol. 17:209-230.
35. Takashiba, S., T. E. Van Dyke, S. Amar, Y. Murayama, A. W. Soskolne, and L. Shapira. 1999. Differentiation of monocytes to macrophages primes cells for lipopolysaccharide stimulation via accumulation of cytoplasmic nuclear factor B. Infect. Immun. 67:5573-5578.
36. Tuvim, M. J., R. Adachi, S. Hoffenberg, and B. F. Dickey. 2001. Traffic control: Rab GTPases and the regulation of interorganellar transport. News Physiol. Sci. 16:56-61.
37. Vendrell, J. P., A. M. Conge, M. Segondy, S. Lombroso, M. F. Huguet, A. Bertrand, F. Janbon, and A. Serre. 1992. In vitro antibody secretion by peripheral blood mononuclear cells as an expression of the immune response to Brucella spp. in humans. J. Clin. Microbiol. 30:2200-2203.
38. Vieira, O. V., R. J. Botelho, and S. Grinstein. 2002. Phagosome maturation: aging gracefully. Biochem. J. 366:689-704.
39. Watarai, M., S. Makino, Y. Fujii, K. Okamoto, and T. Shirahata. 2002. Modulation of Brucella-induced macropinocytosis by lipid rafts mediates intracellular replication. Cell. Microbiol. 4:341-355.
40. Watarai, M., S. Makino, M. Michikawa, K. Yanagisawa, S. Murakami, and T. Shirahata. 2002. Macrophage plasma membrane cholesterol contributes to Brucella abortus infection of mice. Infect. Immun. 70:4818-4825.
41. Young, E. J. 1995. An overview of human brucellosis. Clin. Infect. Dis. 21:283-289.
42. Young, E. J., M. Borchert, F. L. Kretzer, and D. M. Musher. 1985. Phagocytosis and killing of Brucella by human polymorphonuclear leukocytes. J. Infect. Dis. 151:682-690.(Bryan H Bellaire, R. Mart)