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Traffic 2008; 9: 678–694 Blackwell Munksgaard # 2008 The Authors Journal compilation # 2008 Blackwell Publishing Ltd doi: 10.1111/j.1600-0854.2008.00718.x Brucella Intracellular Replication Requires Trafficking Through the Late Endosomal/Lysosomal Compartment Tregei Starr, Tony W. Ng, Tara D. Wehrly, Leigh A. Knodler and Jean Celli* Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA *Corresponding author: Jean Celli, [email protected] Upon entry into mammalian cells, the intracellular pathogen Brucella abortus resides within a membrane-bound compartment, the Brucella-containing vacuole (BCV), the maturation of which is controlled by the bacterium to generate a replicative organelle derived from the endoplasmic reticulum (ER). Prior to reaching the ER, Brucella is believed to ensure its intracellular survival by inhibiting fusion of the intermediate BCV with late endosomes and lysosomes, although such BCVs are acidic and accumulate the lysosomal-associated membrane protein (LAMP-1). Here, we have further examined the nature of intermediate BCVs using confocal microscopy and live cell imaging. We show that BCVs rapidly acquire several late endocytic markers, including the guanosine triphosphatase Rab7 and its effector Rab-interacting lysosomal protein (RILP), and are accessible to fluid-phase markers either delivered to the whole endocytic pathway or preloaded to lysosomes, indicating that BCVs interact with late endosomes and lysosomes. Consistently, intermediate BCVs are acidic and display proteolytic activity up to 12 h postinfection. Expression of dominant-negative Rab7 or overexpression of RILP significantly impaired the ability of bacteria to convert their vacuole into an ER-derived organelle and replicate, indicating that BCV maturation requires interactions with functional late endosomal/ lysosomal compartments. In cells expressing dominantnegative Rab7[T22N], BCVs remained acidic, yet displayed decreased fusion with lysosomes. Taken together, these results demonstrate that BCVs traffic along the endocytic pathway and fuse with lysosomes, and such fusion events are required for further maturation of BCVs into an ER-derived replicative organelle. Key words: Brucella, endocytic pathway, live cell imaging, pathogenesis, phagosome maturation, Rab7, vacuole acidification Received 30 September 2007, revised and accepted for publication 31 January 2008, published online 18 March 2008 Bacteria of the genus Brucella are the etiological agent of brucellosis, a worldwide zoonosis that affects a wide variety of mammals including humans (1). Brucellae are 678 www.traffic.dk intracellular pathogens that enter various cell types during the infection process, including macrophages and epithelial cells (1). Once internalized, Brucella resides within a membrane-bound compartment, the Brucella-containing vacuole (BCV), a modified phagosome in which the bacterium survives and eventually proliferates. Brucella intracellular survival strategies have been elucidated from analysis of BCV trafficking in either macrophage or epithelial cell models (2–4). Based on immunofluorescence and electron microscopy studies in both cell types, it is known that newly formed BCVs interact with early compartments of the endocytic pathway before acquiring and retaining the late endosomal/lysosomal glycoprotein LAMP-1 for up to 12 h post-infection (p.i.) (2,4). Further maturation of BCVs into replicative organelles is characterized by the progressive exclusion of LAMP-1 from the vacuolar membrane, a process that is likely initiated when these vacuoles intercept the secretory pathway at endoplasmic reticulum (ER) exit sites (5), subsequently undergoing fusion with the ER. This process leads to the biogenesis of a replicationpermissive ER-derived organelle (2,4) and is dependent upon the functions of the Brucella type IV secretion system VirB (2,3,6–8). In primary macrophages, monocytes and macrophage-like cells, the majority of internalized bacteria are killed during the first 4–8 h p.i. (2,9,10). The small population of bacteria that survive this initial killing event eventually replicate (2). This disparity in the fate of intracellular Brucella has complicated interpretations of BCV trafficking in phagocytic cells. Although there is evidence that replication-proficient bacteria traffic through a LAMP-1-positive vacuole before reaching the ER (2), it remains possible that these LAMP-1-positive BCVs contain bacteria that will eventually be killed and the replicationproficient bacteria are contained within the small fraction of LAMP-1-negative BCVs. Yet, a fraction of LAMP-1-positive BCVs interact with the ER, while a small percentage of LAMP-1-negative BCVs do not (2), suggesting that LAMP-1 is acquired by replication-proficient BCVs. Despite the early and sustained accumulation of LAMP-1 on BCV membranes, the lack of detection of other late endocytic markers on maturing BCVs has led to the proposal that BCVs have little to no interaction with late endocytic compartments. Instead, it is believed that bacteria rapidly segregate themselves from the endocytic pathway to avoid fusion with terminal lysosomes (2,4). In support of this model, fluorescence microscopy studies of fixed samples have shown that BCVs do not acquire fluid-phase markers from preloaded lysosomes (9), and no Brucella Trafficking Through Lysosomes significant accumulation of lysosomal luminal enzymes, such as cathepsin D, is detectable in BCVs in either macrophages or epithelial cells (2–4). Additionally, electron microscopy studies in the macrophage-like cell line J774A.1 have concluded that BCVs do not significantly fuse with gold-labeled BSA-loaded lysosomes nor do they intercept incoming endocytic traffic (11). However, the lack of detection of luminal antigens or fluid-phase markers by fluorescence or electron microscopy after fixation and permeabilization may simply result from the loss of soluble antigens or markers during fixation procedures, as recently demonstrated for fluorescent dextran in Salmonellacontaining vacuoles (12). In NIH3T3 fibroblast cells, BCV trafficking and bacterial replication are affected by the expression of the constitutively active allele of the small guanosine triphosphatase (GTPase) Rab5, which controls fusion with early endocytic compartments, but not by that of the constitutively active Rab7 (Rab7[Q67L]), which controls fusion with late endosomes and lysosomes (13). However, expression of Rab7[Q67L] does not abrogate the functionality of late endocytic compartments (14), which could potentially skew the conclusions drawn from these experiments. Altogether, the previous studies support a model of BCV trafficking through early endocytic compartments, but because of the technical limitations associated with these studies, a role for late endosomes/ lysosomes cannot be excluded. The current model of BCV segregation from the endocytic pathway does not account for the rapid and sustained acidification of BCVs, which have a luminal pH similar to that found in lysosomal compartments (pH 4–5) (10,15,16), arguing for significant interactions with late endosomal/lysosomal compartments. Moreover, BCV acidification is required for bacterial survival and replication (10,15) and has been linked to the intracellular induction of the virB operon (17), suggesting that it is an essential trait of maturing BCVs. Hence, the available data on the nature of intermediate BCVs and their interactions with intracellular compartments are ambiguous and deserve further investigation. Here, we have used a combination of immunofluorescence microscopy and live cell imaging to further characterize the nature of intermediate BCVs and investigate their potential interactions with late endocytic compartments in established models of Brucella infection of murine bone marrow-derived macrophages (BMMs) and HeLa cells. We show that BCVs acquire various late endocytic markers, including Rab7 and its effector Rabinteracting lysosomal protein (RILP), as well as fluid-phase markers preloaded into lysosomes, demonstrating that BCVs traffic along the endocytic pathway and fuse with lysosomes. More importantly, we show that BCV trafficking and bacterial replication are affected when the functionality of late endocytic compartments is compromised, demonstrating that fusion with these compartments during the intermediate stages of vacuolar maturation is Traffic 2008; 9: 678–694 requisite to trafficking to the ER and subsequent intracellular replication of the bacteria. Results BCV acidification is required for proper trafficking and replication in macrophages and HeLa cells A key process in the maturation of a newly formed phagosome into a degradative phagolysosome is its progressive acidification through sequential interactions with compartments of the endocytic pathway (16). The rapid acidification of phagosomes containing Brucella suis and its importance for Brucella survival and eventual replication in J774A.1 macrophage-like cells (10,15) prompted us to further examine the role of BCV acidification in Brucella abortus intracellular trafficking. Using a model of murine BMM infection with B. abortus (2), we first tested the effect of blocking phagosomal acidification on Brucella intracellular survival. BMMs were left untreated or treated with the vacuolar adenosine triphosphatase (ATPase) inhibitor bafilomycin A1 (BAF), infected with the virulent B. abortus strain 2308 and intracellular viable bacteria were quantified at various times p.i. The number of intracellular viable bacteria in untreated BMMs initially decreased, consistent with previous observations (2), and then increased from 12 to 24 h as a result of bacterial replication (Figure 1A,C). When BAF was added to BMMs 1 h prior to infection and maintained up to 8 h p.i., the number of recoverable bacteria decreased over 24 h, indicating that internalized bacteria did not survive in BAF-treated BMMs (Figure 1A,C). By contrast, treatment of infected BMMs with BAF from 8 to 24 h p.i. did not significantly affect the ability of intracellular Brucella to survive and replicate, as evidenced from the kinetics of intracellular replication in these cells or untreated BMMs (Figure 1A). Taken together, these results confirm and extend previous observations (15) that BCV acidification is essential to B. abortus survival and replication and is required during early, but not late, trafficking events in phagocytic cells. To expand upon these findings, we examined the vacuolar trafficking of B. abortus in BAF-treated BMMs by monitoring the presence of LAMP-1 on BCVs. As previously reported (2), LAMP-1 was rapidly acquired and then progressively excluded from BCVs in untreated cells (Figure 1B,C), concomitant with bacterial replication. By contrast, LAMP-1 in BAF-treated BMMs was retained on BCVs up to 24 h p.i., even after removal of the drug (Figure 1B,C), indicating that BCVs were unable to mature into ERderived, replicative organelles (2). Hence, inhibition of phagosomal acidification during the early stages of BCV maturation leads to a defect in trafficking of bacteria toward the ER. As a consequence, bacteria are degraded within LAMP-1-positive BCVs. The acidification of BCVs in macrophage-like cells (10,15) suggests a sustained interaction of early vacuoles with late 679 Starr et al. Figure 1: Early acidification of BCVs is required for proper trafficking, survival and replication of Brucella in BMMs. A) Effect of inhibition of BCV acidification upon bacterial survival and replication. BMMs were left untreated (open circles), treated with 100 nM BAF from 1 h prior to infection to 8 h p.i. (closed squares) or from 8 to 24 h p.i. (open squares), infected with Brucella abortus 2308 and the viability of intracellular bacteria was monitored over 24 h by enumeration of colony-forming units (CFU). Data are means SD from a representative experiment performed in triplicate. B) Effect of inhibition of BCV acidification upon BCV maturation. BMMs were left untreated (open circles) or treated with 100 nM BAF from 1 h prior to infection to 8 h p.i. (closed circles), infected with DsRedm-expressing B. abortus 2308 and processed for immunostaining of LAMP-1. BCV maturation was monitored over 24 h by measuring colocalization of bacteria with LAMP-1 using confocal microscopy. Data are means SD from three independent experiments. C) Representative confocal micrographs of untreated (control) or BAF-treated BMMs infected with DsRedm-expressing B. abortus 2308 at 4 h p.i. (left-hand panels) and 24 h p.i. (right-hand panels). LAMP-1 was detected using Alexa Fluor 488-conjugated secondary antibodies (shown in green), and DsRedm-expressing bacteria appear in red. Arrows indicate BCVs in whole and close-up images. Scale bars are 10 and 2 mm. compartments of the endocytic pathway, consistent with the demonstrated accumulation of LAMP-1 on BCV membranes for up to 12 h p.i. (Figure 1B) (2–4). To test this hypothesis, we decided to further examine vacuolar trafficking of Brucella. Because of the dual intracellular fate 680 of Brucella in phagocytes, a macrophage infection model is not appropriate to study the vacuolar trafficking of replication-proficient bacteria, which constitute only a small fraction of the intracellular organisms. To circumvent this caveat, we used the established model of Brucella Traffic 2008; 9: 678–694 Brucella Trafficking Through Lysosomes infection of the HeLa epithelial cell line (3–5,8,13,18), first examining the bactericidal activities of these cells toward internalized Brucella. In contrast to BMMs, HeLa cells did not show any significant bactericidal activities during the first 12 h p.i. (Figure 2A), indicating that this model allows for the analysis of only replication-proficient trafficking events. As in BMMs (Figure 1A), treatment of HeLa cells with BAF prior to infection prevented Brucella intracellular growth, while BAF treatment after 8 h p.i. did not show any significant effect on bacterial replication (Figure 2B). However, HeLa cells failed to kill bacteria in BAF-pretreated cells because intracellular viable numbers remained unchanged over 24 h (Figure 2B). Compared with untreated cells where BCVs initially acquired and then excluded LAMP-1, BCVs in BAF-pretreated HeLa cells remained LAMP-1 positive and bacterial replication was inhibited (Figure 2C,D). Hence, inhibition of phagosomal acidification in HeLa cells recapitulated the effects on Brucella trafficking and replication observed in BMMs, with the exception of the late bactericidal effect observed in BMMs pretreated with BAF. BCVs acquire multiple markers of late endosomes/ lysosomes during maturation The above data suggested that perturbation of the endocytic pathway affected BCV trafficking and bacterial replication. To determine the level of interaction of BCVs with the endocytic pathway, particularly late endocytic compartments, we next examined by confocal microscopy the recruitment of various markers of late endocytic compartments to BCVs containing Brucella constitutively expressing a monomeric red fluorescent protein (DsRedm) at early (2 h p.i.), intermediate (6 h p.i.) and late (24 h p.i.) stages of the bacterial intracellular cycle. As previously reported, BCVs were enriched in LAMP-1 at 2 and 6 h p.i. but then excluded this marker (Figure 3A,D), concomitant with the onset of replication (Figure 3D). Interestingly, BCVs also accumulated, and then lost, the late endosomal/multivesicular body marker CD63, with kinetics similar to LAMP-1 (Figure 3B,D), demonstrating that late endocytic markers other than LAMP-1 are transiently recruited to the BCV. Furthermore, in HeLa cells expressing a green fluorescent protein (GFP)–Rab7 fusion, we clearly detected the vacuolar recruitment of Rab7 (Figure 3C) during early and intermediate stages of BCV maturation but not during the Brucella replication phase (Figure 3D). The kinetics of Rab7 accumulation and loss correlated with that of LAMP-1 and CD63 (Figure 3D). In non-EGF-treated HeLa cells, LAMP-1 and Rab7 recruitment to BCVs followed the same kinetics as that in EGF-treated cells (Figure S1), ruling out that such trafficking events are because of the use of EGF to promote Brucella entry into non-phagocytic cells. We conclude that BCVs transiently accumulate numerous late endocytic proteins during their early and intermediate stages of maturation, implying that BCVs interact and fuse with late endocytic compartments. Traffic 2008; 9: 678–694 BCVs are accessible to late endocytic traffic during maturation To further delineate the extent of BCV interactions with the endocytic pathway, we used high-resolution live cell confocal microscopy to assess whether BCVs are accessible to incoming endocytic traffic. HeLa cells were preloaded overnight with Alexa FluorÒ 546-conjugated dextran, infected with GFP-expressing Brucella in the absence of dextran and then reincubated with dextran for the remainder of the experiment. Under these conditions, the entire endocytic pathway is labeled with dextran. We found that early (3 h p.i.) and intermediate (6 h p.i.), but not late (12 h p.i.), BCVs were dextran positive (Figure 4A,B and data not shown; see also Movies S1 and S2), demonstrating the transfer of luminal content between endocytic compartments and BCVs. Quantification of such fusion events revealed that the majority of BCVs were accessible to fluid-phase markers during early (3 h p.i.) and intermediate (6 h p.i.) trafficking stages, with 69 4.6 and 66 16% of BCVs being dextran positive at 3 and 6 h p.i., respectively (Figure 4C). Thereafter, the percentage of dextran-positive BCVs progressively decreased to 20 10% by 12 h p.i. At this stage in the intracellular cycle, most bacteria were clustered in dextran-negative replicative BCVs (Figure 4B,C). Hence, BCVs are accessible to fluid-phase markers delivered from endocytic compartments during the early and intermediate stages of maturation but lose their fusogenicity with endocytic vesicles concomitant with the biogenesis of the replicative vacuole. Given our finding that BCVs transiently fuse with endocytic compartments and recruit late endocytic markers, we next reassessed whether BCVs also fuse with the lysosomal compartment during maturation. For this purpose, HeLa cells were loaded with Alexa Fluor 546-conjugated dextran the day prior to infection and the loaded dextran was chased overnight to lysosomes. Cells were then infected and further incubated in the absence of fluorescent dextran. We clearly detected dextran-positive BCVs interacting with dextran-loaded lysosomal vesicles at 3 and 6 h p.i. (Figure 5A, arrowhead and data not shown; see also Movie S3 and Figure S1). In fact, the majority of BCVs accumulated dextran at 3 and 6 h p.i. (75 13 and 81 9% respectively; Figure 5D). This clearly demonstrates that Brucella interacts with lysosomes and that lysosome fusion is not prevented during early/intermediate BCVs maturation. However, by 12 h p.i., most of the BCVs were replicative and no longer dextran positive (27 6%; Figure 5B,D; see also Movie S4). At this time-point, loaded lysosomes were also seen to dock to, but not fuse with, BCVs (Figure 5B, arrowhead). In conclusion, our live cell imaging studies support a model of Brucella trafficking along the endocytic pathway to the lysosomal compartment before establishing a non-fusogenic, replication-permissive niche. To independently confirm that BCVs interact with lysosomes, we examined whether they acquire lysosomal 681 Starr et al. Figure 2: Early acidification of BCVs is required for proper trafficking, survival and replication of Brucella in HeLa cells. A) Intracellular viability and replication of Brucella in murine BMMs and HeLa cells. BMMs (open circles) or HeLa cells (closed circles) were infected with Brucella abortus 2308 as described in the Materials and Methods, and the viability of intracellular bacteria was monitored over 24 h by enumeration of CFU. Data are means SD from a representative experiment performed in triplicate. B) Effect of inhibition of BCV acidification upon bacterial survival and replication. HeLa cells were left untreated (open circles), treated with 100 nM BAF from 1 h prior to infection to 8 h p.i. (closed squares) or from 8 to 24 h p.i. (open squares), infected with B. abortus 2308 and the viability of intracellular bacteria was monitored over 24 h by enumeration of CFU. Data are means SD from a representative experiment performed in triplicate. C) Effect of inhibition of BCV acidification upon BCV maturation. HeLa cells were left untreated (open circles) or treated with 100 nM BAF from 1 h prior to infection to 8 h p.i. (closed circles), infected with DsRedm-expressing B. abortus 2308 and processed for immunostaining of LAMP-1. BCV maturation was monitored over 24 h from colocalization of bacteria with LAMP-1 using confocal microscopy. Data are means SD from three independent experiments. D) Representative confocal micrographs of untreated (control) or BAF-treated HeLa cells infected with DsRedm-expressing B. abortus 2308 at 4 h p.i. (left-hand panels) and 24 h p.i. (right-hand panels). LAMP-1 was detected using Alexa Fluor 488-conjugated secondary antibodies (shown in green), and DsRedm-expressing bacteria appear in red. Arrows indicate BCVs in whole and close-up images. Scale bars are 10 and 2 mm. CFU, colony-forming units. properties such as proteolytic activity. For this purpose, we used DQ-Red BSA, a fluorescent probe for proteolytic activity based on BODIPY fluorescence dequenching upon proteolysis of conjugated BSA (19). Cells were preloaded with DQ-Red BSA overnight, infected in the presence of the fluorescent probe and analyzed by live cell confocal microscopy. In HeLa cells infected with GFP-expressing Brucella, the majority of BCVs clearly displayed DQ-Red fluorescence at 6 h p.i. (79 3.2% of positive BCVs; Figure 5C, arrowhead and Figure 5D), demonstrating vacuolar proteolytic activities, while replicative BCVs at 12 h 682 p.i. did not display significant fluorescence (12 6.5% of positive BCVs; Figure 5C,D). Consistently, the percentage of DQ-Red-positive BCVs paralleled that of vacuoles acquiring dextran from lysosomes during the first 12 h p.i. (Figure 5D). In addition, BCVs were acidic at 4 and 10 h p.i. (Figure 9A), but not at 24 h p.i. during extensive replication, consistent with previous results obtained in J774A.1 cells (10,15). To evaluate the extent of BCV fusion with lysosomes, we quantified dextran accumulation in vacuoles containing Traffic 2008; 9: 678–694 Brucella Trafficking Through Lysosomes Figure 3: Maturing BCVs acquire various late endocytic markers. A) Representative confocal micrographs of HeLa cells infected with DsRedm-expressing Brucella abortus 2308 (red) and immunostained for A) LAMP-1 (green) and B) CD63 (green). HeLa cells were infected, processed for immunofluorescence labeling at 2, 6 and 24 h p.i. and analyzed by confocal microscopy as described in the Materials and Methods. C) Representative confocal micrographs of HeLa cells expressing GFP–Rab7 that were infected with DsRedm-expressing B. abortus 2308 (appears in red) and immunostained for LAMP-1 using Cyanin 5-conjugated secondary antibodies (appears in blue). HeLa cells were transfected with a derivative of pEGFP-C1 expressing GFP–Rab7, infected, processed for immunostaining at 2, 6 and 24 h p.i. and analyzed by confocal microscopy as described in the Materials and Methods. Scale bars are 10 and 2 mm. D) Quantification of late endocytic markers acquired by BCVs. Recruitment of LAMP-1 (open squares), CD63 (open circles) and GFP–Rab7 (closed circles) on BCVs was scored by confocal microscopy at 2, 6, 12 and 24 h p.i. Arrows indicate the area magnified in the whole images or the positive BCVs in the insets. Data are means SD from three independent experiments. either live or heat-killed bacteria. The latter can be considered as a control for the maximal fusion with lysosomes. At 3 h p.i., the average pixel intensity of dextran fluorescence in BCVs containing live Brucella was reduced by nearly 70% compared with BCVs containing heat-killed bacteria (Figure 5E), suggesting reduced delivery. This indicates that BCV fusion with lysosomes, although evident, is limited and controlled by the bacteria. Altogether, these results demonstrate that early and intermediate BCVs (i) acquire various late endosomal/lysosomal proteins, (ii) are accessible to, but limit the delivery of, fluidphase markers from lysosomes and (iii) are acidic and display proteolytic activities. This provides strong evidence that biogenesis of these bacterial vacuoles involves transient interactions with the endocytic pathway, including lysosomes, prior to generating an ER-derived replicative niche. Traffic 2008; 9: 678–694 Perturbation of late endosome/lysosome functions impairs BCV trafficking and bacterial replication Having established that BCVs interact with the lysosomal compartment prior to biogenesis of the ER-derived replicative organelle and bacterial replication, one obvious question was to determine whether such trafficking events are essential for biogenesis of the replicative vacuole and bacterial proliferation. To address this, we overexpressed mutant forms of Rab7 in HeLa cells and examined their effects on BCV trafficking and bacterial replication. In cells expressing the GTP-bound, constitutively active Rab7[Q67L], BCVs recruited both LAMP-1 and Rab7[Q67L] at early (2 h p.i.) and intermediate (6 h p.i.) stages of trafficking, and bacteria multiplied in a LAMP-1negative, Rab7[Q67L]-negative compartment at 24 h p.i. (Figure 6A). This indicates that expression of the constitutively active Rab7 did not affect BCV trafficking or 683 Starr et al. Figure 4: Intermediate, but not replicative, BCVs are accessible to fluid-phase markers. HeLa cells were preloaded with Alexa Fluor 546–dextran, infected with GFP-expressing Brucella abortus 2308 in absence of dextran and then incubated with dextran until analysis in order to label all endocytic compartments. At 3, 6, 9 and 12 h p.i., cells were visualized by live cell confocal microscopy, and approximately 5-min long time-lapse recordings were performed at each time-point. A) Consecutive frames of a representative time-lapse recording of an Alexa Fluor 546–dextran-loaded HeLa cell (appears in red) infected with GFP-expressing Brucella (appears in green) at 6 h p.i. The arrow indicates a dextran-positive BCV. The arrowhead indicates a dextran-positive endocytic vesicle docking to and fusing with the BCV. Scale bar, 1 mm. B) Consecutive frames of a representative time-lapse recording of an Alexa Fluor 546–dextran-loaded HeLa cell (appears in red) infected with GFP-expressing Brucella (appears in green) at 12 h p.i. The arrow indicates a cluster of replicative bacteria within dextrannegative BCVs. Scale bar, 1 mm. C) Quantification of BCV accessibility to dextran, expressed as the percentage of dextran-positive BCVs over time. Data are means SD from three independent experiments. The total numbers of BCVs analyzed per time-point were 361 at 3 h p.i., 257 at 6 h p.i., 389 at 9 h p.i. and 438 at 12 h p.i. replication and is consistent with previously described results in NIH3T3 cells where intracellular growth was not affected by Rab7[Q67L] expression (13). The kinetics of Rab7[Q67L] recruitment to BCVs was indistinguishable from that of wild-type Rab7 and LAMP-1 over time (Figures 6B and 3D), confirming the recruitment of Rab7 to LAMP-1 BCVs during maturation. By contrast, the GDPbound, dominant-negative allele, Rab7[T22N], whose expression affects Rab7-dependent membrane trafficking, lysosome positioning and functions (14), was not recruited to BCVs (Figure 6C,D), indicating that Rab7 recruitment to BCVs is dependent upon its GTP-binding activity. Expression of Rab7[T22N] did not prevent LAMP-1 accumulation on BCVs (Figure 6C,D), suggesting that LAMP-1 recruit684 ment to BCVs is Rab7 independent or that the residual activity of endogenous Rab7 in the cell is sufficient for LAMP-1 recruitment to BCVs. To extend these results and confirm the relevance of Rab7 recruitment to BCVs, we examined whether the Rab7 effector RILP (20,21) was also recruited. In cells overexpressing a GFP–RILP fusion, GFP–RILP clearly labeled maturing BCVs (Figure 6E and Figure S2). Because RILP is only recruited to membranes by GTP-bound Rab7 (20,21), this indicates the presence of activated, endogenous active Rab7 on BCVs. Interestingly, at 24 h p.i., Brucella remained enclosed within LAMP-1-positive BCVs in a small but significant fraction of cells expressing GFP–Rab7[T22N] and did not Traffic 2008; 9: 678–694 Brucella Trafficking Through Lysosomes Figure 5: Intermediate, but not replicative, BCVs fuse with the lysosomal compartment. HeLa cells were preloaded with Alexa Fluor 546–dextran that was chased for 16 h to lysosomes before infection with GFP-expressing Brucella abortus 2308 was performed in absence of dextran. At 3, 6, 9 and 12 h p.i., cells were visualized by live cell confocal microscopy, and approximately 5-min long time-lapse recordings were performed at each time-point. A) Consecutive frames of a representative time-lapse recording of an Alexa Fluor 546– dextran-loaded HeLa cell (appears in red) infected with GFP-expressing Brucella (appears in green) at 6 h p.i. The arrow indicates a dextranpositive BCV. The arrowhead indicates a dextran-positive lysosome docked to the BCV. Scale bar, 1 mm. B) Consecutive frames of a representative time-lapse recording of an Alexa Fluor 546–dextran-loaded HeLa cell (appears in red) infected with GFP-expressing Brucella (appears in green) at 12 h p.i. The arrow indicates a cluster of replicative bacteria within dextran-negative BCVs. The arrowhead indicates a dextran-positive lysosome that docks to a BCV without delivering its content. Scale bar, 1 mm. C) Representative live cell confocal micrographs of HeLa cells loaded with DQ-Red BSA and infected for 6 h (upper panels) or 12 h (lower panels) with GFPexpressing B. abortus 2308. Intermediate BCVs at 6 h p.i. accumulated DQ-Red fluorescence (arrowhead), while replicative BCVs at 12 h p.i. did not. D) Quantification of BCV accessibility to lysosomal dextran and intravacuolar proteolytic activity, expressed as the percentage of dextran- or DQ-Red-positive BCVs over time. Data are means SD from three independent experiments. The numbers of dextranpositive BCVs analyzed per time-point were 295 at 3 h p.i., 365 at 6 h p.i., 540 at 9 h p.i. and 838 at 12 h p.i. The numbers of DQ-Redpositive BCVs analyzed per time-point were 267 at 3 h p.i., 254 at 6 h p.i., 352 at 9 h p.i. and 374 at 12 h p.i. E) Quantification of intravacuolar dextran accumulated in BCVs containing live or heat-killed Brucella. Values are normalized fluorescence pixel intensities and are means SD from three independent experiments. Asterisk indicates a statistically significant difference (Student’s t-test, p < 0.01). replicate (Figure 6C,D). This correlated with a significant decrease in bacterial replication because 37 5.1% of GFP–Rab7[T22N]-expressing cells were replication permissive compared with 50 3.4, 59 5.2 and 58 6.1% of GFP-, GFP–Rab7- and GFP–Rab7[Q67L]-expressing cells, respectively (Figure 6G; p < 0.01). Hence, perturbation of Rab7-dependent functions affects BCV maturation and Traffic 2008; 9: 678–694 subsequent bacterial replication. Overexpression of GFP– RILP induced the collapse of the late endosomal/lysosomal compartment to a juxtanuclear location close to the microtubule-organizing center [MTOC; (20,21); Figure 6E]. This is because of the increased recruitment of the dynein– dynactin motor complexes to late endosomes/lysosomes and their subsequent enhanced centripetal movements 685 Starr et al. (21). In these cells, Brucella remained enclosed in LAMP-1positive, RILP-positive, clustered BCVs at 24 h p.i. and did not display significant signs of replication (Figure 6E,F). Only 25 5.3% of RILP-overexpressing cells supported bacterial growth compared with 50 3.4% in GFPexpressing cells (Figure 6G; p < 0.01). It remains possible that the dramatic displacement and aggregation of late endosomal/lysosomal compartments because of RILP overexpression prevents BCV conversion into a replicative organelle. These results nonetheless indicate that perturbation of Rab7-dependent functions prevents proper trafficking of BCVs and subsequent bacterial replication. Therefore, Brucella replication requires trafficking through a functional late endosomal/lysosomal compartment to successfully generate a replicative organelle. To confirm these findings, we examined whether BCVs in cells expressing dominant alleles of Rab7 or overexpressing RILP acquired ER membrane markers, a readout of replicative organelle biogenesis (2,4,5). In untransfected or Rab7[Q67L]-expressing cells, BCVs acquired calreticulin from 12 h p.i. and onwards (Figure 7A,B), concomitant with bacterial replication (Figure 7A), indicating that BCVs had fully matured into an replication-permissive, ERderived vacuole. By contrast, BCVs in Rab7[T22N]- or RILP-expressing cells never acquired calreticulin (Figure 7A,B), demonstrating that they did not convert into ERderived, replicative organelles. Therefore, perturbation of Rab7-dependent functions prevents biogenesis of the Brucella replicative organelle. Rab7-dependent functions are required for BCV maturation in RAW264.7 cells To confirm in phagocytic cells the observations made in HeLa cells, we examined Rab7 recruitment to BCVs and the effect of expression of Rab7 dominant alleles upon Brucella intracellular growth in RAW264.7 macrophagelike cells. RAW264.7 cells were transiently transfected to express GFP–Rab7, infected with DsRedm-expressing Brucella and Rab7 and LAMP-1 recruitment was quantified. By 6 h p.i., most BCVs were replication permissive (data not shown), and Rab7 clearly decorated the majority of these BCVs (82 1.0%; Figure 8A,B), indicating that replication-proficient intermediate BCVs interact with late endocytic compartments during maturation in phagocytic cells. Moreover, expression of GFP–Rab7[T22N] in RAW264.7 cells significantly decreased bacterial replication at 24 h p.i. because only 8.9 3.3% of GFP– Rab7[T22N]-transfected cells supported bacterial growth compared with 47 13% in untransfected cells (p < 0.05; Figure 8C,D). By contrast, expression of GFP–Rab7 (34 11%) or GFP–Rab7[Q67L] (33 5.5%) did not significantly affect bacterial replication compared with control cells (p > 0.2; Figure 8C,D). Taken together, these results demonstrate a role for Rab7-dependent functions in BCV maturation and bacterial replication in phagocytic cells, consistent with our data from epithelial cells. 686 Decreased BCV–lysosome fusion correlates with defective vacuolar trafficking Because of the essential role of acidification in BCV maturation and the similar defective BCV trafficking observed in epithelial cells expressing Rab7[T22N] or treated with BAF (Figures 1B,C, 2C,D and 6C,D), we postulated that BCV acidification in Rab7[T22N]-expressing cells was impaired, which consequently affects vacuolar trafficking of bacteria. To test this hypothesis, BCV acidity was assessed by treating GFP-expressing Brucellainfected HeLa cells at 4, 10 and 24 h p.i. with 5 mg/mL acridine orange for 10 min followed by live cell imaging analysis for red fluorescent BCVs. Acridine orange red fluorescence emission was specific for acidic compartments in uninfected cells as it was completely abolished by BAF pretreatment for 1 h and progressively restored upon BAF washout (data not shown). In both untransfected and Rab7[T22N]-transfected cells, the majority of BCVs displayed red fluorescence at 4 and 10 h p.i. (Figure 9A), indicating that these BCVs remain acidic during the early and intermediate maturation stages. BCV acidity was completely abolished in cells pretreated with BAF and analyzed at 4 h p.i. (data not shown). At 24 h p.i., replicative BCVs in control cells were not acidic, while BCVs in cells expressing Rab7[T22N] remained acidic, consistent with their defect in maturation (Figure 6C,D). Thus, the defect in BCV maturation observed in cells expressing GFP–Rab7[T22N] cannot solely be explained by a lack of acidification. Given that Rab7 controls fusion with the late endosomal/ lysosomal compartments, we next examined whether stalled BCVs were defective for accessibility to fluid-phase markers delivered from lysosomes by measuring dextran accumulation in BCVs by quantitative live cell imaging. Dextran accumulation in BCVs was significantly reduced in cells expressing GFP–Rab7[T22N] or cells treated with BAF (65 10 and 42 3.5% of the control, respectively; Figure 9B,C) compared with untreated cells, indicating that failed BCV maturation correlates with decreased intermediate BCV–lysosome fusion. Discussion Intravacuolar, intracellular pathogens have devised various strategies to avoid degradation along the endocytic pathway (22). Whether they avoid interactions with the endocytic pathway, like Legionella pneumophila and Chlamydia species (23), or arrest the maturation of their phagosome at various stages, such as Mycobacterium tuberculosis (22), they ultimately avoid fusion with degradative lysosomes. Such is also the case for Brucella, whose intracellular survival is commonly considered to rely upon inhibition of fusion with lysosomes (2,3,9,11,18,24). This view of Brucella intracellular trafficking is based upon cumulative data from immunofluorescence and electron microscopy of fixed samples. In these experiments, Traffic 2008; 9: 678–694 Brucella Trafficking Through Lysosomes Figure 6: Perturbation of the late endosomal/lysosomal compartment functions and positioning impairs BCV maturation and bacterial replication. A, C and E) Representative confocal micrographs of HeLa cells expressing GFP–Rab7[Q67L] (A), GFP–Rab7[T22N] (C) and GFP–RILP (E) that were infected with DsRedm-expressing Brucella abortus 2308 (appears in red) and immunostained for LAMP-1 using Cyanin 5-conjugated secondary antibodies (appears in blue). Transfected, infected HeLa cells were processed for immunostaining at 2, 6 and 24 h p.i. and analyzed by confocal microscopy as described in the Materials and Methods. Arrows indicate the area magnified in the whole images or the positive BCVs in the insets. Scale bars, 10 or 2 mm. B, D and F) Effect of overexpression of GFP–Rab7[Q67L] (B), GFP–Rab7[T22N] (D) or GFP–RILP (F) on BCV maturation. Colocalization of bacteria with the Rab7 dominant alleles or RILP (closed circles) and LAMP-1 (open circles) was scored by confocal microscopy at 2, 6, 12 and 24 h p.i. Data are means SD from four independent experiments. G) Quantification of bacterial replication in HeLa cells overexpressing Rab7 alleles or RILP. HeLa cells expressing GFP only (control), GFP–Rab7, GFP–Rab7[Q67L], GFP–Rab7[T22N] or GFP–RILP were infected with DsRedm-expressing B. abortus 2308 for 24 h, and intracellular replication was scored by fluorescence microscopy. Infected cells were considered to support bacterial replication when at least 10 bacteria were present in replicative clusters. Data are means SD from five independent experiments. Asterisks indicate a statistically significant difference compared with control conditions (Student’s t-test, p < 0.01). Traffic 2008; 9: 678–694 687 Starr et al. Figure 7: Perturbation of the late endosomal/lysosomal compartment functions and positioning impairs biogenesis of replicative BCVs. A) Representative confocal micrographs of untransfected HeLa cells (control) or HeLa cells expressing GFP– Rab7[Q67L], GFP–Rab7[T22N] or GFP–RILP that were infected with DsRedm-expressing Brucella abortus 2308 and immunostained for calreticulin using Cyanin 5-conjugated secondary antibodies. Left-hand panels show GFP fluorescence in transfected cells. Right-hand panels show overlays of Brucella (appears red) and calreticulin (pseudocolored in white) fluorescence. Boxes on micrographs indicate the area magnified in the insets. Arrows indicate intracellular bacteria. Scale bars, 10 or 2 mm. B) Quantification of calreticulin recruitment to BCVs. Infected HeLa cells were processed for immunostaining at 6, 12 and 24 h p.i. and analyzed for colocalization between bacteria and ER by confocal microscopy as described in the Materials and Methods. Data are means SD from three independent experiments. detected because of the limited sensitivity of the methodologies used. For example, permeabilization steps associated with fixation and immunostaining procedures can lead to extraction of fluid-phase markers or luminal antigens from their compartments and loss of corresponding signals, an artifact that was recently quantified by Drecktrah et al., where fixation and immunofluorescence processing of HeLa cells preloaded with fluorescent dextran led to a >90% decrease of the dextran-associated fluorescence (12). conclusions were drawn from the failure to deliver lysosomal luminal content to the BCV in the form of cathepsin D (2,3,18,24) and preloaded fluid-phase markers such as gold-labeled BSA (11) or fluorescent dextran (9). Yet, there remained the possibility that fusion events were not 688 Here, we have reassessed the trafficking of replicationproficient BCVs using a model of B. abortus infection of epithelial HeLa cells and high-resolution live cell confocal microscopy. Unlike what was previously believed, we show that intermediate BCVs traffic along the endocytic pathway and interact with late endocytic compartments, including lysosomes. Such interactions are transient and occur prior to BCV interaction and fusion with the ER and subsequent conversion into replicative vacuoles. Our results are consistent with the transient accumulation of the late endosomal/lysosomal marker LAMP-1 on maturing BCVs (2,18), a hallmark of BCV trafficking that has remained difficult to explain in the context of the current model of rapid BCV segregation from the endocytic pathway. We also clearly show BCV recruitment of other late endosomal/lysosomal markers such as CD63, Rab7 and RILP. These findings also solve a long-lasting controversy on Brucella intracellular trafficking, whereby the model of rapid segregation from the endocytic pathway and inhibition of fusion with lysosomes did not accommodate the fact that early BCV acidification is essential to bacterial survival (15). Our findings on the nature of the intermediate BCV are also consistent with the unusual trafficking of opsonized B. abortus observed in human monocytes where the bacteria remain and eventually replicate within an ER-negative, LAMP-1-positive compartment (25). By analogy with our results, such a compartment resembles an intermediate BCV that would eventually become replication permissive. It remains unclear why these intracellular bacteria do not reach the ER, although opsonization of bacteria may alter their intracellular trafficking. Traffic 2008; 9: 678–694 Brucella Trafficking Through Lysosomes Figure 8: Rab7-dependent functions are required for Brucella replication in RAW264.7 cells. A) Representative confocal micrograph of a GFP–Rab7-transfected RAW264.7 cell that was infected with DsRedm-expressing Brucella abortus 2308 (appears in red) for 6 h and immunostained for LAMP-1 using Cyanin 5conjugated secondary antibodies (appears in blue). Arrows indicate the area magnified in the whole image or the positive BCVs in the insets. Scale bars, 10 or 2 mm. B) Quantification of Rab7- and LAMP-1positive BCVs in GFP–Rab7-transfected RAW264.7 cells that were infected with DsRedm-expressing B. abortus 2308 for 6 h. Data are means SD from three independent experiments. C) Representative confocal micrographs of either GFP– Rab7[T22N]- or GFP–Rab7[Q67L]-transfected RAW264.7 cells that were infected with DsRedm-expressing B. abortus 2308 (appears in red) for 24 h and immunostained for LAMP-1 using Cyanin 5conjugated secondary antibodies (appears in blue). Arrows indicate the area magnified in the whole image or the positive BCVs in the insets. Scale bars, 10 or 2 mm. D) Quantification of bacterial replication in RAW264.7 cells overexpressing Rab7 alleles. RAW264.7 cells expressing no transgene (control), GFP–Rab7, GFP– Rab7[Q67L] or GFP–Rab7[T22N] were infected with DsRedm-expressing B. abortus 2308 for 24 h, and intracellular replication was scored by fluorescence microscopy. Infected cells were considered to support bacterial replication when at least 10 bacteria were present in replicative clusters. Data are means SD from three independent experiments. The asterisk indicates a statistically significant difference between control and Rab7[T22N]-expressing cells (Student’s t-test, p ¼ 0.03). Although BCV fusion with lysosomes was obvious in live cells, it was not as extensive as fusion events that occurred with vacuoles containing heat-killed Brucella. This indicates that live Brucella limit fusion of their vacuole with lysosomes, possibly to avoid accumulation of lysosomal content to bactericidal levels. The potential loss of luminal antigens associated with immunostaining, the tight luminal space of BCVs and the limited fusion of BCVs with lysosomes together explain why previous studies have failed to detect significant amounts of luminal lysosomal enzymes, such as cathepsin D, inside BCVs (2,18). In support of our findings, intermediate BCVs also displayed proteolytic activities, as judged by the intravacuolar detection of DQ-Red BSA fluorescence, which indicated delivery of proteolytic enzymes into maturing BCVs. Recruitment of active Rab7 to BCVs strongly argues for the interaction of intermediate Traffic 2008; 9: 678–694 BCVs with the late endosomal/lysosomal compartment as this small GTPase controls traffic to, fusion with and positioning of this compartment (14). These results contradict previous observations that Rab7 is not recruited to BCVs in macrophages (2). However, this previous study assessed Rab7 recruitment to BCVs using immunostaining of endogenous Rab7. The absence of detectable antibody signal on BCVs may be explained by the difficulties detecting low steady-state levels of endogenous antigens that rapidly cycle on and off intracellular compartments, such as Rab GTPases. Not only Rab7 was recruited to BCVs in both HeLa and RAW264.7 cells but also its activity was required for the proper maturation of the BCV into a replication-permissive, ER-derived organelle, indicating that Brucella traffic through late endocytic compartments is requisite for 689 Starr et al. Figure 9: Perturbation of late endosomal/lysosomal compartments decreases BCV fusion with lysosomes. A) Quantification of BCV acidity in HeLa or GFP–Rab7[T22N]-overexpressing HeLa cells. Cells were infected with GFP-expressing Brucella abortus 2308, and BCV acidity was evaluated after loading cells with acridine orange using live cell imaging analysis at 4, 10 and 24 h p.i. Acidic BCVs displayed acridine orange red fluorescence. Data are means SD from three independent experiments. B) Single frames of representative time-lapse recordings of untreated, GFP–Rab7[T22N]-expressing or BAF-treated HeLa cells that were preloaded with Alexa Fluor 546–dextran to label lysosomes and infected with GFP-expressing Brucella for 6 h. Left-hand panels show both GFP and Alexa Fluor 546–dextran fluorescence, while right-hand panels show dextran fluorescence only. Note that the bacterial GFP signal was stronger than the GFP–Rab7[T22N] signal, easily allowing the detection of bacteria in transfected cells. Arrows indicate dextran-positive BCVs. C) Quantification of intravacuolar dextran accumulated in BCVs in cells expressing GFP–Rab7[T22N] or treated with BAF compared with untransfected or untreated cells, respectively. The fluorescence pixel intensities measured were normalized to controls in each independent experiment, and data are means SD from three independent experiments. Asterisks indicate a statistically significant difference compared with control conditions (Student’s t-test, p < 0.05). intracellular replication. These results clearly establish late endosomal BCVs as the intermediate niche for replicationproficient bacteria and rule out the possibility that, in macrophages, LAMP-1-positive BCVs only contain bacteria that are routed to a degradative phagolysosome. ChavesOlarte et al. have reported that overexpression of the constitutively active Rab7[Q67L] allele in stably transfected NIH3T3 fibroblasts did not affect Brucella intracellular growth or trafficking, which led the authors to conclude that Rab7 is not required for Brucella intracellular trafficking (13). However, the effect of the dominantnegative allele of Rab7, Rab7[T22N], on Brucella intracellular fate was not examined in this study. Our results using transient transfection of HeLa or RAW264.7 cells with GFP–Rab7[Q67L] concur with this previous study. Yet, our experiments with Rab7[T22N] in HeLa and RAW264.7 cells clearly demonstrate a significant role for this GTPase in BCV trafficking and bacterial replication. This is in agreement with previous work showing that overexpression of Rab7[Q67L] does not significantly affect functionality of late endocytic compartments, while that of Rab7[T22N] decreases endocytic functions by about 50% (14). Overexpression of the Rab7 effector GFP–RILP, which induces a dramatic collapse of late endosomes and lysosomes around the MTOC because of enhanced recruitment of dynein–dynactin motor complexes (21), also affected BCV trafficking and prevented bacterial replication, confirming that intermediate BCVs traffic through late endocytic compartments. The effect of RILP overexpression suggests that proper positioning, in addition to functioning, of this compartment might be necessary for efficient BCV trafficking. However, overexpression of 690 Rab7[T22N] did not significantly affect BCV positioning with respect to the MTOC (data not shown), even though it is known to increase the retrograde movement of late endosomes in uninfected cells (14). This suggests that BCV positioning in the cell does not only rely upon Rab7dependent mechanisms. In cells expressing Rab7[T22N], most BCVs failed to traffic to the ER and generate replication-permissive organelles. Such a trafficking defect was also observed in cells pretreated with the v-ATPase inhibitor BAF, which blocks acidification and subsequent maturation of phagosomes along the endocytic pathway. BAF treatment prevented bacterial replication in both macrophages and epithelial cells. As reported by Porte et al. (15), early, but not late, inhibition of vacuolar acidification affected Brucella intracellular fate, a timing consistent with the kinetics of BCV interaction with late endocytic compartments. This suggests that proper BCV maturation requires a specific sequence of timed intracellular events. The similar effects of BAF treatment and Rab7[T22N] expression on Brucella trafficking suggest a role for intravacuolar pH in both cases because both reportedly affect intraluminal pH of endocytic compartments (14,16). However, defects in endosome maturation resulting from these treatments may also be invoked. Indeed, late BCVs remained acidic in cells expressing Rab7[T22N], ruling out the theory that phagosomal pH is solely responsible for BCV trafficking defects. Instead, both BAF treatment and overexpression of Rab7[T22N] decreased BCV fusion with lysosomes in HeLa cells. It is thus possible that environmental cues other than acidification resulting from BCV interaction with Traffic 2008; 9: 678–694 Brucella Trafficking Through Lysosomes late endosomes/lysosomes are necessary for further trafficking toward the ER. Further work detailing the physiochemical environment within the intermediate BCV is needed to address these questions. Our work provides evidence that intermediate BCVs interact with the endocytic pathway and experience limited fusion with lysosomes to generate a transient, intravacuolar environment conducive to further trafficking of the BCV toward the ER (Figure 10). Because the VirB type IV secretion system is required for subsequent trafficking events, such as the biogenesis of the ER-derived replicative organelle (2,3,8), it is tempting to speculate that intermediate BCVs provide environmental cues required for VirB expression and/or activation and that treatments affecting proper maturation of the intermediate BCV affect VirB functions. In support of this hypothesis, intracellular B. abortus virB gene induction is maximal at 4 h p.i. in Figure 10: Revised model of Brucella intracellular trafficking. A) Upon entry, newly formed BCVs traffic along the endocytic pathway, sequentially interacting with early compartments of the endocytic pathway (shown in yellow) and late endosomes and multivesicular bodies (MVB; shown in green) before fusing with lysosomes (shown in red). Such interactions are required for further trafficking (B), whereby intermediate BCVs interact with ER exit sites (ERES) and subsequently fuse with the ER (shown in blue) (C) to generate ER-derived, replicative BCVs (D). BCVs containing VirB-defective mutants that cannot sustain interactions with the ER (2) and/or limit fusion with the lysosomal compartment and mature into a bactericidal vacuole (E). Traffic 2008; 9: 678–694 J774A.1 cells (26) when bacteria are within intermediate BCVs. Moreover, expression of the VirB type IV secretion system encoding genes in B. suis can be induced in vitro at acidic pH, and BAF reduces intracellular virB promoter activity in J774A.1 cells (17). Consistent with this hypothesis, Brucella mutants in the VirB type IV secretion system in untreated cells exhibit trafficking defects (2,3,8) similar to those we observed for wild-type bacteria in BAF-treated cells or cells expressing Rab7[T22N]. This suggests that the trafficking defects because of perturbation of late endosomal/lysosomal compartments may be because of an impaired expression, or activation, of VirB. Although we could reproduce the inhibitory effect of BAF on B. abortus virB gene induction (17) in both BMMs and HeLa cells using quantitative reverse transcriptase–polymerase chain reaction of virB4 (Figure S2, see also Supplementary Materials and Methods) or virB11 messenger RNAs (data not shown), our attempts at measuring VirB expression in cells expressing dominant alleles of Rab7 were unsuccessful. Despite this, our data do support a model whereby transient fusion of BCVs with lysosomes provides the intravacuolar cues required for proper expression of VirBassociated functions, which in turn allows for trafficking to the ER and replication. The demonstrated interaction of intermediate BCVs with the late endosomal/lysosomal compartment suggests that Brucella is capable of resisting a potentially degradative environment, at least transiently. The fact that VirBdeficient mutants of Brucella do not traffic further than intermediate BCVs and are progressively killed (2) indicates that such an organelle can acquire bactericidal functions over time. It also raises the possibility that, once expressed and assembled, the VirB apparatus translocates effector molecules that support bacterial survival in the intermediate BCV, possibly by limiting fusion with lysosomes. Such a process would allow bacteria to redirect the trafficking of their vacuole to the ER in a VirB-dependent manner. Cyclic b-1,2-glucan synthesized by Brucella has also been proposed to control avoidance of lysosome fusion based on experiments using delivery of cathepsin D as evidence for BCV–lysosome (24). It will be interesting to assess the relative contributions of cyclic b-1,2-glucan and the type IV secretion system using our live cell assays, which are extremely sensitive in measuring the extent of BCV–lysosome fusion. Ultimately, the identification of substrates of the VirB apparatus and their characterization will be required to clarify if, and how, the VirB apparatus participates in the trafficking transition of the BCV from the endocytic pathway to the ER. Materials and Methods Bacterial strains and plasmids The bacterial strains used in this study were the smooth virulent B. abortus strain 2308 and two derivatives expressing either the GFP from plasmid pJC43 (5) or the DsRedm from plasmid pJC44. To construct pJC44, a fusion 691 Starr et al. between the aphA3 gene promoter driving GFP expression on pJC43 and the DsRedm gene from pDsRedm (Clontech) was generated using the primers pairs JC213 (50 -TGCAGGAATTCCCAGCGAACC-30 )/JC214 (50 -TTAATAAACCTCCTTTCGGATCCG-30 ) and JC215 (50 -AAGGAGGTTTATTAAATGGACAACACCGAGGACGTC-30 )/JC216 (50 -GAATTCTAGAGTCGCGGCCGCTC-30 ), respectively. Both fragments were subsequently fused by overlap extension polymerase chain reaction amplification using primers JC213 and JC216. The resulting fragment was cloned into pCR2.1TOPO (Invitrogen), and its sequence was confirmed. The fusion fragment was then excised using EcoRI and XbaI sites present on primers JC213 and JC216 and subcloned into the same restriction sites on pBBR1-MCS2 (27) to give pJC44. This plasmid was then introduced by electroporation into the wild-type strain 2308. All bacteria were grown in tryptic soy broth (TSB; Sigma) or on tryptic soy (TS) agar plates (TSA; Sigma), supplemented with kanamycin (50 mg/mL) to select the pBBR1-MCS2 derivatives pJC43 and pJC44. For infection of eukaryotic cells, 2 mL of TSB was inoculated with a few bacterial colonies from a freshly streaked TSA plate and grown at 378C for 16 h to early stationary phase. Heatkilled bacteria were prepared by incubating an aliquot of an early stationary phase culture at 808C for 30 min. Cell culture and infections To obtain BMMs, bone marrow cells were isolated from femurs of 6–10 week old, C57BL/6J female mice (Jackson Laboratories) and differentiated into macrophages for 5 days at 378C and 7% CO2 in 1 g/L glucose DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen), 10% L-conditioned medium and 2 mM L-glutamine in non-tissue culture-treated Petri dishes. After 5 days, loosely adherent BMMs were washed with PBS, harvested by incubation in chilled cation-free PBS on ice for 10 min, resuspended in complete medium and replated in 24-well cell culturetreated plates (1 105 BMMs/well). BMMs were further incubated at 378C under 7% CO2 atmosphere for 48 h, replenishing with complete medium 24 h before infection. HeLa cells [American Type Culture Collection (ATCC) clone CCL-2] were cultured at 378C under 7% CO2 atmosphere in DMEM supplemented with 10% fetal calf serum (FCS) and 2 mM L-glutamine and seeded 24 h before infection either on 12-mm glass coverslips in 24-well plates (immunofluorescence; 1 105/well) or on WillCo-dishÒ glass bottom 35-mm dishes (live cell imaging; 1 105/dish; WillCo Wells BV). RAW264.7 cells (ATCC number TIB-71) were cultured at 378C under 7% CO2 atmosphere in DMEM supplemented with 10% FCS and 2 mM L-glutamine and seeded 48 h before infection on 12-mm glass coverslips in 24-well plates (immunofluorescence; 3 105/well). For transfections, HeLa or RAW264.7 cells were transfected 1 day prior to infection using either the FuGene 6ä transfection reagent (HeLa; Roche) or the FuGene HDä transfection reagent (RAW264.7; Roche) according to the manufacturer’s instructions. For infections, bacterial cultures were diluted in complete medium and added to chilled cells at a theoretical multiplicity of infection of 25 (BMMs), 100 (RAW264.7 cells) or 1000 (HeLa cells). To improve Brucella uptake by HeLa cells and allow statistically significant analyses, EGF at a final concentration of 50 ng/mL (recombinant human EGF; Calbiochem) was added to the cells at the time of infection, which induced transient membrane ruffling. EGF-mediated uptake did not alter trafficking of internalized bacteria compared with untreated HeLa cells (Figure S1). Bacteria were centrifuged onto cells at 400 g for 10 min at 48C, and infected cells were incubated for either 20 min (BMMs and RAW264.7 cells) or 30 min (HeLa cells) at 378C under 7% CO2 atmosphere following a rapid warm up in a 378C water bath to synchronize bacterial uptake. Infected cells were then washed five times with DMEM to remove extracellular bacteria, incubated for either an additional 30 (BMMs and RAW264.7 cells) or 60 min (HeLa cells) in complete medium before medium containing 100 mg/mL gentamicin was added for 60 min (BMMs and RAW264.7 cells) or 90 min (HeLa cells) to kill extracellular bacteria. Thereafter, infected cells were maintained in gentamicin-free medium. When required, BAF (AG Scientific) was added to cells at a final concentration of 100 nM and replenished every 6 h. 692 Intracellular replication assays To evaluate intracellular replication of Brucella, BMMs or HeLa cells seeded in 24-well plates were infected as described above, and the number of viable intracellular bacteria per well was determined in triplicate for each time-point. Infected cells were washed three times with sterile PBS and then lysed with 1 mL of 0.1% Triton-X-100 in water for 2 min at room temperature. Serial dilutions were rapidly plated onto TSA plates, and plates were incubated for 3 days at 378C before enumeration of colonyforming units. Immunofluorescence microscopy Infected cells grown on 12-mm glass coverslips in 24-well plates were washed three times with PBS, fixed with 3% paraformaldehyde, pH 7.4, at 378C for 20 min, washed three times with PBS and then incubated for 10 min in 50 mM NH4Cl in PBS in order to quench free aldehyde groups. Samples were blocked and permeabilized in 10% horse serum and 0.1% saponin in PBS for 30 min at room temperature. Cells were labeled by inverting coverslips onto drops of primary antibodies diluted in 10% horse serum and 0.1% saponin in PBS and incubating for 45 min at room temperature. Primary antibodies used were rat anti-mouse LAMP-1 clone 1D4B, mouse anti-human LAMP-1 clone H4A3, mouse anti-human CD63 clone H5C6 (developed by J. T. August and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biological Sciences, The University of Iowa, Iowa City, IA, USA) and rabbit polyclonal anti-calreticulin antibodies (Affinity BioReagents). Bound antibodies were detected by incubation with 1:500 dilutions of either Alexa Fluor 488 donkey anti-mouse, anti-rat antibodies or Cyanin 5-conjugated goat anti-rabbit or donkey anti-rat antibodies (Jackson ImmunoResearch Laboratories) for 45 min at room temperature. Cells were washed twice with 0.1% saponin in PBS, once in PBS, once in H2O and then mounted in Mowiol 4-88 mounting medium (Calbiochem). Samples were observed on a Nikon Eclipse E800 epifluorescence microscope equipped with a Plan Apo 60/1.4 objective for quantitative analysis or a Carl Zeiss LSM 510 confocal laser scanning microscope for image acquisition (Carl Zeiss Micro Imaging). Confocal images of 1024 1024 pixels were acquired as projections of three consecutive slices with a 0.38-mm step and assembled using ADOBE PHOTOSHOP CS (Adobe Systems). Live cell imaging To assess BCV accessibility to fluid-phase markers, HeLa cells were preincubated for 16 h in complete medium containing 100 mg/mL Alexa Fluor 546-conjugated dextran (10 000 MW; Molecular Probes) and then infected with GFP-expressing bacteria in the absence of dextran until uptake was completed. Thereafter, fluorescent dextran was added to the medium until sample analysis. To assess BCV fusion with lysosomes, HeLa cells were preincubated for 12 h in complete medium containing 200 mg/mL Alexa Fluor 546-conjugated dextran (10 000 MW; Molecular Probes), which was subsequently chased for 12 h in complete medium before infection to ensure all dextran had trafficked to lysosomes. Cells were then infected with GFPexpressing bacteria in absence of dextran. Because GFP fluorescence was destroyed by heat treatment, heat-killed bacteria were stained using SYTO-9 dye according to the manufacturer’s instructions (Invitrogen). To evaluate proteolytic activities in BCVs, HeLa cells were preincubated for 16 h in complete medium containing 50 mg/mL DQ-Red BSA (Molecular Probes), which was maintained all throughout the subsequent infection process. At the time of analysis, cells were washed with CO2-independent medium (Invitrogen) and incubated with CO2-independent medium supplemented with 10% FBS, 4 mM L-glutamine and 1 mM Trolox (Calbiochem) during live cell imaging. Time-lapse recording was performed immediately on a Carl Zeiss LSM 5 live confocal live cell imaging microscope fitted with a Pecon heated stage insert and LCI Plan 63/1.45 NA objective using 488 and 532 nm solid-state lasers for sequential excitation. Images (1024 1024 pixels) were acquired using the Carl Zeiss LSM 5 LIVE 4.0 SP2 software. Individual frames were assembled using ADOBE PHOTOSHOP CS, and Traffic 2008; 9: 678–694 Brucella Trafficking Through Lysosomes time-lapse movies were assembled as Quick Time movies using PREMIERE PRO software (Adobe Systems). ADOBE To quantify dextran acquisition by BCVs, randomly chosen BCVs were imaged, regions of interest (ROI) corresponding to BCVs were defined and the associated dextran fluorescence measured using the Carl Zeiss LSM 5 LIVE 4.0 SP2 software. Because of differences in the planes of focus between the BCVs analyzed, the total fluorescence intensity measured was normalized to the ROI area (in pixels) to generate average fluorescence intensity values per pixel that were comparable between vacuoles. Data were expressed as the means standard deviations of average pixel intensities from three independent experiments. A standard Student’s two-tailed t-test was used to assess significance between conditions (p < 0.05). Acknowledgments We are grateful to Cecilia Bucci for the generous gift of Rab7 alleles and RILP, Olivia Steele-Mortimer, Jessica Edwards and Audrey Chong for critical reading of the manuscript, Steve Porcella and Kimmo Virtaneva for their help with quantitative reverse transcriptase–polymerase chain reaction experiments, Anita Mora and Austin Athman for assistance with graphics and the Rocky Mountain Laboratories Genomics Unit for DNA sequencing. This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases. Supplementary Materials Supplementary Materials and Methods Quantitative real-time polymerase chain reaction. Figure S1: BCV interaction with late endosomal/lysosomal compartments in non-EGF-treated HeLa cells. A) Representative confocal micrographs of either untransfected, GFP–Rab7- or GFP–RILP-transfected HeLa cells that were infected for 6 h with DsRedm-expressing Brucella abortus 2308 in the absence of EGF and immunostained for LAMP-1 using either Alexa Fluor 488- (untransfected cells) or Cyanin 5-conjugated secondary antibodies. Arrows indicate the area magnified in the whole images or the positive BCVs in the insets. Scale bars, 10 or 2 mm. B) Quantification of LAMP-1 Rab7 and RILP on BCVs at 2, 6, 12 and 24 h p.i. in non-EGF-treated HeLa cells. The kinetics of acquisition and loss of these markers are indistinguishable from those in EGF-treated HeLa cells (compare with Figure 3D). Data are means SD of three independent experiments. C) Single frames of representative time-lapse recordings of Alexa Fluor 546– dextran-loaded HeLa cells (appears in red) infected in the absence of EGF with GFP-expressing Brucella (appears in green) at 6 or 12 h p.i.. The arrows indicate either dextran-positive (6 h p.i.) or -negative (12 h p.i.) BCVs. Intermediate, but not replicative, BCVs acquire dextran through fusion with lysosomes. Scale bar, 10 mm. Figure S2: Inhibition of intracellular induction of virB genes by BAF. A) The time–course of intracellular expression of virB4 inside BMMs. Untreated BMMs (closed circles) or BMMs treated with BAF from 1 h prior to infection to 8 h p.i. (open circles) were infected with Brucella abortus 2308 and processed for TaqMan analysis of virB4-specific mRNAs at 1, 4, 8 and 24 h p.i. Values are expressed as relative mRNA fold changes compared with levels measured at 1 h p.i. and are means SEM of a representative experiment out of three. B) Effect of BAF upon virB4 induction inside BMMs or HeLa cells. BAF-treated or untreated BMMs or HeLa cells were infected with B. abortus 2308 and processed for TaqMan analysis of virB4-specific mRNAs at 0 and 4 h p.i. Values are expressed as relative mRNA fold changes between 0 and 4 h p.i. and are means SEM of a representative experiment out of two. mRNA, messenger RNA. Traffic 2008; 9: 678–694 Movie S1: Time-lapse confocal images of a HeLa cell loaded with Alexa Fluor 546-conjugated dextran and infected with GFP-expressing Brucella abortus 2308 for 6 h, showing an intermediate BCV accessible to dextran delivered from endosomes. The left-hand image shows an overlay of both dextran- (red) and bacteria- (green) associated fluorescence, while the righthand image shows dextran-associated fluorescence only. Seventy-five consecutive frames were acquired through sequential excitation of the sample using 488 nm and then 543 nm laser lines, with no interval between acquisitions. Movie S2: Time-lapse confocal images of a HeLa cell loaded with Alexa Fluor 546-conjugated dextran and infected with GFP-expressing Brucella abortus 2308 for 12 h, showing replicative BCVs that are not accessible to dextran delivered from endosomes. The left-hand image shows an overlay of both dextran- (red) and bacteria- (green) associated fluorescence, while the right-hand image shows dextran-associated fluorescence only. Seventy-five consecutive frames were acquired through sequential excitation of the sample using 488 nm and then 543 nm laser lines, with no interval between acquisitions. Movie S3: Time-lapse confocal images of a HeLa cell whose lysosomes were preloaded with Alexa Fluor 546-conjugated dextran before infection with GFP-expressing Brucella abortus 2308 for 6 h. An intermediate BCV is accessible to dextran delivered from lysosomes. The left-hand image shows an overlay of both dextran- (red) and bacteria- (green) associated fluorescence, while the right-hand image shows dextran-associated fluorescence only. Seventy-five consecutive frames were acquired through sequential excitation of the sample using 488 nm and then 543 nm laser lines, with no interval between acquisitions. Movie S4: Time-lapse confocal images of a HeLa cell whose lysosomes were preloaded with Alexa Fluor 546-conjugated dextran before infection with GFP-expressing Brucella abortus 2308 for 12 h. Replicative BCVs are not accessible to dextran delivered from lysosomes. The left-hand image shows an overlay of both dextran- (red) and bacteria- (green) associated fluorescence, while the right-hand image shows dextran-associated fluorescence only. Seventy-five consecutive frames were acquired through sequential excitation of the sample using 488 nm and then 543 nm laser lines, with no interval between acquisitions. 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