INFECTION AND IMMUNITY, Oct. 2002, p. 5816–5821 Vol. 70, No. 100019-9567/02/$04.00�0 DOI: 10.1128/IAI.70.10.5816–5821.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Coxiella burnetii Localizes in a Rab7-Labeled Compartment withAutophagic Characteristics

Walter Beron,1* Maximiliano G. Gutierrez,1 Michel Rabinovitch,2 and Maria I. Colombo1*Instituto de Histología y Embriología, Facultad de Ciencias Medicas, Universidad Nacional de Cuyo-CONICET,

Mendoza 5500, Argentina,1 and Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulistade Medicina, UNIFESP Sao Paulo, Sao Paulo, Brazil2

Received 29 March 2002/Returned for modification 17 May 2002/Accepted 17 June 2002

The obligate intracellular bacterium Coxiella burnetii, the agent of Q fever in humans and of coxiellosis inother animals, survives and replicates within large, acidified, phagolysosome-like vacuoles known to fusehomo- and heterotypically with other vesicles. To further characterize these vacuoles, HeLa cells were infectedwith C. burnetii phase II; 48 h later, bacteria-containing vacuoles were labeled by LysoTracker, a marker ofacidic compartments, and accumulated monodansylcadaverine and displayed protein LC3, both markers ofautophagic vacuoles. Furthermore, 3-methyladenine and wortmannin, agents known to inhibit early stages inthe autophagic process, each blocked Coxiella vacuole formation. These autophagosomal features suggest thatCoxiella vacuoles interact with the autophagic pathway. The localization and role of wild-type and mutatedRab5 and Rab7, markers of early and late endosomes, respectively, were also examined to determine the roleof these small GTPases in the trafficking of C. burnetii phase II. Green fluorescent protein (GFP)-Rab5 andGFP-Rab7 constructs were overexpressed and visualized by fluorescence microscopy. Coxiella-containing largevacuoles were labeled with wild-type Rab7 (Rab7wt) and with GTPase-deficient mutant Rab7Q67L, whereas nocolocalization was observed with the dominant-negative mutant Rab7T22N. The vacuoles were also decoratedby GFP-Rab5Q79L but not by GFP-Rab5wt. These results suggest that Rab7 participates in the biogenesis ofthe parasitophorous vacuoles.

Phagocytosis is responsible for the internalization of micro-organisms, damaged cells, and inert particles (1, 29). Afterinternalization, the particles become sequestered in mem-brane-bound organelles, called phagosomes, which undergo aprocess of maturation that involves acidification and severalfusion events. At early time points after phagocytosis, phago-somes acquire markers of early endosomes such as Rab5 andmannose receptors (1, 4, 15). Subsequently, phagosomes grad-ually lose these markers and acquire late endosomal markerssuch as mannose-6-phosphate receptors, Rab7, lysosome-asso-ciated membrane glycoproteins (LAMPs), and cathepsin D (1,4, 15). Finally, phagosomes fuse with secondary lysosomes,acquiring higher concentrations of hydrolytic enzymes andLAMPs and a lower pH (14).

Because many microbes are killed and degraded in the pha-gosomal compartment, the phagocytic pathway is an importantcomponent of host defense against microorganisms. However,some intracellular pathogens inhabit vacuoles that interactwith compartments of the biosynthetic pathway, while othersescape from the phagosomes or remain in vacuoles which nei-ther acidify nor fuse with lysosomes (for a review see reference31). In contrast, Coxiella burnetii bacteria live and replicate inacidified compartments with phagolysosomal characteristics(23). Lysosomal membrane markers and enzymes, as well as

molecules internalized by fluid phase endocytosis, are easilyfound in vacuoles containing C. burnetii (23), and. the vacuolesalso fuse with other components of the phagocytic/endocyticsystem (20, 42, 43). The acid environment appears to be es-sential for C. burnetii replication since raising the lysosomal pHwith lysosomotropic amines or proton pump V-ATPase inhib-itors reduces the growth of C. burnetii (22, 24).

Lysosomes represent the hydrolytic compartment not only forextracellular substrates (e.g., microorganisms) but also for turn-over of cellular components including organelles. Autophagy isprobably the main mechanism for degradation of long-lived pro-teins and the only mechanism for turnover of organelles includingmitochondria and peroxisomes (41). Cytoplasmic portions andorganelles are sequestered in a membranous structure (i.e., au-tophagosome) that interacts with the endocytic pathway and fi-nally fuses with the lysosomes, where the incorporated materialsare degraded (17, 18). Recently, it has been shown that someintracellular parasites avoid interactions with the endocytic path-way and replicate rapidly in vacuoles with autophagosome fea-tures (11, 35, 46).

Rab GTPases belong to the Ras superfamily of small GTPasesand play crucial roles in membrane trafficking of eukaryotic cells,regulating tethering, docking, and fusion events among differentcompartments (48). Rab proteins localized in different intracel-lular compartments modulate specific vesicular-transport events(48). Rab5, a member of the Rab family, localizes on early endo-somes and newly formed phagosomes (14–16), regulating mem-brane trafficking events in both the endocytic and phagocyticpathways (2, 3, 7, 44). Rab7, in turn, controls transport and fusionamong degradative endocytic compartments such as late endo-somes and lysosomes (19, 45). Rab7 appears to be a key compo-nent for the maintenance of the perinuclear lysosomal compart-

* Corresponding author. Mailing address: Instituto de Histología yEmbriología (IHEM)-CONICET, Facultad de Ciencias Medicas, Uni-versidad Nacional de Cuyo, Casilla de Correo 56, Centro Universita-rio, Parque General San Martín, 5500 Mendoza, Argentina. Phone:54-261-4205115, ext. 2675. Fax: 54-261-4494117. E-mail for WalterBeron: wberon@fmed2.uncu.edu.ar. E-mail for María I. Colombo:mcolombo@fmed2.uncu.edu.ar.

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ment by controlling the aggregation and fusion of late endosomesand lysosomes (8). Interestingly, some pathogens reside in phago-somes that exclude Rab7 from their membranes (13, 39, 44)whereas others reside in phagosomes that recruit this protein (10,30). Nevertheless, both kinds of phagosomes are arrested in theirmaturation.

Given the diversity of the intracellular compartments tar-geted and customized by microorganisms, it is likely that mod-ulation of the endocytic and phagocytic pathways allows patho-gens to survive and to multiply in their host cells. There isindeed evidence that parasites can disrupt the maturation ofthe phagosomal pathway by interference with the distributionand function of the Rab protein (2, 3, 10, 13, 30, 39, 44).

Although C. burnetii multiplies in vacuoles with lysosomalfeatures, the molecular mechanisms by which the bacteriumcontrols the biogenesis of the replicative vacuoles are largelyunknown. In the present study we further characterized C.burnetii parasitophorous vacuoles. In HeLa cells infected withC. burnetii phase II for 48 h, vacuoles containing these intra-cellular parasites and labeled with LysoTracker, a marker ofacidic compartments, were found to accumulate monodan-sylcadaverine (MDC) and the LC3 protein, both specific mark-ers of autophagic vacuoles (5, 26, 32). Also, 3-methyladenine(3-MA), an inhibitor of autophagy (40), blocked the develop-ment of Coxiella-containing vacuoles. These results suggestthat parasitophorous vacuoles interact with the autophagicpathway. Furthermore, we show that vacuoles harboring C.burnetii are heavily labeled with Rab7 and with the GTPase-deficient Rab5 mutant. Interestingly furthermore, overexpres-sion of the dominant-negative Rab7 mutant altered the forma-tion of the replicative vacuole. These results suggest that Rab7likely participates in the biogenesis of the C. burnetii-contain-ing vacuoles.

MATERIALS AND METHODS

Materials. Minimal essential medium (�-MEM) and fetal bovine serum (FBS)were obtained from Gibco Laboratories (Grand Island, N.Y.). Rhodamine 6Gand LysoTracker were from Molecular Probes (Eugene, Oreg.). All other chem-icals were from Sigma Chemical Co. (St. Louis, Mo.). Recombinant Sindbisviruses expressing green fluorescent protein (GFP)-tagged Rab5, Rab7, and theirmutants were kindly provided by Philip D. Stahl (Washington University, St.Louis, Mo.). Plasmids encoding enhanced GFP (EGFP)-Rab7 and its mutantswere kindly provided by Bo van Deurs (University of Copenhagen, Copenhagen,Denmark).

Cell cultures. HeLa cells were grown in T25 flasks at 37°C in a 5% CO2

atmosphere in �-MEM supplemented with 10% FBS, 2.2 g of sodium bicarbon-ate/liter, 2 mM glutamine, and 0.1% penicillin-streptomycin. Stably transfectedCHO cells overexpressing the EGFP-LC3 protein (33) were grown in T25 flasksat 37°C in a 5% CO2 atmosphere in �-MEM supplemented with 10% FBS, 2.2 gof sodium bicarbonate/liter, 2 mM glutamine, 0.1% penicillin-streptomycin, and0.2 mg of Geneticin/ml.

Propagation of C. burnetii phase II. The clone 4 phase II Nine Mile strain ofC. burnetii, which is infective for cells but not for animals, was provided by TedHackstadt (Rocky Mountain Laboratories, National Institute for Allergy andInfectious Diseases, National Institutes of Health, Hamilton, Mont.) and washandled in a biosafety level II facility (21). Infective inocula were prepared asdescribed previously (47). Nonconfluent Vero cells were cultured in T25 flasks at37°C in �-MEM supplemented with 5% FBS, 0.22 g of sodium bicarbonate/liter,and 20 mM HEPES, pH 7 (MfbH). Cultures were infected with C. burnetii phaseII suspensions for 2 to 6 days at 37°C in an air atmosphere. After being frozenat �70°C, the flasks were thawed, and the cells were scraped and passed 20 timesthrough a 27-gauge needle connected to a syringe. Cell lysates were centrifugedat 800 � g for 10 min at 4°C. The supernatants were centrifuged at 24,000 � gfor 30 min at 4°C, and pellets containing C. burnetii were resuspended with 0.5 mlof phosphate-buffered saline (PBS), aliquoted, and frozen at �70°C.

Infection of cells with C. burnetii. HeLa cells plated in T25 flasks were washedseveral times with PBS and detached with trypsin-EDTA. After resuspensionwith MfbH, cells were plated on coverslips distributed in six-well plates. Forinfection, a 50-�l aliquot of the C. burnetii suspension was diluted with 550 �l ofMfbH, and 100 �l of this dilution was added to each well. Except when indicatedotherwise, cells were incubated for 48 h at 37°C in an air atmosphere. The sameprotocol was used for the infection of CHO cells overexpressing pEGFP-LC3.

Expression of Rab proteins and their mutants in HeLa cells by using theSindbis virus system. Coxiella-infected HeLa cell monolayers grown in 35-mm-diameter dishes were incubated with recombinant Sindbis viruses to overexpressGFP-Rab proteins in 300 �l of PBS containing 1% FBS. Virus absorptionproceeded at room temperature for 1 h. The medium was replaced by 2 ml ofMfbH, and the cells were incubated overnight at 37°C. Cells were mounted andimmediately analyzed by fluorescence microscopy using an inverted microscope(Eclipse TE 300; Nikon) equipped with a charge-coupled device camera (Orca I;Hamamatsu). Images were processed with MetaMorph, version 4.5, software(Universal Images Corporation).

Transfection with pEGFP plasmids. HeLa cells were grown in D-MEM sup-plemented with 10% FBS, 2.2 g of sodium bicarbonate/liter, 2 mM glutamine,and 0.1% penicillin-streptomycin in a 5% CO2 incubator at 37°C. The cells weretransfected with the pEGFP vector alone or pEGFP plasmids encoding wild-typeRab7 (Rab7wt), Rab7T22N, a dominant-negative mutant, and the activeRab7Q67L mutant by using Lipofectamine (GIBCO-BRL) according to themanufacturer’s instructions. After 48 h of transfection cells were infected with C.burnetii. The cells were analyzed and processed as described above.

RESULTS

The replication compartment of C. burnetii phase II displaysautophagosomal markers. To examine the compartmentwhere C. burnetii phase II survives and multiplies, HeLa cellswere infected for 48 h with the bacteria. As previously shown(42), Coxiella-rich large vacuoles can be easily distinguished byphase-contrast microscopy of infected cells (Fig. 1, left). Thesevacuoles have been shown to be labeled by antibodies againstthe LAMP-1 protein and to contain the lysosomal hydrolaseacid phosphatase (23). To further characterize the bacterial-

FIG. 1. The acidotropic probe LysoTracker red accumulates intovacuoles containing C. burnetii. HeLa cells were incubated with C.burnetii suspended in MfbH for 48 h at 37°C in an air atmosphere.Afterwards, Coxiella-infected HeLa cells were incubated for 15 min atroom temperature with LysoTracker red and analyzed by phase-con-trast (PC; left) and fluorescence (right) microscopy. Arrows, vacuolescontaining C. burnetii (CV) loaded with LysoTracker red (top); arrow-heads, clustering of small vacuoles labeled with LysoTracker redaround the CV (bottom). N, nucleus.

VOL. 70, 2002 LOCALIZATION OF Rab7 ON COXIELLA VACUOLES 5817

replication compartment, we labeled Coxiella-infected HeLacells with LysoTracker, a marker for acidic compartments. Asshown in Fig. 1 (right) and in agreement with previous obser-vations (28), the large parasitophorous vacuoles were intenselylabeled. We have also observed small vesicles labeled by Lyso-Tracker next to large Coxiella-containing vacuoles.

To investigate the possible involvement of an autophagicpathway, we next incubated the cells with MDC, an autofluo-rescence-specific marker of autophagosomes (5, 32). The Cox-iella replication niche was labeled by MDC (Fig. 2A,top), in-dicating that this compartment has characteristics of anautophagosomal vacuole. The labeling was heterogeneous

since some vacuoles accumulated more marker than others.Interestingly, clusters of MDC-labeled vesicles were also ob-served in close proximity to parasitophorous vacuoles (Fig. 2A,bottom). It has been proposed that autophagosomes originatefrom an as yet undefined region of the endoplasmic reticulum(ER). Therefore, we were interested in determining if Coxiellacolocalizes with ER markers. Cells were labeled with rhoda-mine 6G, and a typical reticular structure was observed. How-ever, no label surrounding the vacuoles was detected, onlyempty spaces that look like “holes” where the large Coxiella-containing vacuoles are located (Fig. 2C, right).

Enzymes of the phosphatidylinositol 3-kinase (PI3K) familyhas been implicated in the regulation of a multitude of intra-cellular events (12, 27). It has been shown that wortmannin(WM) inhibits autophagy by blocking PI3K activity (6, 32).Furthermore, 3-MA is a specific inhibitor of autophagy thatseems to also block PI3K (6, 40). Therefore, to further inves-tigate the involvement of autophagy in C. burnetii infection, wetested the effect of WM and 3-MA on the development of thelarge vacuoles. Table 1 shows that both compounds were in-hibitory. To confirm the relationship between Coxiella-contain-ing vacuoles and the autophagic pathway, we studied the lo-calization of LC3, a protein that interacts specifically with theautophagosomal compartment (26). However, since the trans-fection and expression of pEGFP-LC3 in HeLa cells were low,CHO cells, which are easily infected by C. burnetii (42, 43),were used instead. As shown in Fig. 2B (right), after 48 h ofinfection of stably transfected CHO-LC3 cells, the limitingmembrane of the Coxiella-containing vacuole was strongly la-beled with protein GFP-LC3. These results clearly indicatethat Coxiella interacts with the autophagic pathway.

C. burnetii localizes in vacuoles labeled with Rab7. It is wellestablished that Rab proteins function in the tethering and dock-ing of vesicles to their target compartment, leading to membranefusion. Rab protein activity is regulated by the binding and hy-drolysis of GTP, by multiple effectors, and by the signal transduc-tion pathway (48). GTPases have the ability to cycle regularlybetween GTP- and GDP-bound states. Their on/off regulatoryfunction is restricted to the membrane compartments where theyare localized. A useful strategy to explore the function of Rabproteins is the use of GTP-binding-defective (dominant-negative)and GTPase-defective (active) mutants.

To study the interaction of the endocytic compartments withvacuoles containing C. burnetii, we examined the distribution ofRab5 and Rab7, early and late endosomal markers, respectively,on Coxiella-containing vacuoles formed in HeLa cells. Cells in-

FIG. 2. C. burnetii-containing vacuole colocalizes with MDC andLC3, specific markers of the autophagic pathway. Coxiella-infectedHeLa cells were incubated for 10 min at 37°C with MDC. After beingwashed, cells were analyzed by phase-contrast (PC; left) and fluores-cence (right) microscopy. (A) Arrows, Coxiella-containing vacuoles(CV) loaded with MDC; arrowheads, clustering of small vacuoleslabeled with MDC (bottom). N, nucleus. (B) Stably transfected CHOcells overexpressing EGFP-LC3 were infected with Coxiella as de-scribed in Materials and Methods. Cells were analyzed by phase-contrast (left) and fluorescence (right) microscopy. (C) Coxiella-in-fected HeLa cells were incubated for 20 min at 37°C with rhodamine6G (Rh6G). After being washed, cells were analyzed by phase-contrast(left) and fluorescence (right) microscopy. Arrows show that the Cox-iella-containing vacuoles are not decorated by the ER marker.

TABLE 1. Effect of autophagy inhibitors on the biogenesis ofvacuoles containing C. burnetiia

Inhibitor (concn)

Cells containing CVb

No.(total cells) %

Control 239 (918) 27WM (100 nM) 167 (945)* 183-MA (2 mM) 96 (759)* 12

a HeLa cells infected with C. burnetii during 12 h were subsequently incubatedwith the inhibitors. After 24 h, the cells were analyzed by light microscopy.

b CV, Coxiella-containing vacuoles. �, significantly different (P � 0.001) fromcontrol cells as calculated by chi-square test.

5818 BERON ET AL. INFECT. IMMUN.

fected with Coxiella for 48 h were superinfected with wild-typeand mutant forms of recombinant GFP-Rab5 or -Rab7 constructsof Sindbis virus for another 12 h. As shown in Fig. 3, the largeCoxiella-containing vacuoles were clearly decorated with GFP-Rab7wt (top) or with GTPase-defective mutant GFP-Rab7Q67L(bottom). These results suggest that C. burnetii resides in a com-partment labeled by Rab7, indicating that this compartment hascharacteristics of late endosomes and lysosomes.

Interestingly, the Coxiella-containing vacuoles were also in-tensively decorated with the GTPase-deficient Rab5 mutant(i.e., GFP-Rab5Q79L), but they were not stained in cells trans-fected with Rab5wt or dominant-negative mutant GFP-Rab5S34N (Fig. 4). The presence of the Rab5-positive mutanton Coxiella-containing vacuoles might be explained by the ob-servation that Rab5cQ79L allows the interaction between en-dosome or phagosome and lysosome compartments (36).

Since the Coxiella-containing vacuoles were heavily labeledwith GFP-Rab7wt, we next examined the distribution of GFP-Rab7T22N, a dominant-negative mutant. For technical rea-sons we were unable to generate recombinant Sindbis virusable to overexpress GFP-Rab7T22N. Therefore, we used adifferent approach to overexpress this protein. HeLa cells weretransiently transfected with plasmid pEGFP-Rab7wt and mu-tants. The expression and distribution of EGFP-Rab proteinswere similar to those observed by other authors (8). In theHeLa cells overexpressing EGFP-Rab7wt or dominant-posi-tive mutant EGFP-Rab7Q67L, the GFP signal was associatedwith vesicles partly distributed throughout the cytoplasm andconcentrated in the perinuclear region. As expected, domi-nant-negative mutant GFP-Rab7T22N showed a diffuse andwidespread distribution (data not shown).

Transiently transfected cells were subsequently infected withC. burnetii, and after 48 h the cells were analyzed by fluores-cence microscopy. As shown in Fig. 5, top, similar to the resultsobtained with the recombinant Sindbis virus, in HeLa cellstransfected with pEGFP encoding Rab7Q67L, large Coxiella-containing vacuoles were formed and a noteworthy ring-

shaped EGFP signal on the vesicles was observed. In cellstransfected with EGFP-Rab7wt, the EGFP-tagged proteinshowed a distribution essentially similar to that exhibited bymutant Rab7Q67L (data not shown). Conversely, in cells over-expressing the dominant-negative EGFP-Rab7T22N mutant,the protein presented a diffuse pattern and it was not localizedon the vacuole-limiting membrane (Fig. 5, bottom). Further-more, the formation of replication vacuoles seems to be de-layed or partly impaired since the transfected cells had few andsmall Coxiella-containing vacuoles. These results suggest thatRab7-mediated vesicular transport is required for the forma-tion and maintenance of the vacuoles that shelter C. burnetii.

DISCUSSION

In this study we present evidence that the compartmentinhabited by Coxiella displays some of the features of autopha-gosomes, such as uptake of MDC and presence of the proteinLC3 (5). Furthermore, vacuolar development was reduced by3-MA and WM, known inhibitors of the autophagic pathway(6, 40). Since parasitophorous vacuoles that shelter Coxiellaalso display lysosomal markers such as LAMPs and lysosomalenzymes, it is possible that C. burnetii resides in an autoph-agolysosome-like compartment. This is not an unusual obser-vation for an intracellular parasite since Brucella abortus seemsto reside within compartments resembling autophagosomes(35). C. burnetii has two cell variants, the large-cell variant

FIG. 3. Rab7 decorates the C. burnetii-containing vacuole. Coxiella-infected HeLa cells were incubated for 1 h with recombinant Sindbisviruses encoding the GFP-Rab7wt fusion protein or active mutantGFP-Rab7Q67L. After overnight incubations at 37°C in an air atmo-sphere, cells were analyzed by phase-contrast (PC; left) and fluores-cence (right) microscopy. GFP-Rab7wt and GFP-Rab7Q67L decoratelarge Coxiella-containing vacuoles (top and bottom, respectively).

FIG. 4. The constitutively active form of Rab5 (Rab5Q79L) local-izes with the C. burnetii-containing vacuole. Coxiella-infected HeLacells were incubated for 1 h with recombinant Sindbis viruses encodingthe GFP-Rab5wt fusion protein, negative mutant GFP-Rab5S34N, oractive mutant GFP-Rab5Q79L. After overnight incubations at 37°C inan air atmosphere, cells were analyzed by phase-contrast (PC; left) andfluorescence (right) microscopy. Coxiella-containing vacuoles did notcolocalize with GFP-Rab5wt and GFP-Rab5S34N proteins (top andmiddle, respectively). Interestingly, the Coxiella-containing vacuolemembrane was strongly labeled by GFP-Rab5Q79L (bottom).

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(LCV), the more sensitive to environmental stress of the two,and the small-cell variant (SCV), which is considered the dor-mant, stress-resistant, and less metabolically active form (23).Therefore, it is possible that Coxiella may transit through theautophagic pathway to initially avoid the harsh environment oflysosomes, perhaps during the transition from SCV to LCV.Indeed, Howe and Mallavia (25) have shown that phagosomescontaining C. burnetii exhibited a significant delay in fusionwith lysosomes. These authors have also shown, in an in vitroassay, increased protein synthesis in C. burnetii incubated atpH 5.5 (endosomal pH) compared to that observed at pH 4.5(lysosomal pH). These results suggest that C. burnetii mayprefer a slightly less acidic compartment perhaps with charac-teristics of a late endosomal or autophagosomal compartment.The interaction between the autophagic compartments and theCoxiella-containing vacuole which has some lysosomal charac-teristics could allow Coxiella to obtain metabolites for its in-tracellular growth and development.

In this report we have shown for the first time that thespacious vacuoles generated by Coxiella are heavily labeled byRab7, a late endocytic marker. The presence of Rab7 antigenin the vacuolar membranes of C. burnetii phase II-infectedmouse primary macrophages has been observed (S. Paul and S.Gomes, personal communication). Other intracellular para-sites also appear to reside in vacuoles that become positive forRab7 (10, 13, 30, 38, 39, 44). It has been shown that Mycobac-terium tuberculosis and Legionella pneumophila phagosomesacquire staining for Rab7 and for the constitutively active mu-tant form of Rab7 (Rab7Q67L) in HeLa cells overexpressingthese proteins (10). Also Salmonella enterica serovar Typhi-murium seems to inhabit a compartment enriched in lysosomalglycoproteins and Rab7, acquired apparently by fusion withnonlysosomal vesicles (30). The functional importance of Rab7in regulating membrane trafficking in the endocytic pathwayhas been established by the demonstration that expression of a

dominant-negative Rab7 mutant interrupts the normal endo-cytic flow from early to late endosomes (19, 36). In addition,overexpression of a GFP-tagged Rab7 dominant-negative mu-tant has been shown to lead to dispersal of the lysosomalcompartment (8). We have reported here not only that theCoxiella-containing vacuoles are labeled by Rab7 but also thatthis protein seems to regulate vacuole biogenesis since smallerparasitophorous vacuoles were observed in HeLa cells overex-pressing dominant-negative mutant Rab7T22N. However, amore exhaustive analysis of the effect of this mutant is neces-sary to determine the role of Rab7 in the biogenesis of theCoxiella replication compartment. Also, further experimentsneed to be done to establish whether Rab7 is actively recruitedto the Coxiella-containing phagosomes or if the protein is ac-quired by fusion with late endosomes. Indeed, we have ob-served large clusters of Rab7-positive vesicles in close proxim-ity to the Coxiella replication compartment.

In agreement with our results it has also been shown that HeLacells incubated with VacA, a toxin produced by Helicobacter py-lori, develop large intracellular vacuoles in a Rab7-dependentfashion through a process that involves fusion between late en-dosomes (34). Interestingly, in cells overexpressing Rab5Q79L,VacA induces the formation of large vesicles positive for bothRab7 and Rab5Q79L. We have also observed that the largeCoxiella-containing vacuoles were labeled by Rab5Q79L but notby Rab5wt, suggesting that the mutant protein is mistargeted orremains associated with the Coxiella replication compartment.Clemens et al. (9) have observed that large vacuoles stainedpositively for Rab5c and LAMP-1 are formed in HeLa cells over-expressing Rab5cQ79L. In contrast, in cells overexpressingRab5cwt this colocalization was not observed. Furthermore, M.tuberculosis, which normally resides in a compartment that ac-quires Rab5 but excludes Rab7 and LAMP-1 and that avoidsfusion with latex bead phagosomes (44), resides in a compartmentthat gets LAMP-1 and that fuses with latex bead phagosomes inHeLa cells overexpressing the Rab5cQ79L mutant. These resultssuggest that Rab5cQ79L allows the interaction between endo-some or phagosome and lysosome compartments. Indeed, in arecent report it has been shown that Rab5aQ79L-positive vesiclescolocalize with lysosomal membrane proteins and lysosomal en-zymes (37). Therefore, an altered traffic between lysosomes andendosomes might explain the presence of Rab5Q79L on the largelysosome-like Coxiella replication compartment. However, sinceby fluorescence microscopy we are unable to distinguish thesmallest Coxiella-containing vacuoles, we cannot discard the pos-sibility that Rab5wt might be present in the tiny vesicles generatedat the early time points of infection.

In summary, in the present report we have further charac-terized the Coxiella replication compartment and we haveclearly shown that Rab7 is present in the parasite-containingvacuoles. Since C. burnetii replicates only when is phagocy-tosed and transported to the phagolysosome, dissecting thistransport pathway at the molecular level will help us to find atherapeutic approach for this infectious agent.

ACKNOWLEDGMENTS

We specially thank Bo van Deurs for the generous gifts of plasmidsencoding the Rab7 protein and its mutants. We also thank Luis S.Mayorga for critical reading of the manuscript.

FIG. 5. The dominant-negative Rab7 mutant does not localize withthe Coxiella replication compartment. HeLa cells transfected withpEGFP plasmids encoding Rab7T22N, a dominant-negative mutant,or active mutant Rab7Q67L were incubated with C. burnetii underinfection conditions (see Materials and Methods). Cells were analyzedby phase-contrast (PC; left) and fluorescence (right) microscopy. Theactive Rab7Q67L mutant clearly labels the Coxiella-containing vacuolemembrane (arrows, top), whereas the inactive Rab7T22N mutant doesnot (arrows, bottom).

5820 BERON ET AL. INFECT. IMMUN.

This work was partly supported by grants from CONICET (PIP0695/98), Agencia Nacional de Promocion Científica y Tecnologica(PICT97 no. 01-00111-02072, PICT99 no. 1-6058), and CIUNC (Uni-versidad Nacional de Cuyo) to M.I.C. and W.B.

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Editor: J. T. Barbieri

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