Intracellular trafficking of Parachlamydia acanthamoebae

Blackwell Science, LtdOxford, UKCMICellular Microbiology 1462-5814Blackwell Publishing Ltd, 200574581589Original ArticleIntracellular trafficking of ParachlamydiaG. Greub et al.

Cellular Microbiology (2005) 7(4), 581–589

doi:10.1111/j.1462-5822.2004.00488.x

Intracellular trafficking of Parachlamydia acanthamoebae Gilbert Greub,1,2* Jean-Louis Mege,2 Jean-Pierre Gorvel,3 Didier Raoult2 and Stéphane Méresse3 1 Institute of Microbiology, Faculty of Biology and Medicine, University of Lausanne, Switzerland. 2 Unité des Rickettsies, Faculté de Médecine, Université de la Méditerranée, Marseille, France. 3 Centre d’Immunologie de Marseille-Luminy, Marseille, France. Summary Parachlamydia acanthamoebae is an obligate intracellular bacterium that naturally infects free-living amoebae. It is a potential agent of pneumonia that resists destruction by human macrophages. However, the strategy used by this Chlamydia-like organism in order to resist to macrophage destruction is unknown. We analysed the intracellular trafficking of P. acanthamoebae within monocyte-derived macrophages. Infected cells were immunolabelled for the bacteria and for various intracellular compartments by using specific antibodies. We analysed the bacteria colocalization with the different subcellular compartments by using epifluorescence and confocal microscopy. Bacterial replication took place 4–6 h post infection within acidic vacuoles. At that time, P. acanthamoebae colocalized with Lamp-1, a membrane marker of late endosomal and lysosomal compartments. A transient accumulation of EEA1 15 min post infection, and of rab7 and the mannose 6phosphate receptor 30 min post infection confirmed that P. acanthamoebae traffic through the endocytic pathway. The acquisition of Lamp-1 was not different after infection with living and heat-inactivated bacteria. However, 24.5% and 79.5% of living and heat-inactivated P. acanthamoebae, respectively, colocalized with the vacuolar proton ATPase. Moreover, P. acanthamoebae did not colocalized with cathepsin D, a lysosomal hydrolase, suggesting that P. acanthamoebae interferes with maturation of its vacuole. Thus, P. acanthamoebae survives to

Received 3 August, 2004; revised 20 October, 2004; accepted 26 October, 2004. *For correspondence. E-mail gilbert.greub@ hospvd.ch; Tel. (+41) 21 314 49 79; Fax (+41) 21 314 40 60. © 2005 Blackwell Publishing Ltd

destruction by human macrophages probably by controlling the vacuole biogenesis. Introduction First described in 1997, Parachlamydia acanthamoebae is an obligate intracellular bacterium naturally infecting free-living amoebae (Amann et al., 1997). The human pathogenicity of this Chlamydia-like organism is supported by a growing body of evidence (reviewed in Greub and Raoult, 2002a). Thus, serological hints suggested the role of P. acanthamoebae in an epidemic of fever associated with an humidifier in a print-shop (Birtles et al., 1997), and its roles as an agent of community-acquired (Marrie et al., 2001) and inhalation pneumonia (Greub et al., 2003a). Moreover, the amplification of parachlamydial DNA from monocytes, sputa and bronchoalveolar lavages taken from patients with bronchitis or pneumonia further supported the pathogenic potential of some Parachlamydiaceae (Ossewaarde and Meijer, 1999; Corsaro et al., 2001; 2002; Greub et al., 2003b). Parachlamydia acanthamoebae share more than 80% sequence similarity for the 16S rRNA-encoding gene with Chlamydophila pneumoniae and Chlamydia trachomatis, two established human pathogens (Fritsche et al., 2000; Greub and Raoult, 2002a). Like other Chlamydiales, it presents two developmental stages, the elementary body – an infective stage – and the reticulate body – a metabolically active dividing form (Greub and Raoult, 2002b). It also presents a further infective stage, the crescent body, only present within the Parachlamydiaceae (Greub and Raoult, 2002b). Differentiation of the infective stages in reticulate bodies and multiplication of the reticulate bodies occurs within amoebal vacuoles (Greub and Raoult, 2002b). A similar life cycle of P. acanthamoebae has been observed within monocyte-derived human macrophages, where multiplication of reticulate bodies occurs within vacuoles (Greub et al., 2003c). P. acanthamoebae is thus able to resist to the microbicidal effectors of human macrophages. This is probably related to subversion of the endocytic pathway generally intended to lyse the internalized microorganisms within phagolysosomes. However, the strategy used by P. acanthamoebae to resist destruction by macrophages is unknown. Given the phylogenic relationship of P. acanthamoebae with Chlamydiaceae, it may like Chlamydia spp. escape the endocytic

582 G. Greub et al. pathway and replicate in a vacuole in close contact with the trans-Golgi network (Hackstadt et al., 1995; Wolf and Hackstadt, 2001). Nevertheless, as P. acanthamoebae diverged from Chlamydiaceae more than 700 millions years ago (Greub and Raoult, 2003), it may use another strategy to resist to human macrophages and, like Legionella, multiplie within endoplasmic reticulum-associated vacuoles (Scidmore-Carlson and Hackstadt, 2000; Roy and Tilney, 2002). It may, also like Mycobacterium sp., interfere with phagosome maturation and replicate within endocytic vacuoles that did not acquired the vacuolar proton ATPase (v-ATPase) responsible for the acidic pH of lysosome (Sturgill-Koszycki et al., 1994) or, like Tropheryma whipplei, replicate within acidic phagosomes, lacking lysosomal hydrolases such as cathepsin D (Ghigo et al., 2002). We analysed the intracellular trafficking of P. acanthamoebae within monocyte-derived macrophages and defined the characteristics of the Parachlamydia-containing vacuoles (PCVs), early after infection and later, at time of bacterial replication. To reveal early interactions between PCVs and the host cell, we developed a shortened infection procedure allowing a rapid and synchronized bacterial invasion. Four to six hours after this infection procedure, we observed bacterial replication that leads to the lysis of the macrophages as early as 8 h post infection. At time of replication, there was no colocalization of P. acanthamoebae with markers of the endoplasmic reticulum and Golgi, but with markers of late endocytic compartments such as Lamp-1 and rab7. The intracellular fate of P. acanthamoebae within endocytic-derived vacuoles was confirmed by the rapid association of the bacteria with the early endocytic marker EEA1. Although the rate of colocalization of the v-ATPase with living bacteria was significantly lower than that of heat-inactivated bacteria (that are degraded within mature lysosome), the vacuole of replication was found to be acidic. The absence of colocalization of P. acanthamoebae with hydrolases such as cathepsin D suggests that P. acanthamoebae may survive to macrophages by preventing the maturation of the PCV in a phagolysosome. This strategy, clearly different from that used by Chlamydiaceae, may reflect the long history of intracellular parasitism of Chlamydiales estimated to begun more than 1 billions years ago, and the early divergence of Parachlamydiaceae from Chlamydiaceae (Greub and Raoult, 2003).

Results Infection procedure We developed a shortened infection procedure, consisting of a centrifugation step followed by a 15 min incubation time at 37∞C. Electron microscopy showed that, using this

infection procedure, P. acanthamoebae is internalized as early as 15 min post infection (Fig. 1A), and replicates within vacuoles 4–6 h post infection. At that time, clusters of about 10 Parachlamydia may be seen within replicative vacuoles (Fig. 1B). The number of Parachlamydia per macrophages increased about four times within the first 6 h (Fig. 1C). Replicative vacuole At time of replication (4–6 h post infection), we determined the nature of the PCV by analysing the presence of various specific markers for intracellular organelles. We first analysed the distribution of mitochondria, which surround inclusions of cells infected by some Chlamydiaceae. Mitochondria were not recruited nor associated with the replicative vacuole (data not shown). Next, we analysed the association of the replicative PCV with compartments of the exocytic pathway. The bacteria did not colocalized either with markers of the endoplasmic reticulum (calnexin) or with markers of the Golgi (GM 130) (data not shown). The presence of molecules specific of the endocytic pathway was examined and revealed that P. acanthamoebae colocalized with the late endosomal and lysosomal integral glycoprotein Lamp-1 (Fig. 2). The endocytic nature of the replicative vacuole was confirmed by the presence of the late endocytic marker v-ATPase on the PCV (Fig. 2). These results showed that P. acanthamoebae replicates in a vacuole having acquired markers of the endocytic pathway but not of the exocytic pathway. Intracellular trafficking As vacuole biogenesis may influence the intracellular survival of P. acanthamoebae, we defined the characteristics of the PCV and its maturation during the course of an infection. We examined the presence on the PCV of markers specific of the successive stations of the endocytic pathway. EEA1 is an early endosome-specific peripheral membrane protein which colocalizes with the small GTPbinding protein rab5 (Mu et al., 1995). In cells fixed immediately after the 15 min incubation in the presence of P. acanthamoebae, we observed the presence of EEA1 on 22 ± 7% of PCVs. EEA1 was rapidly removed from PCVs as the percentage of vacuoles positive for this marker dropped to 7 ± 4% and 2 ± 1% at 30 and 60 min post infection, respectively (Fig. 3). We next examined the presence of the late endosome markers rab7 and the cationindependent mannose 6-phosphate receptor (CI-MPR). Both molecules were detected on the PCV and their presence peaked at 30 min post infection (Fig. 3). These transient accumulations of early and late endocytic markers on PCVs before the acquisition of Lamp-1 confirmed that © 2005 Blackwell Publishing Ltd, Cellular Microbiology, 7, 581–589

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Fig. 1. Parachlamydia replicates within human macrophages. A. P. acanthamoebae (arrow) internalized by monocyte-derived macrophages, as early as 15 min post infection. Electron microscopy, bar represents 1 mm. B. Clusters of about 10 Parachlamydia (arrows) may be seen within replicative vacuole 4–6 h post infection. Confocal microscopy, bar represents 5 mm. The dashed line represent the border of the cell. C. Using a 15 min infection procedure, replication of P. acanthamoebae occurs as early as 4–6 h post infection. Intracellular bacteria were enumerated at different time post infection, and the fold increase was calculated by normalization of the number of bacteria according to the number of bacteria 15 min post infection. The results shown are representative of three independent experiments. Results are the means ± SEM of triplicate experiments.

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(Fig. 2). As control heat-killed P. acanthamoebae were internalized in monocyte-derived macrophages and were degraded through the endocytic pathway, we compared the rates of colocalization of Lamp-1 with living and heatinactivated bacteria. These rates were not different 6 h post infection, being 89.5% and 92.5% respectively. However, 24.5% and 79.5% of living and heat-inactivated P. acanthamoebae, respectively, colocalized with the vATPase. We used the fixable acidotropic probe LysoTracker to monitor acidic organelles in infected macrophages. This dye displayed a major overlap with PCVs, suggesting that the replicative vacuole was acidic (Fig. 4). We next investigated the presence in vacuole of cathepsin D as a marker for the lysosomal soluble content. P. acanthamoebae did not colocalized with cathepsin D (Fig. 4), suggesting that the PCV does not fuse with lysosomes and confirming that P. acanthamoebae interferes with the maturation of its vacuole. Interestingly, heat-inactivated bacteria did not colocalized with cathepsin D and harboured preserved morphological properties as long as 48 h post infection.

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In this paper, we show that P. acanthamoebae is trafficking in monocyte-derived macrophages within vacuoles associated to the endocytic pathway (Fig. 5). However, contrary to heat-inactivated bacteria, which as a rule colocalize with the v-ATPase, living bacteria apparently modify the vacuole biogenesis, preventing the fusion of the PCV with the hydrolases-rich lysosomal compartment and partially inhibiting the acquisition of the v-ATPase, a multisubunit complex that is involved in the acidification of the endocytic pathway. The replicative vacuole of P. acanthamoebae and its biogenesis were studied by immunofluorescence. A shortened infection procedure was used to obtain a synchronous infection of macrophages and to allow the study of the PCV early after infection. With this shortened infection procedure, internalization was observed less than 15 min

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Fig. 2. Parachlamydia is trafficking and replicates within Parachlamydia-containing vacuoles (PCVs) associated with the endocytic pathway, as demonstrated by the colocalization (arrows) of P. acanthamoebae with Lamp-1 (top) and, though to a lesser extent, with the v-ATPase (bottom). The graphs show the percentages of bacteria enclosed in vacuoles positive for Lamp-1 or v-ATPase at various time post infection. The results are the means ± SEM of triplicate experiments. Right panels are representative confocal images showing the colocalization of P. acanthamoebae with Lamp-1 and the v-ATPase 4 h post infection (p.i.). Confocal microscopy, bar represents 10 mm.

post infection, replication took place about 4–6 h post infection and first evidence of Parachlamydia-induced apoptosis (Greub et al., 2003c) was already observed 8 h post infection. The internalized bacteria are initially present in a vacuole related to the endocytic pathway, as demonstrated by their positivity for EEA1 15 min post infection. The presence of EEA1 on the membrane of PCV early after infection probably results from recruitment of EEA1 from the cytosol or by fusion of the nascent vacuole with early endosome, similarly to what is observed for Salmonella (Steele-Mortimer et al., 1999). P. acanthamoebae does not remain within early endosomes, as demonstrated by the transient acquisition of rab7 and of the CI-MPR 30 min post infection. Even if we can not exclude that the fate of P. acanthamoebae may be different in non-phagocytic cells, we showed that the rapid recycling of rab7 and of the CI-MPR, the concomitant acquisition of Lamp-1 and the exclusion of lysosomal hydrolases are the hallmarks of the maturation of the nascent PCV in monocyte-derived macrophages. The association of the PCV with markers of the endocytic pathway such as Lamp-1 was also observed after an infection procedure performed without centrifugation (data not shown). The replicative vacuole of P. acanthamoebae is acidic. This may have therapeutic implications, as some antibiotics are ineffective at low pH (Maurin et al., 1992). Living bacteria exhibit, however, a lower rate of colocalization with the v-ATPase than that observed with heat-

inactivated bacteria suggesting that the pH of the vacuole of replication was less acidic than that of lysosomes. Such inhibition of the acquisition of v-ATPase by the internalized bacteria is a mechanism that has been observed for Mycobacterium spp. (Sturgill-Koszycki et al., 1994). Preventing the acquisition of lysosomal hydrolases such as cathepsin D is a mechanism also observed for Tropheryma whipplei, which replicates within acidic phagosomes, lacking cathepsin D (Ghigo et al., 2002). However, the strategy of P. acanthamoebae to subvert innate immune cells is much closer to that used by Salmonella (Meresse et al., 1999a,b; Steele-Mortimer et al., 1999) than to any other bacteria, as both Parachlamydia and Salmonella replicate within acidic Lamp-1-positive cathepsin-negative vacuoles. Parachlamydia acanthamoebae is the first member of the order Chlamydiales, which is shown to traffic in association with vacuoles of the endocytic pathway. This is of importance, as Chlamydiaceae appear to completely bypass the early endocytic pathway (Scidmore-Carlson and Hackstadt, 2000), and replicate within an inclusion that is trafficked to the peri-Golgi region where it fuses with exocytic vesicles (Hackstadt et al., 1995). Early synthesis of chlamydial proteins apparently regulate the traffic of the chlamydial inclusion (Scidmore et al., 1996), by recruiting some rab GTPases (rab1, rab4, rab11), which are key regulators of membrane trafficking. The interactions of different rab GTPases with the chlamydial inclusion membrane were proposed as a possible mechanism © 2005 Blackwell Publishing Ltd, Cellular Microbiology, 7, 581–589

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Fig. 3. Parachlamydia-containing vacuoles (PCVs) transiently acquire early and late endocytic markers. The graphs show the percentages of bacteria enclosed in vacuoles positive for EEA1, rab7, or the cation-independent mannose 6-phosphate receptor (CI-MPR) at various time post infection (p.i.). The results are the means ± SEM of duplicate experiments. Right panels are representative confocal images; bar represents 5 mm.

explaining the various tropism of Chlamydiaceae (Rzomp et al., 2003). Contrary to the Chlamydiaceae, P. acanthamoebae is successively associated with the early endosome GTPase rab5, as demonstrated by the recruitment on PCVs of its effector protein EEA1, and with rab7, a marker of late endosome. The mechanism used by P. acanthamoebae to modify the trafficking or the fusogenic properties of the PCV remains to be defined. As the heat-inactivated bacteria did not colocalized with cathepsin D, P. acanthamoebae should be able to passively modulate its intracellular fate. As for Mycobacterium, such interactions might be facilitated by special composition of the bacterial membrane, and by the observed tight association of the vacuole membrane with the bacterial cell wall. In addition, the type III secretion system, shown to be present in the genome of the Parachlamydia-related strain UWE25 (Horn et al., 2004), enabling the translocation of bacterial effector into © 2005 Blackwell Publishing Ltd, Cellular Microbiology, 7, 581–589

the host cell, might also be present in P. acanthamoebae. If this is the case, this secretion system might modulate the PCV biogenesis. Such an active system might explain the different traffic of living and heat-inactivated bacteria. This secretion might occurs or be triggered by the contact of elementary or reticulate bodies with the vacuole membrane. A better knowledge of the biology of P. acanthamoebae is thus needed to understand in the future how this Chlamydia-like organism is able to subvert the biogenesis of its phagosome. However, the fact that P. acanthamoebae, contrary to Chlamydiaceae, traffic in an endosomeassociated vacuole underlines the important biological differences between these two representing families of the order Chlamydiales that diverged more than 700 millions years ago (Greub and Raoult, 2003), and is an additional example of evolutive strategy developed by intracellular bacteria to resist to macrophages.

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Fig. 4. The Parachlamydia-containing vacuole (PCV) is acidic and does not accumulate the lysosomal hydrolase cathepsin D. Absence of colocalization of the PCV with lysosomal hydrolase such as cathepsin D 6 h post infection (p.i.) (top). Acidification of the PCV (bottom), as demonstrated by the accumulation of the LysoTracker within PCVs 6 h post infection. Confocal microscopy, bar represents 10 mm for cathepsin D and 5 mm for the LysoTracker.

Experimental procedures Parachlamydia culture and purification Parachlamydia acanthamoebae strain Hall coccus was grown at 32∞C within Acanthamoeba polyphaga strain Linc AP-1 in 150 cm2 cell culture flasks (Corning, NY) with 30 ml of peptone yeast-extract glucose broth (PYG) (Greub et al., 2004). After 6 days of incubation, cultures were harvested and the broth was centrifuged at 180 g for 10 min to eliminate most amoebae. The supernatant was then centrifuged at 6600 g for 30 min. The bacterial pellet was suspended in 10 ml of Dulbecco’s PBS (GibcoBRL, Life Technologies, Paisley, Scotland) and purified by centrifugation on a 10% sucrose (Sigma) barrier, as described (Greub et al., 2003c). To improve purification, the pellet was suspended in PBS, loaded onto a discontinuous Gastrografine (Schering, Lys-Lez-Lannoy, France) gradient and ultracentrifuged at 140 000 g. P. acanthamoebae, which clustered in a large lower band, were collected, centrifuged at 5800 g, and washed in PBS twice before freezing at -80∞C. Parachlamydia were titred using a lysis test (Greub et al., 2003d). Briefly, 200 ml of 5 ¥ 105 ml-1 A. polyphaga in Page’s modified Neff’s amoeba saline (PAS) (Greub et al., 2004) were distributed in a 96-well Costar microplate (Corning). Parachlamydia were serially diluted from 10-1 to 10-14 in another microplate. Each dilution (10 ml) was inoculated into six wells of the Acanthamoeba microplate, which was read daily

to determine the highest dilution that led to amoebal lysis. Heatinactivated Parachlamydia were obtained by heating the bacteria at 100∞C for 1 h and were stored at -80∞C.

Macrophages Blood from healthy volunteers was drawn in EDTA anticoagulated tubes. Peripheral blood mononuclear cells (PBMC) were separated by centrifugation at 720 g for 20 min on Ficoll (Eurobio, Les Ulis, France), and suspended in RPMI-Hepes supplemented with 200 mM L-glutamine (Gibco-BRL) and 10% fetal calf serum (Gibco-BRL). Then, 106 PBMCs per millilitre were incubated for 1 h at 37∞C on 12-mm-diameter round coverslips in flat-bottom 24-well cell culture plates (Corning) or in 50 cm3 flasks (Corning). After washing, adherent cells were considered monocytes because more than 95% of them were CD14+ and exhibited phagocytic characteristics. Monocytes were further differentiated into macrophages by incubation at 37∞C in the presence of fetal calf serum, as previously described (Greub et al., 2003c).

Antibodies and probes Human anti-EEA1 was a gift from Dr Ban-Hock Toh, Monash Medical School, Melbourne, Australia. Monoclonal anti-human © 2005 Blackwell Publishing Ltd, Cellular Microbiology, 7, 581–589

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Fig. 5. Model of the intracellular trafficking of Parachlamydia within monocyte-derived macrophages. After internalization, P. acanthamoebae resides within a Parachlamydia-containing vacuole (PCV) that interacts with early and late endosomes, as demonstrated by the transient acquisition of EEA1 15 min post infection (p.i.) and, rab7 and the CI-MPR 30 min post infection. The replicative vacuole of P. acanthamoebae acquired Lamp-1. However, the maturation of the vacuole is modulated by the bacteria that completely prevents the acquisition of lysosomal hydrolases (cathepsin D) and partially prevents the acquisition of the v-ATPase. The crescent bodies, a developmental stage specific of the Parachlamydiaceae and described by electron microscopy (Greub and Raoult, 2002b), could be seen by confocal microscopy 4–8 h post infection. P. acanthamoebae were revealed by rabbit anti-Parachlamydia antibodies and Donkey anti-rabbit FITC antibodies. In red, the v-ATPase-positive vacuoles may be seen. TR, transferrin receptor. Confocal microscopy, bar represents 5 mm. © 2005 Blackwell Publishing Ltd, Cellular Microbiology, 7, 581–589

588 G. Greub et al. Lamp-1, developed by T.J. August, was obtained from the Development Studies Hybridoma Bank maintained by the University of Iowa, Department of Biological Studies, Iowa City. Rabbit antibody against rab7 has been described previously (Meresse et al., 1997). Mouse anti-v-ATPase (OSW2) was a kind gift of Dr Satoshi B. Sato (Kyoto University). Rabbit anti-CI-MPR was a gift from Dr Bernard Hoflack. Rabbit anti-cathepsin D was obtained from Dako Corporation (Carpinteria, CA). Rabbit anti-Calnexin was purchased from Stressgen Biotechnologies (Victoria, BC, Canada). Mouse anti-GM130 was obtained from BD Biosciences (Franklin Lakes, NJ). Mouse anti-human mitochondria were obtained from Chemicon International (Temecula, CA). The fluorescent basic amine probe LysoTracker Red was obtained from Molecular Probes (Leiden, the Netherlands). Bacteria were revealed by immunofluorescence using home-made polyclonal mouse or rabbit anti-Parachlamydia antibodies (Greub et al., 2003a,c). The secondary antibodies used were FITC-conjugated donkey anti-rabbit IgG, FITC-conjugated donkey anti-mouse IgG, FITC-conjugated donkey anti-goat IgG, Texas red-conjugated donkey anti-mouse IgG and Texas red-conjugated donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Infection procedure Macrophages were seeded with living or heat-inactivated bacteria (bacterium-to-cell ratio, 100:1) in supplemented RPMI medium without antibiotics, centrifuged at 1793 g for 10 min at 20∞C, and incubated for 15 min at 37∞C. Then, cells were washed with RPMI-Hepes, and further incubated for different periods of time at 37∞C in a 5% CO2 atmosphere.

Immunofluorescence and confocal microscopy Infected cells were washed in PBS after 15 min, 1 h, 2 h, 4 h, 6 h and 8 h and fixed with 3% paraformaldehyde. Fixed cells were washed three times in PBS and permeabilized by incubation in PBS containing 0.1% saponin (Research Organics, Cleveland, OH). Saponin (0.1%) was included in all subsequent incubation steps. Primary and secondary antibodies were diluted in PBS containing 0.1% saponin and 10% horse serum. Coverslips were incubated with primary antibodies for 30 min at room temperature, washed in PBS containing 0.1% saponin and then incubated with FITC- or Texas red-coupled donkey antirabbit, anti-goat or anti-mouse antibodies. Coverslips were mounted onto glass slides using Mowiol (Aldrich, Steinheim, Germany). Cells were observed with a Leica TCS 4DA confocal microscope. The laser emission lines used were 488 nm and 568 nm. Files were analysed using Adobe Photoshop software, and images were merged using RVB format. To determine the percentage of bacteria that colocalized with the different intracellular markers, a minimum of 100 intracellular bacteria were counted. The number of bacteria per macrophage was determined by counting three times the number of Parachlamydia within about 100 macrophages.

Electron microscopy Infected cells in Corning flasks were washed after 15 min, 4 h and 8 h with PBS, fixed with 4% glutaraldehyde, and prepared

as previously described (Greub and Raoult, 2002b). Grids were examined with a Morgagni 268D electron microscope (Philips Eindhoven, the Netherlands).

Acknowledgements We thank the Swiss National Science Foundation for funding the Postodoctoral Fellowship of Gilbert Greub, who received an ‘Advanced Researcher Fellowship’.

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