1 Unité de Biologie des Interactions Cellulaires, Institut Pasteur, CNRS URA 2582, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France
2 CNRS UMR 144 Institut Curie, 12 rue Lhomond, 75005 Paris, France
Author for correspondence (e-mail: adautry{at}pasteur.fr)
Accepted 24 February 2005
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Summary |
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Key words: Chlamydia, Phagocytosis, Intracellular bacteria, PIP 5-kinase, PIP2
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Introduction |
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The ADP-ribosylation factor 6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and the actin cytoskeleton at the plasma membrane (Donaldson, 2003). It is involved in membrane trafficking during receptor-mediated endocytosis, endosomal recycling and exocytosis of secretory granules (D'Souza-Schorey et al., 1995
; D'Souza-Schorey et al., 1998
; Radhakrishna and Donaldson, 1997
; Vitale et al., 2002
). It is also implicated in the formation of actin-rich membrane protrusions and ruffles (Radhakrishna et al., 1996
). Like all small GTP-binding proteins, ARF6 cycles between an inactive GDP-bound state and an active form when bound to GTP. GTP-ARF6 acts through the activation of downstream effectors, including lipid-modifying enzymes such as phospholipase D (PLD) and phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase) (Honda et al., 1999
; Massenburg et al., 1994
). PIP 5-kinase, which is responsible for generating phosphatidylinositol 4,5-bisphosphate (PIP2), has been implicated in the phagocytosis of IgG-coated erythrocytes, probably through the regulation of actin cytoskeletal proteins by PIP2 (Coppolino et al., 2002
). Altogether, changes in membrane lipid composition and structure may mediate ARF6 alterations of the cortical actin cytoskeleton and regulation of membrane traffic and signal transduction. Interestingly, in the case of phagocytosis of red blood cells by macrophages, ARF6 was shown to control membrane recruitment at the sites of phagocytosis rather than actin polymerization (Niedergang et al., 2003
). In the case of Yersinia pseudotuberculosis, it was shown that a PIP2-dependent pathway regulated by ARF6 is associated with bacterial internalization, suggesting that it controls actin polymerization (Wong and Isberg, 2003
).
Chlamydiae are obligate intracellular parasites of eukaryotic cells, which constitute an important group of pathogens responsible for a variety of acute and chronic diseases including trachoma, pelvic inflammatory disease, pneumoniae and their sequelae (Gregory and Schaffner, 1997; Kuo et al., 1995
; Stamm, 1999
). The bacteria attach to epithelial host cells via a relatively weak electrostatic interaction with heparan sulfate proteoglycans (Su et al., 1996
) and a more specific binding to an unidentified secondary receptor (Carabeo et al., 2002
). Chlamydia concentrate in lipid membrane microdomains (Simons and Toomre, 2000
) and this is necessary for bacterial entry (Jutras et al., 2003
; Stuart et al., 2003
).
Here, we show that endogenous ARF6 undergoes a rapid and transient activation after infection by Chlamydia caviae and plays a critical role in bacterial uptake by regulating the actin cytoskeleton reorganization at the sites of entry. Moreover, the bacterial entry site forms a remarkable calyx-like membrane protrusion, where ARF6 and its effectors differentially localize around the bacteria.
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Materials and Methods |
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The bacteria were labelled with FITC as described (Subtil et al., 2004). Bacteria labelling with CyTM5 was performed with FluoroLinkTMCy5 reactive dye (Amersham Pharmacia, UK) in phosphate-buffered saline pH 7.8 (PBS) for 1 hour on ice. Labelled bacteria were washed twice with SPG (0.5 ml), centrifuged at 18,000 g, and finally spun for 5 minutes at 700 g to eliminate large patches. The supernatant was aliquoted and stored at 80°C until use.
Pull-down assay
Expression of the ARF-binding domain (ARF-BD) of ARHGAP10 fused with GST was induced in E. coli with 1 mM IPTG for 2 hours at 37°C. The fusion protein was purified by affinity chromatography on glutathione-Sepharose beads (Amersham Pharmacia Biotech) and stored in 20 mM Tris-HCl, pH 7.4, 2 mM EDTA, 100 mM NaCl, 10% glycerol and 2 mM ß-mercaptoethanol (Dubois et al., 2001).
HeLa cells (5x106 per time point) grown overnight at subconfluence in serum-free conditions were detached with PBS-EDTA and resuspended in DMEM medium. Cells were mixed with the bacteria in 0.1 ml DMEM, centrifuged for 20 seconds at 16,000 g and immediately transferred to 37°C (t=0). For the control, the cells were mixed with an equivalent amount of a preparation from mock-infected cells. After incubation for different times, the cells were lysed on ice for 15 minutes in lysis buffer (150 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 7.5). Cell lysates were centrifuged at 16,000 g at 4°C for 10 minutes. Equal volumes of supernatant for each time point were incubated for 90 minutes with 40 µg ARF-BD of ARHGAP10 (T. Dubois and P.C., unpublished results) in the presence of 0.5% BSA and glutathione-Sepharose. The resin was washed extensively with washing buffer (150 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 50 mM Tris-HCl, pH 7.5), boiled in SDS-PAGE sample buffer, and bound proteins were resolved on SDS gels followed by western blot analysis using an anti-ARF6 antibody.
Transfection
HeLa cells, which were less than 50% confluent, were transfected with ARF6 WT, ARF6 T27N, ARF6 QS-EI, ARF6 N48I, Lyn-phosphatase-CFP, PIP 5-kinase or PLC1 PH-GFP constructs using Fugene reagent (Roche Applied Science) or by electroporation (900 µF, 200 V, EasyJect Eurogentec, Belgium) and grown in 24-well plates for 18 hours.
RNA interference
The siRNA targeting PIP 5-kinase ß was as described (Padron et al., 2003). The sequence of the siRNA targeting ARF6 was 5'-AAGCUGCACCGCAUUAUCAAU-3'. Control cells were transfected with an irrelevant siRNA. RNA oligonucleotides were synthesized by Dharmacon Research.
HeLa cells were plated at 50% confluence in DMEM supplemented with 10% FCS. The following day, 0.2 nmoles of siRNA were introduced into 4x106 cells by electroporation at 500 µF and 300 V (Easyject, Eurogentec).The medium was changed after an overnight incubation. In the case of ARF6, siRNA was re-introduced at 48 hours, following the same procedure. 48 hours after the first transfection (or 72 hours in the case of ARF6), the cells were infected. For studying the effect of RNA interference on protein expression, lysates from control and ARF6 siRNA-treated cells were prepared. The cells were lysed on ice for 15 minutes in lysis buffer (150 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 7.5). Cell lysates were centrifuged at 16,000 g at 4°C for 10 minutes and analysed by western blotting using monoclonal anti-ARF6 antibody.
Chlamydia GPIC Infection
Cells transfected for 18 hours or treated with siRNA were washed twice in phosphate-buffered saline (PBS) and incubated in complete medium with Chlamydia caviae GPIC (guinea pig inclusion conjunctivitis) at a concentration resulting in 30-70% infected cells, 24 hours post-infection. After 1.5 hours at 37°C, the bacteria were removed from the supernatant and the cells were washed twice in PBS, complete medium was added and the incubation continued for 2.5 hours in the bacteria entry experiments or 17 hours to measure the efficiency of infection.
In the experiments where entry was synchronized for actin patch quantification at the entry sites, transfected cells were infected in 0.25 ml culture medium and immediately centrifuged for 5 minutes at 770 g at room temperature, and finally incubated for 5 minutes at 37°C. When indicated, cells were infected with CyTM5 or FITC-labelled Chlamydia.
At the end of the infection (at the time indicated in each case), the cells were washed twice in PBS and fixed in 4% paraformaldehyde, 120 mM sucrose in PBS for 30 minutes at room temperature. The cells were washed in PBS, incubated for 10 minutes in 50 mM NH4Cl in PBS at room temperature and washed in PBS containing 1 mg/ml bovine serum albumin.
Immunofluorescence
The efficiency of bacterial entry was measured as described (Subtil et al., 2004). The efficiency of entry ranged between 40 and 60%. The association index was calculated as the mean number of cell-associated bacteria (intracellular+surface-associated) per cell in transfected cells/cell-associated bacteria in control non-transfected cells x 100. On average, 5-20 bacteria were associated with control cells. For bacterial entry assay in Lyn-phosphatase-CFP transfected cells, FITC-labelled Chlamydia were used and extracellular bacteria were labelled with anti-Chlamydia antibody followed by a Cy5-coupled antibody.
To measure the percentage of infected cells 20 hours after infection, the cells were fixed in paraformaldehyde as above, permeabilized with 0.05% saponin and the inclusions labelled with FITC-conjugated anti-Chlamydia antibody (Boleti et al., 1999). The number of infected cells was counted in about 300 transfected cells randomly chosen on the coverslips and in the surrounding non-transfected cells; the index of infection was calculated as the number of infected cells/total cells counted. The index obtained for transfected cells was divided by the index obtained for control non-transfected cells and expressed as percentage of control cells.
The quantification of local actin rearrangements visualized 5 minutes post infection at the entry sites (Subtil et al., 2004) were quantified in ARF6 T27N and ARF6 QS-EI transfected cells, and identified using polyclonal anti-ARF6 antibody and Cy3-coupled secondary antibody. Actin was labelled with Alexa 488-coupled phalloidin. The number of local actin rearrangements was counted for each transfected and non-transfected cell in the fields (about 100 cells were counted per experiment) using an epifluorescence microscope (Axiophot, Zeiss, Germany) equipped with a 63x Apochromat objective and a cooled CDD-camera (Photometrics, Tucson, AZ) driven by Metaview software (Universal Imaging, Downington, PA). Results are expressed as the number of patches in transfected cells as a percentage of those in non-transfected cells.
Confocal microscopy
Actin was visualized using Alexa 633, Alexa 488 or Alexa 546-coupled phalloidin. ARF6 and PIP 5-kinase transfected cells were identified with anti-ARF6 or anti-HA antibodies and secondary antibodies. Coverslips were mounted on Mowiol with 100 mg/ml DABCO and examined under a confocal microscope (LSM 510, Zeiss) using a 63x objective. A z-series of optical sections was taken every 0.2 or 0.5 µm. Image deconvolution was performed using Huygens software (SVI, The Netherlands), and three-dimensional image reconstruction was carried out using Imaris and OsiriX software (Bitplane, Switzerland). To prevent bleed-through effects in samples stained with multiple fluorochromes, green and far-red fluorescence emissions (excitations at 488 and 633 nm, respectively) were acquired simultaneously, but separately from red fluorescence (excitation at 546 nm).
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Results |
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ARF6 is activated during bacterial infection
To further study ARF6 involvement, we monitored the activation levels of endogenous ARF6 upon bacterial infection using the ARF-binding domain of ARHGAP10 (ARF-BD) fused to GST, which binds the active GTP-bound form of ARF6 (T. Dubois and P.C., unpublished results). One difficulty in the study of the initial events of bacterial infection resides in the asynchronous attachment of bacteria to host cells. To circumvent this problem, the bacterial inoculum was centrifuged together with cells in suspension for 20 seconds and then incubated at 37°C to allow infection to proceed. At different times, the cells were lysed on ice, and activated ARF6 was pulled-down. The precipitated GTP-bound ARF6 was analysed by western blotting with anti-ARF6 antibody.
Activation of ARF6 was observed 5 minutes after bacterial contact, and decreased after 30 minutes to return to basal levels by 60 minutes after infection (Fig. 3). These experiments show that endogenous ARF6 is sharply and transiently activated at a very early step of bacterial entry.
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Recruitment of PIP 5-kinase at the sites of bacterial entry and local PIP2 production
ARF6 is known to activate lipid-modifying enzymes, PIP 5-kinase and phospholipase D (PLD) (Honda et al., 1999; Massenburg et al., 1994
). Expression of an ARF6 mutant form (ARF6 N48I) unable to activate PLD (Vitale et al., 2002
) did not impair Chlamydia uptake (not shown), suggesting that regulation of PLD by ARF6 is not required for entry. Next, we investigated the role of PIP 5-kinase and first determined the subcellular localization of PIP 5-kinase during bacterial uptake. Cells transfected for 18 hours with a construct encoding an HA-tagged PIP 5-kinase were infected for 5 minutes with FITC-labelled bacteria. PIP 5-kinase was recruited around the bacteria at the plasma membrane (Fig. 5A).
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To determine if PIP 5-kinase was necessary for infection, cells were treated with siRNA specific for PIP 5-kinase before infection (Padron et al., 2003). The number of infected cells was then quantified in treated and control cells after staining the inclusions with anti-Chlamydia antibodies. In PIP 5-kinase siRNA-treated cells the infection was reduced by 50% (Fig. 5C). This decrease is probably underestimated because we quantified infection in the whole siRNA-treated cell population. To determine if PIP2 plays a functional role during Chlamydia entry, the effect of lowering the cellular concentration of PIP2 on bacterial uptake was examined. To that end, the cells were transfected with a plasma membrane-targeted PIP2-specific 5'-phosphatase, Lyn-CFP-Inp54p, which has been successfully used to selectively reduce plasma membrane PIP2 concentration (Raucher et al., 2000
; Wong and Isberg, 2003
). Expression of Lyn-CFP-Inp54p phosphatase resulted in a 40% reduction of uptake efficiency compared with mock-treated cells (Fig. 5D).
Together, the presence of PIP 5-kinase and its product PIP2 at the sites of bacterial entry, as well as the inhibition of entry when the transfected PIP2 phosphatase was expressed, indicate that PIP2 is involved in bacterial entry. It is noteworthy that the inhibition of bacterial entry in the latter conditions (about 40%) is close to the one measured when ARF6 dominant-negative mutant was used (about 50%), further suggesting that PIP 5-kinase is a major downstream effector in the pathway by which ARF6 regulates bacterial entry.
Chlamydia entry sites form a spatially well-organized structure
We have identified at the sites of Chlamydia entry several molecules, which are required for bacterial uptake. As presented above, transiently expressed ARF6 and PIP 5-kinase, appear to accumulate at the plasma membrane at the sites of entry together with PIP2 (Figs 1, 5; Fig. 6B). These accumulations coincided with rounded membrane protrusions that were readily observed by differential interference contrast microscopy on the edges of infected cells (Fig. 5A,B). It is worth noting that endogenous ARF6 was found in the same structure (see supplementary material Fig. S1). The sites of bacterial entry were also enriched in F-actin and ARP 2/3 complex, key components for nucleating the formation of branched networks of actin filaments at the cell cortex (Fig. 6C and Fig. S1 in supplementary material). This indicates that bacterial entry sites are zones of dynamic actin polymerization. ARF6, PIP 5-kinase and PIP2 staining in these areas expanded over 2-3 µm in height, as assessed by confocal sectioning, indicating that they were part of a deep membrane calyx-like structure that engulfs the bacterium (see 3D reconstruction in Fig. 6A).
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Interestingly, the observation of many entry sites revealed that in the mid height of the protrusion, where the bacterium was localized, ARF6, PIP 5-kinase and PIP2 stainings, although partially overlapping, were differentially localized around the bacterium. PIP2 staining was the closest to the bacterium and covered most of the surface of the round structure, being more enriched on the edges (Fig. 5B; Fig. 6B-D). By contrast, ARF6 staining was preferentially enriched on the edges of the round protrusion, leaving a large unstained area in the centre (Figs 1 and 6). Finally, PIP 5-kinase appeared to occupy the largest area that overlapped with PIP2 and ARF6 staining (Fig. 5B and Fig. 6D). At the bottom of the calyx-like structure, ARF6, PIP 5-kinase and PIP2 staining overlapped in an apparently less structured distribution. Moreover, F-actin and ARP2/3 staining appeared enriched in this bottom area, and seemed to form a pedestal that protruded underneath the bacterium (not shown). These data indicate that the contact of Chlamydia with the epithelial cell membrane induces the accumulation and spatial distribution of the regulatory GTPase ARF6 and downstream signalling targets leading to the formation of a calyx-like membrane protrusion that engulfs and internalizes the bacterium.
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Discussion |
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As discussed below, we showed that the main role of ARF6 activation was to control actin polymerization via PIP 5-kinase. ARF6 was recruited to the sites of bacteria entry, where intense actin polymerization occurs upon Chlamydia binding. In cells expressing the ARF6 T27N mutant, the number of actin polymerization sites was reduced to the same extent as bacterial entry. Thus, our data indicate that ARF6 function during Chlamydia entry is mostly to control actin remodelling that is essential for bacterial uptake (Boleti et al., 1999; Coombes and Mahony, 2002
; Subtil et al., 2004
). Furthermore, in experiments using the ARF6 QS-EI mutant, which only prevents ARF6-induced peripheral actin remodelling, the inhibition of bacterial uptake (about 45%) was similar to that observed using the ARF6 dominant-negative mutant (
50%). Therefore, if ARF6 participates in membrane recruitment at the site of Chlamydia entry, it is only to a minor extent. ARF6 has previously been shown to control the phagocytosis of antibody-coated particles by macrophages (Niedergang et al., 2003
; Zhang et al., 1998
). ARF6 activity is required for pseudopod extension by controlling membrane delivery at the site of phagocytosis and not for actin polymerization (Niedergang et al., 2003
). In this case, ARF6 regulates the delivery of endocytic vesicles bearing the vesicle-associated membrane protein 3 (VAMP 3) at the phagocytosis sites. In the case of Chlamydia infection, VAMP 3 did not appear to be recruited at the entry sites (not shown). It is interesting to note that infectious Chlamydiae are very tiny (0.3 µm diameter). It is thus not surprising that membrane recruitment is not required for their entry.
The lipid modifying enzymes, phospholipase D (PLD) and PIP 5-kinase, which produce phosphatidic acid and PIP2 respectively, are well-known effectors of ARF6 (Honda et al., 1999; Vitale et al., 2002
), known to modulate vesicular traffic and actin polymerization. Our results indicate that PLD does not have a major contribution in the ARF6 regulation of Chlamydia uptake. Indeed, expression of a mutant of ARF6 with the mutation N48I that abolishes the ability of ARF6 to stimulate PLD activity (Vitale et al., 2002
) did not affect bacteria entry. On the other hand, PIP 5-kinase was recruited and PIP2 accumulated at the bacterial entry sites. Furthermore, reducing the expression of PIP 5-kinase or the concentration of membrane PIP2 lowered bacterial uptake. Our results clearly demonstrate that PIP 5-kinase is a key ARF6 downstream effector, probably involved in the reorganization of actin cytoskeletal proteins known to interact with and to be regulated by PIP2 (Yin and Janmey, 2003
). A recent study demonstrated that ARF6 participates in the activation of PIP 5-kinase associated with Yersinia pseudotuberculosis uptake (Wong and Isberg, 2003
). Because of the role of PIP2 in stimulating actin polymerization, this result suggested a control by ARF6 of actin polymerization. The putative function of ARF6 in membrane recruitment was not analysed in this case.
We and others recently showed that small GTPases of the Rho family are rapidly activated and recruited at the sites of Chlamydia entry and are necessary for invasion (Carabeo et al., 2004; Subtil et al., 2004
). Therefore, one interesting possibility is that ARF6 and other Rho GTPases act in synergy to induce actin reorganization during internalization of Chlamydia. Their downstream effectors may overlap to some extent, which would explain why the inhibition of uptake observed when cells expressed dominant-negative mutant ARF6 T27N, was about 50%. In addition, the remaining level of endogenous ARF6 may be sufficient to partially allow bacterial internalization. Another possibility is that Chlamydia, as other intracellular bacteria, enter host cells by more than one pathway and ARF 6 controls one of them.
The signalling cascade leading to ARF6 activation upon Chlamydia infection is presently unknown. Small GTPases are known to be targeted by bacterial products injected into host cells. For instance, the Salmonella-secreted toxins SopE and SopE2 act as exchange factors for Rac and Cdc42 (Friebel et al., 2001). Interestingly, a recent report showed that actin polymerization induced by Chlamydia is preceded by the translocation of a bacterial phosphoprotein, which may be part of the signal transduced by the pathogen to trigger its internalization (Clifton et al., 2004
). Secretion of chlamydial protein(s) may therefore be a triggering mechanism for ARF6 activation. Alternatively, ARF6 activation may be a consequence of signalling events transduced after Chlamydia binding to an as yet unidentified host cell receptor and resulting in the activation of a cellular ARF6 specific-GEF. In particular, Chlamydia association with specialized lipid microdomains, in which signalling molecules concentrate, may initiate the transduction events that eventually lead to the bacterial entry (Jutras et al., 2003
).
Remarkably, the contact zone between Chlamydia and the host cell is a highly organized structure. Indeed, a membrane protrusion is induced in contact with the bacteria. ARF6 and downstream signalling molecules accumulate and differentially localize, leading to the formation of an actin-driven calyx-like structure that engulfs and internalizes the bacterium.
Altogether our findings demonstrate that ARF6 activation is responsible for extensive actin remodelling necessary for bacterial uptake. Furthermore, we report that Chlamydia stimulates the formation of a specialized structure to promote its own internalization through the activation of ARF6 and downstream effectors.
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Acknowledgments |
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Footnotes |
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* These authors contributed equally to this work
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References |
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