Download pdf - ARF6 GTPase controls bacterial invasion by actin remodelling

IntroductionIntracellular pathogens have evolved multiple strategies tointerfere with normal cellular processes in order to promotetheir entry and survival within the host. Some strategies sharecommon features with phagocytosis, the process that allowscells to take up relatively large particles as debris, apoptoticcells, opsonised antigens and some pathogens (Underhill andOzinsky, 2002). Phagocytosis is driven by finely regulatedrearrangements of the actin cytoskeleton, under the control ofmembers of the Rho GTPase family (Niedergang and Chavrier,2004). Rho proteins are also the targets of several pathogensto induce their entry in host cells (Boquet and Lemichez,2003).

The ADP-ribosylation factor 6 (ARF6) belongs to the ARFfamily of small GTP-binding proteins. ARF6 regulatesmembrane trafficking and the actin cytoskeleton at the plasmamembrane (Donaldson, 2003). It is involved in membranetrafficking during receptor-mediated endocytosis, endosomalrecycling 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 isalso implicated in the formation of actin-rich membraneprotrusions and ruffles (Radhakrishna et al., 1996). Like allsmall GTP-binding proteins, ARF6 cycles between aninactive GDP-bound state and an active form when bound toGTP. GTP-ARF6 acts through the activation of downstreameffectors, including lipid-modifying enzymes such asphospholipase D (PLD) and phosphatidylinositol 4-

phosphate 5-kinase (PIP 5-kinase) (Honda et al., 1999;Massenburg et al., 1994). PIP 5-kinase, which is responsiblefor generating phosphatidylinositol 4,5-bisphosphate (PIP2),has been implicated in the phagocytosis of IgG-coatederythrocytes, probably through the regulation of actincytoskeletal proteins by PIP2 (Coppolino et al., 2002).Altogether, changes in membrane lipid composition andstructure may mediate ARF6 alterations of the cortical actincytoskeleton and regulation of membrane traffic and signaltransduction. Interestingly, in the case of phagocytosis of redblood cells by macrophages, ARF6 was shown to controlmembrane recruitment at the sites of phagocytosis rather thanactin polymerization (Niedergang et al., 2003). In the caseof Yersinia pseudotuberculosis, it was shown that a PIP2-dependent pathway regulated by ARF6 is associated withbacterial internalization, suggesting that it controls actinpolymerization (Wong and Isberg, 2003).

Chlamydiae are obligate intracellular parasites of eukaryoticcells, which constitute an important group of pathogensresponsible for a variety of acute and chronic diseasesincluding trachoma, pelvic inflammatory disease, pneumoniaeand their sequelae (Gregory and Schaffner, 1997; Kuo et al.,1995; Stamm, 1999). The bacteria attach to epithelial host cellsvia a relatively weak electrostatic interaction with heparansulfate proteoglycans (Su et al., 1996) and a more specificbinding to an unidentified secondary receptor (Carabeo etal., 2002). Chlamydia concentrate in lipid membranemicrodomains (Simons and Toomre, 2000) and this is

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The obligate intracellular bacterium Chlamydia penetratesthe host epithelial cell by inducing cytoskeleton andmembrane rearrangements reminiscent of phagocytosis.Here we report that Chlamydia induces a sharp andtransient activation of the endogenous small GTP-bindingprotein ARF6, which is required for efficient uptake. Wealso show that a downstream effector of ARF6,phosphatidylinositol 4-phosphate 5-kinase and its product,phosphatidylinositol 4,5-bisphosphate were instrumentalfor bacterial entry. By contrast, ARF6 activation ofphospholipase D was not required for Chlamydia uptake.ARF6 activation was necessary for extensive actinreorganization at the invasion sites. Remarkably, these

signalling players gathered with F-actin in a highlyorganized three-dimensional concentric calyx-likeprotrusion around invasive bacteria. These results indicatethat ARF6, which controls membrane delivery duringphagocytosis of red blood cells in macrophages, has adifferent role in the entry of this small bacterium,controlling cytoskeletal reorganization.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/118/10/2201/DC1

Key words: Chlamydia, Phagocytosis, Intracellular bacteria, PIP 5-kinase, PIP2

Summary

ARF6 GTPase controls bacterial invasion by actinremodelling María Eugenia Balañá1, Florence Niedergang2,*, Agathe Subtil1,*, Andrés Alcover1, Philippe Chavrier2 andAlice Dautry-Varsat1,‡

1Unité de Biologie des Interactions Cellulaires, Institut Pasteur, CNRS URA 2582, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France2CNRS UMR 144 Institut Curie, 12 rue Lhomond, 75005 Paris, France*These authors contributed equally to this work‡Author for correspondence (e-mail: [email protected])

Accepted 24 February 2005Journal of Cell Science 118, 2201-2210 Published by The Company of Biologists 2005doi:10.1242/jcs.02351

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necessary for bacterial entry (Jutras et al., 2003; Stuart et al.,2003).

Here, we show that endogenous ARF6 undergoes a rapid andtransient activation after infection by Chlamydia caviae andplays a critical role in bacterial uptake by regulating the actincytoskeleton reorganization at the sites of entry. Moreover, thebacterial entry site forms a remarkable calyx-like membraneprotrusion, where ARF6 and its effectors differentially localizearound the bacteria.

Materials and MethodsCells, bacteria, antibodies, plasmids and other reagentsThe human cervical adenocarcinoma cell line, HeLa 229, was fromthe American Type Culture Collection and was grown in Dulbecco’smodified Eagle’s medium with Glutamax (Life Technologies)supplemented with 10% foetal calf serum (FCS) (complete medium).The GPIC serovar of C. caviae was obtained from Roger Rank(University of Arkansas) and the bacteria were prepared as described(Boleti et al., 1999). Mowiol was from Calbiochem (La Jolla, CA),and saponin, fluorescein isothiocyanate (FITC) and DABCO (1,4-diazabicyclo-[2.2.2]octane) were from Sigma. Alexa 633, Alexa 488and Texas Red-phalloidin and Alexa 488-conjugated goat anti-rabbitantibodies were from Molecular Probes. The mouse anti-Chlamydiaantibodies (unlabelled and FITC-labelled) and the anti-HA tagrat monoclonal antibody were purchased from Argene, Biosoft(Varilhes, France) and from Roche Diagnostics (Mannheim,Germany) respectively. CyTM-3- and CyTM-5-conjugated goatanti-mouse and anti-rabbit antibodies were from Amersham.Tetramethylrhodamine 5-isothiocyanate (TRITC)-conjugated anti-ratantibody was purchased from Jackson ImmunoResearch (WestGrove, PA). Mouse monoclonal anti-ARF6 antibody used for westernblotting was a kind gift from P. Stahl (Washington University Schoolof Medicine, St Louis, MO). Plasmids encoding HA-tagged ARF6WT and HA-tagged ARF6 T27N in the pSR vector and PLCδ PH inthe pSRα EGFP-C1 vector were as described (Raucher et al., 2000).The anti-ARPC5 monoclonal antibody (anti-Arp2/3 antibody)(Olazabal et al., 2002) was a kind gift from Jürgen Wehland(Department of Cell Biology, Braunschweig, Germany). Plasmidscoding for untagged ARF6 WT, ARF6 T27N and ARF6 QS-EI andrabbit polyclonal anti-ARF6 antibody used for immunofluorescence(Song et al., 1998) were kind gifts from Julie Donaldson (NHLBI,National Institutes of Health, Bethesda, MD). Plasmids encodingLyn-phosphatase-CFP in the X4Blue vector (Raucher et al., 2000)and HA-tagged mouse PIP 5-kinase α in the pcDNA vector (Wongand Isberg, 2003) were kind gifts from Ralph Isberg (Tufts UniversitySchool of Medicine, Boston, MA). pCFP-Lyn-phosphatase encodesa PIP2-specific 5′-phosphatase from yeast Inp54p linked tomyristoylation/palmitoylation sequence from Lyn (Raucher et al.,2000; Wong and Isberg, 2003). The HA-tagged ARF6 N48I in thepXS vector (Vitale et al., 2002) was a kind gift from Marie FranceBader (CNRS, Strasbourg, France).

The bacteria were labelled with FITC as described (Subtil et al.,2004). Bacteria labelling with CyTM5 was performed withFluoroLinkTMCy5 reactive dye (Amersham Pharmacia, UK) inphosphate-buffered saline pH 7.8 (PBS) for 1 hour on ice. Labelledbacteria were washed twice with SPG (0.5 ml), centrifuged at 18,000g, 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 assayExpression of the ARF-binding domain (ARF-BD) of ARHGAP10fused with GST was induced in E. coli with 1 mM IPTG for 2 hoursat 37°C. The fusion protein was purified by affinity chromatographyon 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 β-mercaptoethanol (Dubois et al., 2001).

HeLa cells (5�106 per time point) grown overnight atsubconfluence in serum-free conditions were detached with PBS-EDTA and resuspended in DMEM medium. Cells were mixed withthe bacteria in 0.1 ml DMEM, centrifuged for 20 seconds at 16,000g and immediately transferred to 37°C (t=0). For the control, the cellswere mixed with an equivalent amount of a preparation from mock-infected cells. After incubation for different times, the cells were lysedon 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 10minutes. Equal volumes of supernatant for each time point wereincubated for 90 minutes with 40 µg ARF-BD of ARHGAP10 (T.Dubois and P.C., unpublished results) in the presence of 0.5% BSAand glutathione-Sepharose. The resin was washed extensively withwashing buffer (150 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 50mM Tris-HCl, pH 7.5), boiled in SDS-PAGE sample buffer, andbound proteins were resolved on SDS gels followed by western blotanalysis using an anti-ARF6 antibody.

TransfectionHeLa cells, which were less than 50% confluent, were transfectedwith ARF6 WT, ARF6 T27N, ARF6 QS-EI, ARF6 N48I, Lyn-phosphatase-CFP, PIP 5-kinase or PLCδ1 PH-GFP constructs usingFugene reagent (Roche Applied Science) or by electroporation (900µF, 200V, EasyJect Eurogentec, Belgium) and grown in 24-well platesfor 18 hours.

RNA interferenceThe siRNA targeting PIP 5-kinase β was as described (Padron et al.,2003). The sequence of the siRNA targeting ARF6 was 5′-AAG-CUGCACCGCAUUAUCAAU-3′. Control cells were transfected withan irrelevant siRNA. RNA oligonucleotides were synthesized byDharmacon Research.

HeLa cells were plated at 50% confluence in DMEM supplementedwith 10% FCS. The following day, 0.2 nmoles of siRNA wereintroduced into 4�106 cells by electroporation at 500 µF and 300 V(Easyject, Eurogentec).The medium was changed after an overnightincubation. In the case of ARF6, siRNA was re-introduced at 48hours, following the same procedure. 48 hours after the firsttransfection (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 mMNaCl, 10 mM MgCl2, 1% Triton X-100, 0.5% deoxycholate, 0.1%SDS, 50 mM Tris-HCl, pH 7.5). Cell lysates were centrifuged at16,000 g at 4°C for 10 minutes and analysed by western blotting usingmonoclonal anti-ARF6 antibody.

Chlamydia GPIC InfectionCells transfected for 18 hours or treated with siRNA were washedtwice in phosphate-buffered saline (PBS) and incubated in completemedium with Chlamydia caviae GPIC (guinea pig inclusionconjunctivitis) at a concentration resulting in 30-70% infected cells,24 hours post-infection. After 1.5 hours at 37°C, the bacteria wereremoved from the supernatant and the cells were washed twice inPBS, complete medium was added and the incubation continued for2.5 hours in the bacteria entry experiments or 17 hours to measure theefficiency of infection.

In the experiments where entry was synchronized for actin patchquantification at the entry sites, transfected cells were infected in 0.25ml culture medium and immediately centrifuged for 5 minutes at 770g at room temperature, and finally incubated for 5 minutes at 37°C.

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When indicated, cells were infected with CyTM5 or FITC-labelledChlamydia.

At the end of the infection (at the time indicated in each case), thecells were washed twice in PBS and fixed in 4% paraformaldehyde,120 mM sucrose in PBS for 30 minutes at room temperature. The cellswere washed in PBS, incubated for 10 minutes in 50 mM NH4Cl inPBS at room temperature and washed in PBS containing 1 mg/mlbovine serum albumin.

ImmunofluorescenceThe efficiency of bacterial entry was measured as described (Subtil etal., 2004). The efficiency of entry ranged between 40 and 60%. Theassociation index was calculated as the mean number of cell-associated bacteria (intracellular+surface-associated) per cell intransfected cells/cell-associated bacteria in control non-transfectedcells � 100. On average, 5-20 bacteria were associated with controlcells. For bacterial entry assay in Lyn-phosphatase-CFP transfectedcells, FITC-labelled Chlamydia were used and extracellular bacteriawere 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 with0.05% saponin and the inclusions labelled with FITC-conjugated anti-Chlamydia antibody (Boleti et al., 1999). The number of infected cellswas counted in about 300 transfected cells randomly chosen on thecoverslips and in the surrounding non-transfected cells; the index ofinfection was calculated as the number of infected cells/total cellscounted. The index obtained for transfected cells was divided by theindex obtained for control non-transfected cells and expressed aspercentage of control cells.

The quantification of local actin rearrangements visualized 5minutes post infection at the entry sites (Subtil et al., 2004) werequantified in ARF6 T27N and ARF6 QS-EI transfected cells, andidentified using polyclonal anti-ARF6 antibody and Cy3-coupledsecondary antibody. Actin was labelled with Alexa 488-coupledphalloidin. The number of local actin rearrangements was counted foreach transfected and non-transfected cell in the fields (about 100 cellswere counted per experiment) using an epifluorescence microscope(Axiophot, Zeiss, Germany) equipped with a 63� Apochromatobjective 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 asa percentage of those in non-transfected cells.

Confocal microscopyActin was visualized using Alexa 633, Alexa 488 or Alexa 546-coupled phalloidin. ARF6 and PIP 5-kinase transfected cells wereidentified with anti-ARF6 or anti-HA antibodies and secondaryantibodies. Coverslips were mounted on Mowiol with 100 mg/mlDABCO and examined under a confocal microscope (LSM 510,Zeiss) using a 63� objective. A z-series of optical sections was takenevery 0.2 or 0.5 µm. Image deconvolution was performed usingHuygens software (SVI, The Netherlands), and three-dimensionalimage reconstruction was carried out using Imaris and OsiriXsoftware (Bitplane, Switzerland). To prevent bleed-through effects insamples stained with multiple fluorochromes, green and far-redfluorescence emissions (excitations at 488 and 633 nm, respectively)were acquired simultaneously, but separately from red fluorescence(excitation at 546 nm).

ResultsARF6 is recruited at the sites of bacterial entryWe first analysed the localization of ARF6 at the initial steps

of bacterial infection. The sites of entry are characterized bylocal actin polymerization, forming patches around bacteria(Subtil et al., 2004). To study ARF6 localization, HeLa cellsexpressing HA-tagged ARF6 wild type (WT) were infected for5 minutes with FITC-labelled GPIC. ARF6 was recruited at thesites of intense actin polymerization forming around invasivebacteria (Fig. 1). Interestingly, the area in the immediatevicinity of the bacteria was largely unstained with anti-HAantibodies, indicating that ARF6 is located at the peripheryof the entry structure. The same result was obtained whenendogenous ARF6 was stained with an anti-ARF6 antibody innon-transfected cells (Supplementary material Fig. S1).

ARF6 is involved in bacterial entryTo investigate whether ARF6 plays a role in C. caviae GPICinfection we quantified infection in cells transfected with adominant-negative form of ARF6, ARF6 T27N. Untransfectedcells and cells transfected for 18 hours with HA-tagged ARF6T27N or ARF6 WT were infected for 18 hours and fixed.Transfected cells were identified with anti-HA antibodies andbacterial inclusions were stained with anti-Chlamydiaantibodies (Fig. 2A). In cells expressing ARF6 T27N, thenumber of cells containing inclusions was decreased by 50%compared to neighbouring untransfected cells. Cellstransfected with ARF6 WT were infected as efficiently asuntransfected cells (Fig. 2B). The inhibition of infection inARF6 T27N expressing cells was not due to a defect inbinding, as the association of bacteria with HeLa cells was notaffected by expression of either ARF6 construct (Fig. 2C).

We next analysed the ability of cells to internalize bacteria.Cells transfected for 18 hours with ARF6 T27N or ARF6 WTconstructs were infected and fixed 4 hours post-infection.Bacterial internalization was then measured as previouslydescribed (Subtil et al., 2004). Overexpression of dominant-negative ARF6 T27N induced a decrease in the efficiency ofentry (47%) (Fig. 2D). By contrast, overexpression of ARF6WT did not affect bacterial entry. Thus, expression of ARF6T27N inhibited bacterial uptake and cell infection to the sameextent, indicating that ARF6 is involved in chlamydialinfection at the step of entry. To further assess the role of ARF6in infection, we used RNA interference (RNAi) to inhibit itsexpression. Cells were transfected with ARF6 siRNA asdescribed in the Materials and Methods section and theninfected. The inhibition of ARF6 expression was checked bywestern blotting (Fig. 2G). Bacterial uptake was quantified insiRNA-treated and control cells as described above. In siRNA-treated cells, the efficiency of uptake was reduced by 50% (Fig.2E,F). This decrease may be underestimated because it wasmeasured on the whole cell population, including cells inwhich the transfection was probably not sufficient to fullyinhibit ARF6 activity. Altogether, these experiments show thatARF6 activity is necessary for the entry of Chlamydia.

ARF6 is activated during bacterial infectionTo further study ARF6 involvement, we monitored theactivation levels of endogenous ARF6 upon bacterial infectionusing the ARF-binding domain of ARHGAP10 (ARF-BD)fused to GST, which binds the active GTP-bound form ofARF6 (T. Dubois and P.C., unpublished results). One difficulty

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in the study of the initial events of bacterial infection residesin the asynchronous attachment of bacteria to host cells. Tocircumvent this problem, the bacterial inoculum wascentrifuged together with cells in suspension for 20 secondsand then incubated at 37°C to allow infection to proceed. Atdifferent times, the cells were lysed on ice, and activated ARF6was pulled-down. The precipitated GTP-bound ARF6 wasanalysed by western blotting with anti-ARF6 antibody.

Activation of ARF6 was observed 5 minutes after bacterialcontact, and decreased after 30 minutes to return to basal levelsby 60 minutes after infection (Fig. 3). These experiments show

that endogenous ARF6 is sharply and transiently activated at avery early step of bacterial entry.

ARF6 is involved in actin polymerizationARF6 is known to regulate both membrane trafficking and thecortical actin cytoskeleton (Donaldson, 2003). To furtherassess the role of ARF6 in Chlamydia infection, we made useof the ARF6 QS-EI mutant, in which residues Q37 and S38 inthe effector domain are replaced by the equivalent residues ofARF1 (Al-Awar et al., 2000). These mutations prevent ARF6-induced peripheral actin remodelling (Al-Awar et al., 2000).Cells were transfected with the ARF6 QS-EI construct andtheir ability to internalize bacteria was analysed 18 hours later,as described above. As shown in Fig. 4A, expression of ARF6QS-EI induced a decrease in the efficiency of bacterial uptake(45%) suggesting that actin remodelling controlled by ARF6plays a role in bacterial entry.

To investigate whether ARF6 is implicated in the formationof actin patches observed upon infection (Carabeo et al., 2002;Carabeo et al., 2004; Subtil et al., 2004) HeLa cells transfectedwith the ARF6 T27N dominant-negative construct or ARF6QS-EI construct were infected for 5 minutes. The actin patcheswere then stained with phalloidin and quantified. The numberof actin patches observed after 5 minutes of infection wasdecreased by 40% and 45% in cells expressing ARF6 T27Nand ARF6 QS-EI, respectively (Fig. 4B,C). Thus, the ARF6QS-EI mutant has the same effect as the ARF6 dominant-negative mutant, inhibiting bacterial uptake and actin patchformation. These results suggest that the main function ofARF6 in Chlamydia entry is to regulate actin cytoskeletonremodelling.

Recruitment of PIP 5-kinase at the sites of bacterialentry and local PIP2 productionARF6 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) didnot impair Chlamydia uptake (not shown), suggesting thatregulation of PLD by ARF6 is not required for entry. Next, weinvestigated the role of PIP 5-kinase and first determined thesubcellular 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 bacteriaat the plasma membrane (Fig. 5A).

PIP 5-kinase is responsible for generating PIP2, a majorphosphoinositide that regulates diverse cellular processesincluding actin cytoskeleton reorganization. To determine thelocalization of PIP2 during bacterial uptake, we made use ofthe PH domain from phospholipase C δ (PLC δ-PH) that hasa high binding affinity for plasma membrane PIP2 (Raucher etal., 2000). Cells transfected with a GFP-tagged PLCδ-PHconstruct were infected for 5 minutes. As was observed withPIP 5-kinase, PIP2 was concentrated at the sites of bacterialentry. The staining for PIP 5-kinase and PIP2 largelyoverlapped. The overall structure at the sites of bacterial entrycan be distinguished in the interference contrast images (Fig.5B).


Fig. 1. ARF6 recruitment and distribution during bacterial entry. Cellstransfected with HA-tagged-ARF6 WT were infected for 5 minuteswith FITC-coupled C. caviae GPIC (white spots). Cells were fixedand stained with anti-HA antibody and Cy5-labelled secondaryantibody (top panel) and with Alexa 546-coupled phalloidin (middlepanel). The bottom panel is the merged image of ARF6 (red), actin(green) and bacteria (white) staining. A medial confocal section isshown. x-z and y-z optical sections on the positions marked by thecrossed lines are displayed on the top and right of the image. ARF6recruitment is observed at a site of intense actin polymerization,where a bacterium is present (arrows). Bar, 5 µm.

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To determine if PIP 5-kinase was necessary for infection,cells were treated with siRNA specific for PIP 5-kinasebefore infection (Padron et al., 2003). The number ofinfected cells was then quantified in treated and control cellsafter staining the inclusions with anti-Chlamydia antibodies. InPIP 5-kinase siRNA-treated cells the infection was reduced by50% (Fig. 5C). This decrease is probably underestimatedbecause we quantified infection in the whole siRNA-treatedcell population. To determine if PIP2 plays a functional roleduring Chlamydia entry, the effect of lowering the cellularconcentration of PIP2 on bacterial uptake was examined. Tothat end, the cells were transfected with a plasma membrane-targeted PIP2-specific 5′-phosphatase, Lyn-CFP-Inp54p, whichhas been successfully used to selectively reduce plasmamembrane PIP2 concentration (Raucher et al., 2000; Wong andIsberg, 2003). Expression of Lyn-CFP-Inp54p phosphataseresulted in a 40% reduction of uptake efficiency compared withmock-treated cells (Fig. 5D).

Together, the presence of PIP 5-kinase and its product PIP2at the sites of bacterial entry, as well as the inhibition of entrywhen the transfected PIP2 phosphatase was expressed, indicatethat PIP2 is involved in bacterial entry. It is noteworthy that theinhibition of bacterial entry in the latter conditions (about 40%)is close to the one measured when ARF6 dominant-negativemutant was used (about 50%), further suggesting that PIP 5-

kinase is a major downstream effector in the pathway by whichARF6 regulates bacterial entry.

Chlamydia entry sites form a spatially well-organizedstructureWe have identified at the sites of Chlamydia entry severalmolecules, which are required for bacterial uptake. Aspresented above, transiently expressed ARF6 and PIP 5-kinase,appear to accumulate at the plasma membrane at the sitesof entry together with PIP2 (Figs 1, 5; Fig. 6B). Theseaccumulations coincided with rounded membrane protrusionsthat were readily observed by differential interference contrastmicroscopy on the edges of infected cells (Fig. 5A,B). It isworth noting that endogenous ARF6 was found in the samestructure (see supplementary material Fig. S1). The sites ofbacterial entry were also enriched in F-actin and ARP 2/3complex, key components for nucleating the formation ofbranched networks of actin filaments at the cell cortex (Fig. 6Cand Fig. S1 in supplementary material). This indicates thatbacterial entry sites are zones of dynamic actin polymerization.ARF6, PIP 5-kinase and PIP2 staining in these areas expanded

Fig. 2. Inhibition of ARF6 impairs infection. (A-C) ARF6 T27Ninhibits C. caviae GPIC infection. HeLa cells transientlyexpressing the HA-tagged-ARF6 WT or ARF6 T27N wereinfected for 18 hours. Cells were then fixed and stained with anti-HA antibody followed by TRITC-coupled anti-rat antibody todetect transfected cells (arrows). Bacteria were revealed usingFITC-conjugated anti-Chlamydia antibody (A). Arrowheadsindicate bacterial inclusions. The efficiency of infection (B) orbacterial association with cells (C) was calculated as indicated inthe Methods section and the efficiency of infection or associationin transfected cells relative to that in surrounding non-transfectedcells (NT) is plotted. The results are the mean±s.e.m. of fourindependent experiments. (D) ARF6 T27N inhibits bacterialentry. HeLa cells were transfected with the indicated plasmidsand infected on the following day for 4 hours. Extracellularbacteria were labelled with anti-Chlamydia antibody followed bya Cy5-coupled secondary antibody. The cells were thenpermeabilized in 0.05% saponin and incubated with FITC-conjugated anti-Chlamydia antibody to label all bacteria.Transfected cells were visualized with anti-HA antibodyfollowed by TRITC-coupled secondary antibody. The number ofsurface-associated and intracellular bacteria was counted in thetransfected and non-transfected population (n>50 cells) and theefficiency of entry (intracellular/total cell-associated) wascalculated. For each experiment, the efficiency of entry intransfected cells is expressed relative to that in non-transfectedcells. Data are the mean±s.e.m. of four independent experiments.(E-F) Bacterial entry is impaired in ARF6-depleted cells. HeLacells were treated with ARF6 siRNA prior to measuring bacteriaentry as in D. Intracellular bacteria appear green; surface-associated bacteria appear red or yellow. Quantification is shownin F. The efficiency of entry is expressed relative to that in non-treated cells. One representative experiment out of four is shown.(G) Effect of RNAi on ARF6 protein expression. Equal amountsof protein from control or siRNA-treated cell lysates were run onSDS-PAGE and immunoblots were probed with anti-ARF6antibodies. The western blot corresponds to the experimentshown in F.

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over 2-3 µm in height, as assessed by confocal sectioning,indicating that they were part of a deep membrane calyx-likestructure that engulfs the bacterium (see 3D reconstruction inFig. 6A).

Interestingly, the observation of many entry sites revealedthat in the mid height of the protrusion, where the bacteriumwas localized, ARF6, PIP 5-kinase and PIP2 stainings,although partially overlapping, were differentially localizedaround the bacterium. PIP2 staining was the closest to thebacterium and covered most of the surface of the roundstructure, being more enriched on the edges (Fig. 5B; Fig. 6B-D). By contrast, ARF6 staining was preferentially enriched onthe edges of the round protrusion, leaving a large unstainedarea in the centre (Figs 1 and 6). Finally, PIP 5-kinaseappeared to occupy the largest area that overlapped with PIP2and ARF6 staining (Fig. 5B and Fig. 6D). At the bottom ofthe calyx-like structure, ARF6, PIP 5-kinase and PIP2 stainingoverlapped in an apparently less structured distribution.Moreover, F-actin and ARP2/3 staining appeared enriched inthis bottom area, and seemed to form a pedestal that protrudedunderneath the bacterium (not shown). These data indicatethat the contact of Chlamydia with the epithelial cellmembrane induces the accumulation and spatial distributionof the regulatory GTPase ARF6 and downstream signallingtargets leading to the formation of a calyx-like membraneprotrusion that engulfs and internalizes the bacterium.

DiscussionIn this report, we have investigated the entry of an intracellularbacterial pathogen, Chlamydia, in epithelial host cells. Weobserved that endogenous ARF6 was activated upon infectionwith Chlamydia. This activation took place at an earlyinfection step, within a few minutes of infection. When cellsexpressed the dominant-negative mutant ARF6 T27N or weretreated with siRNA targeting ARF6, bacterial entry wasinhibited, showing that ARF6 activation participated in theinternalization step. Overall infection was also inhibited and tothe same extent, suggesting that ARF6 is involved at the bacterialentry step and does not participate in later steps of infection.

As discussed below, we showed that the main role of ARF6activation was to control actin polymerization via PIP 5-kinase.ARF6 was recruited to the sites of bacteria entry, where intenseactin polymerization occurs upon Chlamydia binding. In cellsexpressing the ARF6 T27N mutant, the number of actinpolymerization sites was reduced to the same extent asbacterial entry. Thus, our data indicate that ARF6 functionduring Chlamydia entry is mostly to control actin remodellingthat is essential for bacterial uptake (Boleti et al., 1999;Coombes and Mahony, 2002; Subtil et al., 2004). Furthermore,


Fig. 3. ARF6 activation upon infection. HeLa cells in suspensionwere centrifuged for 20 seconds with or without bacteria (control)and incubated for the indicated times at 37°C as described in theMethods section. The cells were then lysed on ice and incubated withthe ARF binding domain of ARHGAP 10 (ARF-BD) fused to GST,which binds the active GTP-bound form of ARF6. The proteinsassociated with this GST construct were pulled down usingglutathione sepharose and analysed by western blot using anti-ARF6antibodies. Aliquots of total cell lysates were immunoblotted fortotal ARF6, showing that the total amount of protein was identical atall time points. Data are representative of five experiments.

Fig. 4. ARF6 involvement in actin polymerization. (A) ARF6 QS-EIinhibits bacterial entry. HeLa cells were transfected with the ARF6QS-EI construct and infected for 4 hours with C. caviae GPIC on thefollowing day. Extracellular and intracellular bacteria, as well astransfected cells, were labelled as described in Fig. 2D. The numberof surface-associated and intracellular bacteria was counted in thetransfected and non-transfected population (n>25 cells) and theefficiency of entry (intracellular/total cell-associated) was calculated.For each experiment, the efficiency of entry in transfected cells isexpressed relative to that in non-transfected cells. Data are themean±s.e.m. of three independent experiments. (B,C) ARF6 T27Nand QS-EI mutants affect actin polymerization upon bacteria entry.Cells transfected or not with the ARF6 T27N-HA or the ARF6 QS-EI constructs were infected for 5 minutes as described in theMethods section. Actin was visualized with Texas Red-coupledphalloidin, ARF6 T27N-transfected cells were identified with anti-HA antibody and Alexa 488-labelled second antibody and cellstransfected with the ARF6 QS-EI were visualized with anti-ARF6antibody and Alexa 488-coupled second antibody. A representativeexperiment is shown in B. The arrows indicate actin patches. Theactin patches visualized 5 minutes post-infection at the entry siteswere quantified in transfected and non-transfected cells (C). Resultsare expressed as percent of patches in transfected cells comparedwith percent of patches in non-transfected cells. Data are themean±s.e.m. of three independent experiments.

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in experiments using the ARF6 QS-EI mutant, which onlyprevents ARF6-induced peripheral actin remodelling, theinhibition of bacterial uptake (about 45%) was similar to thatobserved using the ARF6 dominant-negative mutant (~50%).Therefore, if ARF6 participates in membrane recruitment at thesite of Chlamydia entry, it is only to a minor extent. ARF6 haspreviously 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 pseudopodextension by controlling membrane delivery at the site ofphagocytosis and not for actin polymerization (Niedergang et

al., 2003). In this case, ARF6 regulates the delivery ofendocytic vesicles bearing the vesicle-associated membraneprotein 3 (VAMP 3) at the phagocytosis sites. In the case ofChlamydia infection, VAMP 3 did not appear to be recruitedat the entry sites (not shown). It is interesting to note thatinfectious Chlamydiae are very tiny (0.3 µm diameter). It isthus not surprising that membrane recruitment is not requiredfor their entry.

The lipid modifying enzymes, phospholipase D (PLD) andPIP 5-kinase, which produce phosphatidic acid and PIP2respectively, are well-known effectors of ARF6 (Honda et al.,

Fig. 5. Recruitment of PIP 5-kinase at the sites ofbacterial entry and local PIP2 production. (A) PIP 5-kinase localizes at the sites of bacteria entry. HeLacells were transfected with HA-tagged PIP 5-kinasefor 18 hours and infected with FITC-coupledChlamydia for 5 minutes. Transfected cells wereidentified with anti-HA antibody followed by anti-rat TRITC antibody (in red). PIP 5-kinase is shownrecruited (arrows) around the bacteria (green) (topleft panel). In the bottom panels a highermagnification of the entry sites is shown; note theabsence of PIP 5-kinase in the central area (bottomright panel) where the bacterium is present (bottomleft panel). Medial confocal optical sections areshown. The overall structure at the sites of bacterialentry can be visualized in the differentialinterference contrast image (arrows in the top rightpanel). Results are representative of at least threeindependent experiments. (B) Distribution of PIP2during bacterial entry. HeLa cells transfected withGFP-PLCδ-PH and HA-tagged PIP 5-kinase wereinfected for 5 minutes as indicated. Cells were fixedand stained with anti-HA antibody followed by anti-rat TRITC to reveal PIP 5-kinase (red). Thefluorescence of GFP-PLCδ-PH reveals the presenceof PIP2 (green) (top panel). HeLa cells transfectedwith GFP-PLCδ-PH were infected as indicated withCy5-coupled Chlamydia for 5 minutes beforefixation. The arrowheads indicate PIP2 found at theentry sites accumulated around a bacterium (red).The differential interference contrast image with theoverall structure is shown (bottom panel). Resultsare representative of at least three independentexperiments. (C) PIP 5-kinase depletion by RNAiimpairs bacterial infection. HeLa cells were treatedfor 48 hours with PIP 5-kinase siRNA and infectedfor 20 hours prior to quantification of the number ofinfected cells. Infected cells were visualized withFITC-coupled anti-Chlamydia antibody. Thenumber of infected cells is expressed relative to thatin non-treated cells. The results presented are themean±s.e.m. of three experiments. (D) Transientexpression of Lyn-phosphatase affects Chlamydiaentry. HeLa cells were transfected to express Lyn-CFP-Inp54p, infected with FITC-coupled bacteriafor 4 hours on the following day and bacteria entrywas quantified. Extracellular bacteria were stainedwith anti-Chlamydia antibody followed by Cy5-coupled secondary antibody. Surface-associated(Cy5- and FITC-positive) and intracellular bacteria(FITC-positive) were counted in the transfected and non-transfected population (n>25 cells) and the efficiency of entry (intracellular/total cell-associated) was calculated. For each experiment, the efficiency of entry in transfected cells is expressed relative to that in non-transfected cells(NT). Data are the mean±s.e.m. of three independent experiments. Bar, 10 µm.

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1999; Vitale et al., 2002), known to modulate vesicular trafficand actin polymerization. Our results indicate that PLD doesnot have a major contribution in the ARF6 regulation ofChlamydia uptake. Indeed, expression of a mutant of ARF6with the mutation N48I that abolishes the ability of ARF6 tostimulate PLD activity (Vitale et al., 2002) did not affectbacteria entry. On the other hand, PIP 5-kinase was recruitedand PIP2 accumulated at the bacterial entry sites. Furthermore,reducing the expression of PIP 5-kinase or the concentrationof membrane PIP2 lowered bacterial uptake. Our results clearlydemonstrate that PIP 5-kinase is a key ARF6 downstreameffector, probably involved in the reorganization of actincytoskeletal proteins known to interact with and to be regulated

by PIP2 (Yin and Janmey, 2003). A recent study demonstratedthat ARF6 participates in the activation of PIP 5-kinaseassociated with Yersinia pseudotuberculosis uptake (Wong andIsberg, 2003). Because of the role of PIP2 in stimulating actinpolymerization, this result suggested a control by ARF6 ofactin polymerization. The putative function of ARF6 inmembrane recruitment was not analysed in this case.

We and others recently showed that small GTPases of theRho family are rapidly activated and recruited at the sites ofChlamydia entry and are necessary for invasion (Carabeo et al.,2004; Subtil et al., 2004). Therefore, one interesting possibilityis that ARF6 and other Rho GTPases act in synergy to induceactin reorganization during internalization of Chlamydia. Their

downstream effectors may overlapto some extent, which wouldexplain why the inhibition ofuptake observed when cellsexpressed dominant-negativemutant ARF6 T27N, was about50%. In addition, the remaininglevel of endogenous ARF6 may besufficient to partially allowbacterial internalization. Anotherpossibility is that Chlamydia, as


Fig. 6. Confocal analysis ofChlamydia entry sites. Sites ofChlamydia entry appear as spatiallywell-organized membrane structuresthat accumulate ARF6 and itsdownstream effectors. (A) HeLa cellstransfected with HA-tagged-ARF6WT were infected for 5 minutes withFITC-coupled Chlamydia (blue).Cells were then stained with anti-HAantibody followed by TRITC-coupledsecond antibody. Inset showsbacterium (blue) surrounded byARF6 staining. In the right-handpanel, a plan of the 3D reconstructedstructure of ARF6 (red) andinternalizing bacterium (blue) isshown. Results are representative ofat least three independentexperiments. (B) HeLa cellstransfected with GFP-PLCδ PH andHA-tagged-ARF6 WT were infectedby Chlamydia for 5 minutes.Transfected cells were detected with aspecific anti-HA antibody followed

by Cy5-coupled secondary antibody (red) and the fluorescence of GFP (green). Highermagnifications of the entry site (arrowheads) are shown in the right-hand panels. Results arerepresentative of at least three independent experiments. (C) HeLa cells transfected with HA-tagged PIP 5-kinase and ARF6 were infected by Chlamydia for 5 minutes. Cells werestained with anti-ARF6 antibody followed by Alexa488-coupled secondary antibody (green),anti-HA antibody followed by Cy5-coupled secondary antibody (red) and with Alexa 633-coupled phalloidin (blue). A higher magnification of the entry site (arrowhead) is shown inthe right-hand panel. (D) Scheme showing the relative localization of ARF6, PIP2 and PIP 5-kinase around the internalizing bacteria. PIP2 staining (green) was the closest to thebacterium and covered most of the surface of the round structure; ARF6 (blue) and PIP 5-kinase (red) staining were preferentially enriched on the edges of the round protrusion,leaving a large unstained area in the centre filled by the bacterium. PIP 5-kinase appears tooccupy the largest area that overlapped with PIP2 and ARF6 staining. Bar, 2 µm (A andright-hand panels in C); 10 µm (B); 5 µm (left-hand panels in C).

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other intracellular bacteria, enter host cells by more than onepathway and ARF 6 controls one of them.

The signalling cascade leading to ARF6 activation uponChlamydia infection is presently unknown. Small GTPases areknown to be targeted by bacterial products injected into hostcells. For instance, the Salmonella-secreted toxins SopE andSopE2 act as exchange factors for Rac and Cdc42 (Friebel etal., 2001). Interestingly, a recent report showed that actinpolymerization induced by Chlamydia is preceded by thetranslocation of a bacterial phosphoprotein, which may be partof the signal transduced by the pathogen to trigger itsinternalization (Clifton et al., 2004). Secretion of chlamydialprotein(s) may therefore be a triggering mechanism for ARF6activation. Alternatively, ARF6 activation may be aconsequence of signalling events transduced after Chlamydiabinding to an as yet unidentified host cell receptor and resultingin the activation of a cellular ARF6 specific-GEF. In particular,Chlamydia association with specialized lipid microdomains, inwhich signalling molecules concentrate, may initiate thetransduction events that eventually lead to the bacterial entry(Jutras et al., 2003).

Remarkably, the contact zone between Chlamydia and thehost cell is a highly organized structure. Indeed, a membraneprotrusion is induced in contact with the bacteria. ARF6and downstream signalling molecules accumulate anddifferentially localize, leading to the formation of an actin-driven calyx-like structure that engulfs and internalizes thebacterium.

Altogether our findings demonstrate that ARF6 activationis responsible for extensive actin remodelling necessary forbacterial uptake. Furthermore, we report that Chlamydiastimulates the formation of a specialized structure to promoteits own internalization through the activation of ARF6 anddownstream effectors.

M.E.B. was supported by a postdoctoral fellowship from Fondationpour la Recherche Médicale. We are grateful to Stéphanie Perrinet fortechnical assistance. We thank T. Dubois for the gift of theARHGAP10 ARF-BD construct; P. Roux and M. Marchand from thePlateforme d’Imagerie Dynamique, Institut Pasteur, for expert helpwith microscopy imaging. We are very grateful to our colleagues,listed in the Materials and Methods section, for the generous gifts ofconstructs and antibodies. This work was supported by theAssociation pour la Recherche sur le Cancer and by Action ConcertéeIncitative Biologie Cellulaire Moléculaire et Structurale.

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