Article |
Address correspondence to Pascale Cossart, Unité des Interactions Bactéries-Cellules, INSERM U604, Institut Pasteur, 75015 Paris Cedex 15, France. Tel.: 33-1-45-68-88-41. Fax: 33-1-45-68-87-06. email: pcossart{at}pasteur.fr
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Abstract |
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Key Words: Listeria; phagocytosis; rafts; E-cadherin; HGF-R/c-Met
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Introduction |
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Cellular invasion by viruses or parasites and the uptake of some intracellular bacteria, such as FimH-expressing enterobacteria, mycobacteria, Salmonella, Chlamydia, and Shigella into professional or nonprofessional phagocytic cells, has been shown to require a specialized host membrane microenvironment organized into lipid domains (Gatfield and Pieters, 2000; Shin et al., 2000; Garner et al., 2002; Lafont et al., 2002; Jutras et al., 2003). In addition, bacterial toxins such as cholera toxin, LLO, and anthrax toxin target lipid domains (Orlandi and Fishman, 1998; Coconnier et al., 2000; Abrami et al., 2003).
Formation of membrane lipid domains is thought to be based on the segregation of different lipid phases within the lipid bilayer. A liquid-ordered phase (raft-like domains) and a liquid-disordered phase (more fluid regions of the membrane) might coexist in biological membranes (Brown and Rose, 1992; Brown and London, 1998; Rietveld and Simons, 1998). The first type of membrane domain, often called lipid rafts, has been extensively studied. A biochemical approach for their analysis is the isolation of detergent-resistant membranes (DRMs) after cellular extraction with cold nonionic detergents, and successive flotation on density gradients (London and Brown, 2000; Giurisato et al., 2003; Schuck et al., 2003). Although this methodology might not preserve the native structural organization of lipid rafts, it has been used to study their composition. DRMs are characterized by a high content of cholesterol and glycosphingolipids compared with the average membrane content. Cholesterol, the major lipid of the membrane, is a critical component that controls lipid phase separation and stabilizes the liquid-ordered phase (London and Brown, 2000). Drugs that modify membrane cholesterol concentration disrupt membrane domain organization, and serve as tools to study lipid domain functions in cellular processes (Kilsdonk et al., 1995). Raft domains comprise different classes of proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins, myristoylated or palmitoylated proteins, cholesterol-linked and some transmembrane proteins (Harder and Simons, 1997; Zacharias et al., 2002; Edidin, 2003). Different raft domains might coexist and be present in distinct areas of the membranes, as for example caveolar (caveolin-containing) rafts and noncaveolar rafts. Functionally, membrane domain segregation provides a mechanism for the selection of molecular effectors into functional units for efficient signaling and sorting processes. Cellular events such as cell trafficking, cell migration, and phagocytosis require membrane domain segregation (Simons and Ikonen, 1997; Seveau et al., 2001; Manes et al., 2003), and key components of their molecular machineries have been isolated in DRMs.
In the present work, we addressed the role of membrane lipid domains in L. monocytogenes entry and focused our work on the study of the interactions between the bacterial invasion proteins internalin and InlB, and their receptors E-cadherin and HGF-R. Whether E-cadherin and the HGF-R requires lipid assemblies to function properly was previously unknown. Our work provides the first demonstration that both E-cadherin and HGF-R require lipid domains to mediate L. monocytogenes entry. Interestingly, depending on the receptor involved, lipid domains are important for different steps of the internalization process.
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Results |
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To definitively establish if the specific interactions of internalin or InlB with their respective receptors required plasma membrane cholesterol, we used latex beads coated with the purified recombinant bacterial invasion proteins. As shown previously (Lecuit et al., 1997; Braun et al., 1998), internalin- and InlB-coated beads are able to induce their specific entry into mammalian cell lines expressing their receptors. As shown in Fig. 2 B, in the absence of any other bacterial factors, internalin- and InlB-mediated uptake also required plasma membrane cholesterol.
Recruitment of raft markers at the internalin and InlB entry sites
To further demonstrate the implication of lipid domains in L. monocytogenes entry, we performed colocalization studies of raft and nonraft markers with bacteria or beads bound to the cell surface. For this purpose, cells were transiently transfected with the Aequorea GFP fused to GPI moiety (GFP-GPI), or with the cyan variant of Aequorea GFP fused to short peptide sequences which are the site for posttranslational modifications: myristoylation and palmitoylation (CFP-MyrPalm); and geranylgeranylation (CFP-GerGer). These chimeras are preferentially targeted to lipid domains at the outer leaflet (GFP-GPI) or at the inner leaflet (CFP-MyrPalm), whereas CFP-GerGer is present at the inner leaflet of the plasma membrane, does not display any preferential association with lipid domains, and was used as a negative control (Zacharias et al., 2002). In addition, we performed ganglioside 1 (GM1) labeling by using the Alexa Fluor 488conjugated B subunit of cholera toxin after cellular fixation (see Materials and methods). Prion protein (PrP) was also used as a raft marker (Naslavsky et al., 1997) and the transferrin receptor as a nonraft marker.
To study the internalin pathway, we infected Lovo epithelial cells with L. innocua, which expresses internalin. Lovo cells are permissive for both internalin- and InlB-mediated entry (Pizarro-Cerda et al., 2004), are more easily transfected, and more efficiently labeled by the B subunit of cholera toxin than the L2071hEcad cells. Our results show that L. innocua expressing internalin recruited at the plasma membrane the raft markers GM1 and CFP-MyrPalm, but not the nonraft marker CFP-GerGer (Fig. 3). The raft markers GFP-GPI, and PrP were also recruited at the bacterial entry site (Fig. 3).
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HGF-R phosphorylation and PI 3 kinase recruitment are not affected by cholesterol depletion
Some of the InlB- and HGF-signaling events occurring downstream from the initial interaction with HGF-R have been identified, including HGF-R tyrosine phosphorylation, recruitment and activation of Gab1, Cbl and Shc, and formation of signaling complexes containing these adapters and the p85 subunit of PI 3-kinase (Ireton et al., 1999). We investigated whether InlB could still induce HGF-R tyrosine phosphorylation and PI 3-kinase recruitment after cholesterol depletion. Vero cells incubated at 37°C for 1 min with soluble InlB (8 nM) were lysed and cell lysates were immunoprecipitated with antiHGF-R or with anti-phosphotyrosine antibodies as described previously (Ireton et al., 1999). As shown in Fig. 9, MßCD treatment did not induce HGF-R phosphorylation or p85 recruitment in Vero cells. This control was important as it has been reported that MßCD treatment could induce by itself PI 3-Kinase activation and the phosphorylation of the epidermal growth factor (Chen and Resh, 2002). As previously reported, in control cells (not incubated with MßCD), soluble InlB induced HGF-R tyrosine phosphorylation and coimmunoprecipitation of the p85
subunit of PI 3-kinase with phosphotyrosine proteins (Ireton et al., 1999). After cholesterol depletion, soluble InlB and HGF still induced tyrosine phosphorylation of HGF-R (Fig. 9 A) as well as the coimmunoprecipitation of the p85
subunit of PI 3-kinase with phosphotyrosine proteins (Fig. 9 B).
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Discussion |
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E-cadherin present within DRMs acts as a receptor for the invasion protein internalin
We conducted functional and biochemical studies to address the role of lipid rafts in internalin-mediated entry. We showed that internalin-dependent adherence and internalin-dependent recruitment of E-cadherin at the entry site were inhibited after cholesterol depletion by MßCD. E-cadherin, -catenin- and E-cadherinassociated internalin were present in the low density DRMs in a cholesterol-dependent manner. In addition, lipid raft markers are recruited at internalin-dependent entry site. Altogether, our results indicate that E-cadherin engagement into lipid domains is critical for internalin-dependent entry of L. monocytogenes. We favor the hypothesis that lipid rafts are important for the initial clustering of E-cadherin molecules during multivalent binding to internalin present on bacteria or beads. This conclusion is based on several observations: (a) for control and MßCD-treated cells, the same amount of E-cadherin is present at the plasma membrane (Fig. 6); (b) soluble internalin associates with E-cadherin present in DRM fractions (Fig. 7); and (c) the internalin-coated beads failed to recruit E-cadherin at the plasma membrane of cholesterol-depleted cells.
It has been reported that in polarized epithelial cells, adherens junctions were not disassembled after cholesterol depletion whereas tight junctions were disrupted (Nusrat et al., 2000). Thus, cholesterol would be important for tight junction but not for adherens junction maintenance. An alternative possibility is that both junctions are raft dependent but that in adherens junction, cholesterol is protected from cholesterol extraction. We favor the hypothesis that similarities exist between adherens junction formation and internalin-mediated phagocytosis and that lipid rafts would be necessary for adherens junction establishment.
HGF-R signaling pathway involves membrane cholesterol
In contrast to what was observed for the E-cadherininternalin interactions, cholesterol depletion did not affect InlB binding to HGF-R nor HGF-R recruitment at the plasma membrane. However, downstream signaling events required membrane cholesterol because InlB-induced membrane ruffles and actin-rich phagocytic cups were not observed after cholesterol depletion. HGF, as observed with soluble InlB, could not either induce the formation of actin-rich membrane ruffles after cholesterol depletion. Some events involved in the InlB- and HGF-mediated signaling have been described previously (Ireton et al., 1999; Shen et al., 2000; Bierne et al., 2001), among which tyrosine phosphorylation of the HGF-R, activation of PI 3-kinase, production of phosphoinositides, and Rac activation. In this work, we report that after cholesterol depletion, tyrosine phosphorylation of the HGF-R as well as PI 3-kinase recruitment to phosphotyrosine proteins were still occurring, indicating that the signaling cascade was interrupted further downstream, in agreement with our finding that HGF-R is present in DRMs. It remains to determine which of the downstream events of InlB- and HGF-mediated signaling pathways, between PI 3-kinase recruitment and F-actin polymerization, is cholesterol dependent.
Cross-talk between HGF-R and E-cadherin
Several reports point to a cross-talk between HGF-R and E-cadherin. The activation of epithelial cells by HGF results in the disruption of E-cadherindependent adherens junctions and in the coendocytosis of E-cadherin and HGF-R (Kamei et al., 1999). HGF stimulation induces MAPK activation and phosphorylation of ß-catenin (Liou et al., 2002). In the case of L. monocytogenes, depending on the target cell, the bacterium exploits HGF-R and E-cadherin independently or in conjunction, to trigger its entry. In Lovo cells (Pizarro-Cerda et al., 2004) and in L2071hEcad (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200406078/DC1), E-cadherin and HGF-R can be corecruited at the phagosomal membrane bacterial entry site. However, the molecular basis of the possible synergy between the two receptors during L. monocytogenes entry has not been elucidated. Our work shows that lipid domain integrity is a common requirement for both receptors to allow bacterial uptake. Altogether, these observations lead to the hypothesis that upon InlB or HGF stimulation, both receptors could be localized into lipid rafts and be cointernalized through these microdomains. Whether this coendocytosis has a synergistic effect on bacterial entry has still to be demonstrated.
Exploitation of lipid rafts in bacterial entry
Over the past years lipid rafts have been reported as implicated in the entry of various bacterial pathogens into professional and nonprofessional phagocytic cells. However, the integrity of lipid domains is not necessarily required for all phagocytic events. Raft integrity is required for nonopsonic uptake of mycobacteria and Brucella by macrophages, whereas after opsonization by antibodies, these pathogens could still infect macrophages despite cholesterol depletion (Peyron et al., 2000). To our knowledge, the only example of cholesterol-independent entry into nonphagocytic cells is that of Yersinia via the invasion protein invasin, which binds to ß1 integrins (Lafont et al., 2002). These results indicate that depleting plasma membrane cholesterol has no general effect and does not always inhibit receptor activity, signaling events, or cytoskeletal rearrangements.
Interestingly, it was proposed that entry via raft microdomains determines the fate of some intracellular pathogens. For example, lipid raft-mediated entry of Brucella and FimH-expressing E. coli, prevents phagolysosomal fusion and permits intracellular survival of the pathogens (Gatfield and Pieters, 2000; Shin et al., 2000; Naroeni and Porte, 2002). Our present work demonstrates that in the case of L. monocytogenes, entry occurs in raft containing microdomains. Whether this determines the fate of the bacteria is unknown. Quite strikingly, the secreted bacterial toxin, LLO, which allows escape from the phagocytic vacuole and also induces signaling pathways, binds to cholesterol in mammalian membranes. It will be of particular interest to study the spatiotemporal interactions of LLO with plasma membrane during L. monocytogenes internalization, and the possible synergy between LLO and bacterial invasion proteins internalin and InlB.
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Materials and methods |
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Mammalian cells and bacterial strains
African green monkey kidney epithelial Vero cells (American Type Culture Collection; CCL-81), L2071 mouse fibroblasts (American Type Culture Collection; CCL1.1), were cultured in DME glutamax (GIBCO BRL) supplemented with 10% FBS. L2071 cells transfected with the human E-cadherin cDNA (L2071-hEcad; Lecuit et al., 2000) were cultured in the presence of 800 µg/ml of G418 (GIBCO BRL). Human epithelial Lovo cells (American Type Culture Collection; CCL-229) were cultured with F-12K nutrient mixture (GIBCO BRL) supplemented with 10% FBS and 2 mM glutamine. Rabbit kidney epithelial Rov9 cells, stably expressing the PrP protein (Vilette et al., 2001) were cultured with MEM glutamax (GIBCO BRL) supplemented with 10% FBS.
Wild-type L. monocytogenes EGD (BUG600) and its isogenic deletion mutants, inlA (BUG947) and
inlB (BUG1047; Dramsi et al., 1995), EGDhly::Tn917 mutant strain obtained by signature-tagged mutagenesis, L. monocytogenes variant EGD
inlB (LRRs-IR-SPA) (BUG 1641; Bierne et al., 2001), and L. innocua transformed with pRB474 harboring the inlA gene (BUG 1489) were grown overnight at 37°C in brainheart infusion agar (Difco), diluted 10 times in brainheart infusion, and cultured until OD600nm = 0.8. Bacteria were washed in culture medium three times before assayed.
The DNA encoding monomeric CFP fused onto short peptides containing consensus sequences for acylation (MyrPalm-CFP) or prenylation (GerGer-CFP) in vector were provided by R.Y. Tsien (University of California, San Diego, La Jolla, CA) and have been described in Zacharias et al. (2002). The GPI moiety of the folate receptor cloned as a GFP-GPI chimera in pJB20 vector was provided by C. Zurzolo and S. Paladino (Institut Pasteur). Cells were transfected with Lipofectin (GIBCO BRL) following the manufacturer's instructions.
Cholesterol depletion and repletion
For cholesterol depletion, cells were washed twice with DME (without serum) and incubated at 37°C for 1 h (L2071hEcad) or 30 min (Vero cells) with MßCD 10 mM in DME. Cells were then washed twice and assayed as described. For cholesterol repletion, L2071hEcad and Vero cells were incubated with a solution of 2.5 mM and 5 mM cholesterol-MßCD for 2 h and 15 min, respectively, then washed and assayed as described.
Gentamicin survival assay
Gentamicin assays were performed in 24-well tissue culture plates (Costar), three wells for each condition. Cells (±105 cell/well) were washed twice with DME without antibiotic, and bacterial suspensions were added to mammalian cells at multiplicity of infection of 50. Cells were incubated with the bacterial suspension at 37°C for 30 min (L2071hEcad) or 1 h (Vero), then washed three times with DME, and overlaid with DME containing gentamicin (10 µg/ml) for 1 h at 37°C. Cells were washed three times in DME and were lysed by adding 0.2% Triton X-100 in PBS. The number of viable bacteria released from the cells was assessed by titration on agar plates. The results were expressed as the number of intracellular bacteria per cell relative to control nondepleted cells. Results are the mean value of at least three independent experiments.
Internalin- and InlB-coated beads adhesion and entry assay
Cells were plated onto glass coverslips in 24-well tissue culture plates. Control and cholesterol-depleted cells were incubated 30 min with internalin- or InlB-coated latex beads at 37°C. Cells were washed three times with DME and fixed with paraformaldehyde solution (3.5% in PBS) for 30 min. Cells were then washed twice with a 0.1 M glycine in PBS solution and incubated in blocking solution (PBS; 10% BSA) at RT for 30 min. Extracellular beads were labeled with anti-internalin or anti-InlB antibodies and with Alexa Fluor 546conjugated goat antimouse secondary antibodies. Total number of beads associated with cells (extracellular plus intracellular beads) was determined by phase-contrast microscopy. The number of extracellular beads was quantified by fluorescence microscopy. Results were the mean of at least three independent experiments and were expressed relative to control nondepleted cells.
Immunolabeling, wide-field microscopy, and fluorescence quantification of E-cadherin expression
For immunolabeling, cells were fixed with a PFA solution (3.5% in PBS) for 30 min, then permeabilized (0.2% Triton X-100 for 5 min in PBS) or not. Cells were then washed twice with a 0.1 M glycine in PBS solution and incubated in blocking solution (PBS; 10% BSA) at RT for 30 min. Antibodies were incubated for 30 min at RT in blocking solution.
For GM1 labeling, cells were fixed with cold PFA for 30 min and labeled with a 40-µg/ml cold solution of Alexa Fluor 488 cholera toxin subunit B for 30 min.
Images were acquired on a fluorescence inverted microscope (Axiovert 135; Carl Zeiss MicroImaging, Inc.) equipped with a cooled charge-coupled device camera (MicroMax 5 MHz; Princetown Instruments) driven by Metamorph Imaging System software (Universal Imaging Corp). For quantification of fluorescence, images were acquired under the same condition (40x oil immersion objective, acquisition time, and microscope setting), were background corrected, and a mask was applied to consider only the fluorescence associated with cells. Fluorescence intensity per cell was calculated by the ratio of the total fluorescence intensity per field over the cell number within the field. The results were expressed relative to control cells. Three independent experiments were performed, 500 cells were analyzed for each experiment.
Scanning EM
For scanning EM analysis, cells were washed in PBS and fixed in 2.5% (vol/vol) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, overnight at 4°C. Cells were washed three times for 5 min (each time) in 0.2 M cacodylate buffer, pH 7.2, fixed after for 1 h in 1% (wt/vol) osmium tetroxide in 0.2 M cacodylate buffer, pH 7.2, and then rinsed with distilled water. Cells were dehydrated through a graded series of 25, 50, 75, and 95% ethanol solutions for 10 min (each time) and washed two times for 10 min (each time) in 100% ethanol. Dehydrated cells were consecutively immersed in 25, 50, and 75% (vol/vol) hexamethyldisilazane in ethanol for 5 min (each time), immersed twice in 100% hexamethyldisilazane for 5 min (each time), and quickly air dried. Coverslips were sputter coated twice with carbon with a BALTEC MED010 evaporator. Samples were examined and photographed with a JEOL JSM 6700F field emission scanning electron microscope operating at 5 Kv.
Phosphotyrosine and HGF-R immunoprecipitations
Vero cells were cultured in 75-cm2 tissue culture flasks (106 cell/flask), serum starved for 5 h, then depleted or not for 30 min with 10 mM MßCD. After two washes, cells were stimulated with 8 nM soluble InlB or with 0.6 nM HGF for 1 min at 37°C. Cell were washed with cold PBS and solubilized by 1 ml of ice-cold immunoprecipitation buffer (1% NP-40, 50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 3 mM sodium orthovanadate, 1 mM PMSF, and 10 µg/ml aprotinin, leupeptin, pepstatin, and chymostatin) according to Ireton et al. (1999). Cell lysates were precleared with protein ASepharose beads for 1 h at 4°C. After protein quantification, cell lysates were immunoprecipitated with 1 µg anti-phosphotyrosine (clone 4G10) or anti HGF-R antibodies (C-28) overnight. Protein A Sepharose was added for 2 h. After washes by centrifugation, samples were boiled for 2 min in buffer containing 50 mM Tris-HCl, pH 7.5, 0.5% SDS, and 5 mM DTT. Protein ASepharose beads were then removed by centrifugation. Samples were analyzed by SDS-PAGE and by Western blotting for PI 3-kinase p85
subunit, phosphotyrosine or HGF-R.
Equilibrium density gradient centrifugation
Cells were cultured on 60-mm Petri dishes (Costar), treated as described were washed twice with cold TKM buffer (50 mM Tris-HCl, ph 7.4, 25 mM KCl, 5 mM MgCl2, and 1 mM EGTA), then were lysed with 500 µl TKM buffer containing, Brij58 (0.5%) or Triton X-100 (0.5% or 0.1%) and protease inhibitor cocktail (1:50), for 30 min on ice according to Ilangumaran (Ilangumaran et al., 1998). Aliquots of cell lysates were used to assess protein concentration. Cell lysates were mixed with an equal volume of 80% sucrose containing 0.5% Brij58, or 0.5% Triton X-100. Centrifuge tubes (Beckman Coulter) were filled successively with 1 ml of 80% sucrose in TKM, 1 ml of 40% sucrose containing the cell lysate, 6 ml of 36% sucrose, and 3.5 ml of 5% sucrose. The gradients were centrifuged in an ultracentrifuge (rotor SW41; Beckman Coulter) at 4°C for 18 h at 38,000 rpm. Fractions (1 ml) were collected from top to bottom, and proteins were precipitated with TCA. Samples corresponding to each fraction were analyzed by SDS-PAGE and Western blotting, using antibodies against E-cadherin, caveolin-2, -catenin, internalin, transferring receptor, and HGF-R/Met.
Online supplemental material
In Fig. S1, Lovo cells were incubated with L. monocytogenes (BUG 1641) for 30 min at 37°C, cells were then washed, fixed, and labeled for E-cadherin and HGF-R/Met. Images were acquired with a 100x objective. Bar, 10 µm. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200406078/DC1.
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Acknowledgments |
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This work was supported by the Pasteur Institute, INSERM, Association pour la Recherche contre le Cancer (ARC), and INRA, and by a fellowship (to S. Seveau) from the ARC, Paris, France. P. Cossart is an international research scholar from the Howard Hughes Medical Institute.
Submitted: 17 June 2004
Accepted: 8 July 2004
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