Molecular Dissection of Internalization of Porphyromonas Gingivalis by Cells Using Fluorescent Beads Coated with Bacterial Membrane Vesicle

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Molecular Dissection of Internalization of Porphyromonas gingivalis by Cells using Fluorescent Beads Coated with Bacterial Membrane Vesicle

Kayoko Tsuda1,2, Atsuo Amano3,4, Kyohei Umebayashi2, Hiroaki Inaba3, Ichiro Nakagawa5,6, Yoshinobu Nakanishi1,7, and Tamotsu Yoshimori2,4 1Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan, 2Department of Cell Genetics, National Institute of Genetics/SOKENDAI, Yata 1111, Mishima-Shizuoka 411-8540, Japan, 3Department of Oral Frontier Biology, Osaka University Graduate School of Dentistry, Suita-Osaka 565-0871, Japan, 4CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan, 5Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, Suita- Osaka 565-0871, Japan, 6PRESTO, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan, and 7Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan

ABSTRACT. Porphyromonas gingivalis is one of the causative agents of adult periodontitis, and has been reported to be internalized by nonphagocytic epithelial cells. However, the mechanism for the internalization remains unclear. In the present study, we addressed this issue using fluorescent beads coated with bacterial membrane vesicles (MVs) that retain surface components of P. gingivalis. We established an assay system in which we could easily quantify the bead internalization to cells. MVs-coated beads were internalized by HeLa cells in kinetics similar to that of living bacteria. The internalization depended on dynamin but not clathrin. The beads were internalized through the actin-mediated pathway that is controlled by phosphatidylinositol (PI) 3-kinase. The dynamics of microtubule assembly and disassembly was also required. Further, the treatment of cells with cholesterol-binding reagents significantly inhibited bead internalization, and the internalized beads were apparently colocalized with ganglioside GM1 and caveolin-1, which suggest the involvement of the lipid raft in the process. These results suggest that P. gingivalis accomplishes its internalization utilizing membrane lipid raft and cytoskeletal functions of the target cells.

Key words: P. gingivalis/membrane vesicles/dynamin/actin/microtubule/lipid raft

Introduction Rottner et al., 2005). Salmonella typhimurium and Shigella flexneri deliver their virulence factors into the cellular cyto- Many bacterial pathogens are known to enter nonphago- plasm with type III secretion system (TTSS), following cytic cells (Cossart and Sansonetti, 2004), and two types of their adherence to the target cells. These virulence factors mechanism mediating bacterial entry into host cells have elicit actin rearrangement, leading to entry triggered by the been extensively studied: trigger and zipper mechanisms bacteria (trigger mechanism). In the zipper mechanism, the (Finlay and Cossart, 1997; Lafont and van der Goot, 2005; bacterial internalization is mediated by cellular actin rearrangement which is induced through specific interactions between bacterial ligands and cell surface receptors. For *To whom correspondence should be addressed: Tamotsu Yoshimori, example, Listeria monocytogenes and Yersinia pseudotu- Department of Cell Genetics, National Institute of Genetics, Yata 1111 berculosis express their specific adhesions, such as inter- Mishima, Shizuoka 455-8540, Japan. nalin and invasin for cellular components and extracellular Tel: +81–559–81–6881, Fax: +81–559–81–6884 E-mail: [email protected] matrix proteins, respectively, and these bindings activate Abbreviations: PI3K, phosphatidylinositol 3-kinase; LPS, lipopolysaccha- the entry mechanisms. Rho family GTPases, such as Rac ride; BSA, bovine serum albumin; GFP, green fluorescent protein; MCD, and Cdc42, are involved in actin rearrangement in both methyl--cyclodextrin; CTxB, cholera toxin subunit B; DMEM, Dul- becco’s modified Eagle’s medium; FBS, fetal bovine serum; RT, room mechanisms (Gruenheid and Finlay, 2003). For several temperature. bacteria, lipid rafts in the plasma membrane have been

81 K. Tsuda et al. suggested as bacterial entry sites (Garner et al., 2002; Materials and Methods Lafont et al., 2002; Seveau et al., 2004; Zobiack et al., 2002). Lipid rafts are membrane subdomains rich in choles- Reagents and plasmids terol, sphingolipids and specific membrane proteins, such as glycosylphosphatidylinositol-anchored proteins (GPI-APs). All Alexa Fluor dyes-conjugated reagents were purchased from It has been suggested that lipid rafts act as platforms in pro- Molecular Probes Inc. (Eugene, OR, USA), EZ-link sulfo-NHS- tein sorting and signal transduction (Simons and Ikonen, LC-biotin was from Pierce (Rockford, IL, USA), mouse mono- 1997). In addition to these roles, lipid rafts may serve as clonal anti-tubulin antibody, cytochalasin D, nocodazole, taxol, entry sites for bacteria. Recently, Veiga and Cossart methyl--cyclodextrin (MCD), filipin III, and nystatin were from reported that Listeria monocytogenes hijacks the clathrin- Sigma (St Louis, MO, USA), latrunculin A and wortmannin were mediated endocytic machinery for its entry into its host cells from Wako (Osaka, Japan). The dominant-negative Eps15 con- (Veiga and Cossart, 2005). However, the details of its struct GFP-Eps1595/295, in which the second and third EH mechanism still remain largely unknown. domains are deleted, was kindly provided by Dr. A. Dautry-Varsat Periodontitis is one of the most common chronic diseases (Institut Pasteur, Paris, France). Using this plasmid, we con- afflicting mankind, and is characterized by the destruction structed GFP-Eps15EH in which all of the three EH domains are of supporting periodontal connective tissue and tooth loss. lacking. GFP-dynamin 2 and GFP-dynamin 2-K44A were gener- The disease has been also implicated in atherosclerosis ous gifts from Dr. K. Nakayama (Kyoto University, Kyoto, Japan). (Dorn et al., 1999; Gibson et al., 2004; Haraszthy et al., Caveolin-1-GFP was a kind gift from Dr. A. Helenius (Swiss 2000) and pre-term delivery of low birth weight infants Federal Institute of Technology, Zurich, Switzerland). (Offenbacher et al., 1996). Porphyromonas gingivalis, a Gram-negative short rod anaerobe, has been identified as a Cell culture and transfection bona fide pathogen of adult periodontitis (Socransky and Haffajee, 1992). P. gingivalis was detected within gingival HeLa cells were grown in DMEM (Sigma) supplemented with tissues of periodontitis patients (Papapanou et al., 1994), 10% FBS (Invitrogen, Carlsbad, CA), 4 mM L-glutamine (Invitro- and was also reportedly internalized by several human gen) and 10 g/mL gentamicin (Sigma). 6×104 HeLa cells seeded epithelial cell lines in vitro (Duncan et al., 1993; Lamont et on coverslips in 24-well plates were transiently transfected with al., 1995). Thus, those bacterial interactions with epithelial 1.0 g of the plasmid by LipofectAMINE 2000 reagent (Invitro- cells are considered to be the key events to establish chronic gen, Carlsbad, CA) according to the manufacturer’s recommenda- periodontal infection (Lamont and Yilmaz, 2002). P. gingi- tions. Transfected cells were further incubated for 20 h and used valis initiates its entry to host cells through the interaction for the assays. P. gingivalis TDC60 (type II fimA) was grown in between cellular 51 integrin molecules and fimbriae, GAM broth (Nissui, Tokyo, Japan) supplemented with 5 g/ml which are filamentous appendages on the bacterial surfaces hemin and 1 g/ml menadione anaerobically (80% N2, 10% H2, (Nakagawa et al., 2002; Yilmaz et al., 2002). However, 10% CO2). the further molecular machinery involved in the event is unclear. A large number of Gram-negative bacteria, includ- Isolation of P. gingivalis extracellular vesicles ing P. gingivalis, have an ability to extracellularly release membrane vesicles (MVs). This takes place during normal P. gingivalis extracellular vesicles were prepared according to the growth, possibly as a result of cell wall turn over (Grenier Rosen method (Rosen et al., 1995). Briefly, 500 ml of cell culture and Mayrand, 1987; Mayrand and Grenier, 1989; Zhou et was subjected to centrifugation at 10,000×g for 20 min. The cul- al., 1998). In fact, the MVs retain full components of outer ture supernatant was filtrated through a 0.2 m-pore-size filter membrane constituents of cell wall including proteins, LPS, (Nalge Nunc, Rochester, NY), and centrifuged at 100,000×g for muramic acid, capsule, and fimbriae (Zhou et al., 1998). 50 min. The precipitates containing extracellular vesicles were MVs isolated from culture medium of P. gingivalis can be suspended in 500 l of PBS (~2.5 g proteins/ml). For heat-inacti- conjugated to polystyrene fluorescent-beads, and the beads vation, the MVs suspension was incubated at 70°C for 30 min. were internalized by epithelial cells in a MVs-dependent manner (Inaba et al., 2006). Use of the MVs-coated beads Preparation of MV-coated beads as a homogenous artificial intruder allows us to quantify the internalization efficiency easily and stably, and would Alexa 365- or 580-conjugated sulfate-modified polystyrene beads potentially avoid the complications arising from living bac- (1.0 m in diameter) were used for the bead internalization assay. terial organisms such as multiplication. First, the beads were washed three times with PBS. The sulfate- Here, we developed a new assay system to measure inter- modified beads (1.45×1010) were resuspended in PBS (50 l) con- nalization of the MVs-coated beads to cultured cells, and taining MVs (1 g/ml), heat-inactivated MVs (1 g/ml) or BSA used it to characterize the entry mechanisms of P. gingivalis (200 g/ml), and incubated at room temperature (RT) for 4 h. The with respect to the host cellular endocytic machinery, MVs- or BSA-coated beads were washed three times with PBS and cytoskeleton, and lipid rafts. resuspended in 1 ml of PBS containing 1% BSA.

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Internalization assays Results

4 8×10 HeLa cells were seeded onto glass coverslips in 24-well Efficient internalization of MVs-coated beads to HeLa plates (Nalge Nunc) the day before the assay. The cells were cells washed twice with DMEM and incubated for 30 min in the presence or absence of indicated reagents. 1.35×107 of MVs-coated To precisely measure the internalization of MVs-coated beads were added to the cells. The plate was centrifuged at 410×g beads to cells, we developed an assay as illustrated in for 1 min at 37°C to promote contact between the beads and the Fig. 1A. HeLa cells were incubated with the MVs-coated, cells. After being incubated at 37°C for 1 h, the plate was trans- magenta-colored beads (1 m in diameter) for 1 h, washed ferred to 4°C. The cells were washed with ice-cold PBS and incu- with PBS, and then the plasma membrane of the host cells bated with sulfo-NHS-LC-biotin (100 g/ml in PBS) at 4°C for 15 as well as extracellular beads attaching cell surface were min. Subsequently, the cells were washed twice and exposed to biotinylated with membrane impermeable biotin. Finally, streptavidin-Alexa Fluor 488 (10 g/ml in PBS) at 4°C for 10 min. Alexa 488 (green) conjugated streptavidin was added to Finally, the cells were washed and fixed with 3% paraformalde- cells to visualize the biotinylated molecules (see Materials hyde (PFA) in PBS for 10 min. and Methods). The result is shown in Fig. 1B. If the beads An Olympus FV1000 laser scanning confocal microscope just adhered to the host cell surface or were incompletely (Olympus, Tokyo, Japan) was used for image collection and analy- internalized, they were accessible to the biotin-streptavidin sis. The images were taken with a 100×objective oil immersion labeling. As a result, they became white by color merge and lens at 800×800 pixel resolution. To quantify the beads internal- were easily distinguished from completely internalized ized by the cells, maximal projection images were made. In each beads with the magenta image. The cell boundaries were field, intra- and extracellular beads were counted manually. At also confirmed by the vertical optical sections. The MVs- least 6 fields and ~200 cells were analyzed for each experiment, coated beads were efficiently internalized by cells for 1 h. and three independent experiments were done. The results were If the beads were coated with BSA or heat-inactivated MVs, normalized by the control experiments. they were hardly internalized (Fig. 1C), indicating that the internalization is dependent on the MVs. Fig. 1D shows Drug treatments time-dependent internalization of MVs-coated beads. The number of the completely internalized beads increased HeLa cells were washed twice with serum-free DMEM and incu- along the incubation time, and reached saturation at 40 min. bated with the following drugs in DMEM at 37°C for 30 min prior The kinetics of internalization was found to be quite similar to the assay. To disorganize the cytoskeletal architecture, 1 g/ml to that of living bacteria reported in previous studies cytochalasin D, 2 M latrunculin A, 25 M nocodazol or 50 M (Yilmaz et al., 2004; Yilmaz et al., 2003).We also examined taxol was used. To analyze the role of cholesterol in bead internal- the localization of 51-integrin which was reported to be ization, 10 mM MCD, 5 g/ml filipin III or 50 g/ml nystatin responsible for the initial binding of P. gingivalis to the was used. To inhibit PI3K activity, 100 nM wortmannin or 50 M cells (Nakagawa et al., 2005; Yilmaz et al., 2002). Con- LY294002 was used. All reagents were included in the medium sistent with those previous reports, recruitment of 51- throughout the internalization experiments. integrin was observed at the bead internalization site (Fig. 1E). Fluorescence microscopy Internalization of the MVs-coated beads is dependent For immunostaining, cells were washed with ice-cold PBS, fixed on dynamin 2 but not on Eps15 with 3% PFA in PBS for 10 min, and then permeabilized with 0.1% Triton X-100 in PBS for 10 min. After being washed twice Eps15 is required for clathrin-dependent endocytosis. This with PBS, the cells were incubated in blocking solution (0.1% gel- protein contains three EH (Eps15-homology) domains, and atin in PBS) for 5 min and subsequently with primary antibodies overexpression of the Eps15 mutant lacking the EH diluted with the blocking solution at RT for 1 h. To stain F-actin, domains interferes with clathrin-coated pit assembly cells were incubated with 0.25 M of Alexa 488-phalloidin in (Benmerah et al., 1999). To examine whether the MVs- PBS/1% BSA at RT for 30 min. For GM1 labeling, cells were coated beads are internalized by cells in a clathrin-dependent fixed with PFA and subsequently labeled with 40 g/ml of Alexa manner, GFP-Eps15EH was transiently transfected to 488-conjugated cholera toxin subunit B (Molecular Probes Inc.) HeLa cells. As expected, uptake of transferrin, a marker for for 30 min on ice. The cells were observed with an Olympus clathrin-dependent endocytosis, was inhibited in the trans- FV1000 laser scanning confocal microscope. fected cells (Fig. 2A). In contrast, the transfected cells were found to internalize the MVs-coated beads (Fig. 2B), indicating that the bead internalization is a clathrin-independent process. Next, we analyzed the involvement of the GTPase dynamin in bead internalization. The dynamin family con-

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Fig. 1. MVs-coated beads are internalized by HeLa cells. (A) A scheme for surface biotinylation to distinguish internalized beads from extracellular ones. HeLa cells were incubated with the MVs-coated beads (magenta) for 1 h and subjected to biotinylation with sulfo-NHS-LC-biotin. Subsequently, the cells were treated with Alexa 488-conjugated streptavidin (green), and then fixed with paraformaldehyde. (B) In cells treated as in (A), confocal optical sections of 0.18 m thickness were taken at 0.5 m intervals. A projection image and vertical (z) optical sections (x-z and y-z planes) are shown. The magenta arrow indicates completely internalized beads. The white arrow indicates beads that just adhered to the host cell surface or were incompletely internalized. Bar, 4 m. (C) Comparison of entry efficiency among MVs-, BSA- and heat inactivated MVs-coated beads. The number of the magenta-colored beads per cell was quantified. The mean values from three independent experiments are shown with SEs. (D) Time-dependent internalization of the MVs-coated beads. At the beginning of the assay, the cells were incubated with the MVs-coated beads for the indicated time periods. (E) Recruitment of 51-integrin to the entry of MVs-coated bead. After 1 h of incubation with the beads, HeLa cells were processed for the anti-51-integrin antibody staining. The boxed area shows that 51-integrin (magenta) is recruited around the MVs-coated bead (cyan). Bar, 5 m.

84 Mechanisms of P. gingivalis-Bead Internalization into Cells

Fig. 2. Overexpression of the dominant-negative mutant dynamin 2 inhibits the internalization of MVs-coated beads but the Eps15 mutant does not. HeLa cells transfected with GFP-tagged Eps15EH (A, B), GFP-Dyn2-WT (C) or GFP-Dyn2-K44A (D) were incubated with Alexa568-conjugated transferrin (A: magenta) or MVs-coated beads (B, C, D: magenta) at 37°C for 1h, washed, fixed and permeabilized. Cells were then stained with DAPI (B, D: cyan). A projection image at mid-height of the cell and vertical (z) optical sections (x-z and y-z planes) are shown (GFP: green). White outlines show perimeters of non-transfected cells. Arrows in (B) and (C) indicate the internalized beads, arrowheads in (D) indicate recruitment of GFP-Dyn2-K44A to the aggregated beads on the cell surface. Bars, 10 m.

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Fig. 3. Internalization of the MVs-coated beads is dependent on both actin and microtubule cytoskeletons. The bead internalization assay described in Fig. 1A and Materials and Methods was carried out in the presence of the indicated drugs. (A) Each image is a projection of 4–5 confocal sections of the middle region (beads: magenta, surface label: green). Bars, 20 m. (B) The number of the magenta-colored beads per cell was quantified, and presented as percentage of control cells, which were not treated with the drugs. The mean values from three independent experiments are shown with SEs. (C) After 40 min of incubation with the MVs-coated beads (cyan), the cells were processed for staining with anti--tubulin (green) and Alexa Fluor 568-conjugated phalloidin (magenta). Images of x-y and x-z planes are shown. Arrowheads indicate colocalization of beads with both F-actin and -tubulin. Arrow indicates colocalization of beads with F-actin. (D) After incubation with the MVs-coated beads in the presence of taxol (upper panels) or nocodazole (lower panels), cells were stained as described in (C). Insets show enlarged images of the boxed areas. Bars, 5 m.

86 Mechanisms of P. gingivalis-Bead Internalization into Cells

Fig. 4. Internalization of the MVs-coated beads is dependent on PI3K. (A) The bead internalization assay described in Fig. 1A and Materials and Methods was carried out in the presence of the PI3K inhibitors, wortmannin and LY294002. Each image is a projection of 4–5 confocal sections of the middle region. Bars, 10 m. (B) The number of the internalized beads (magenta) per cell was quantified, and presented as percentage of control cells, which were not treated with drugs. The mean values from three independent experiments are shown with SEs. tains three members Dyn1, 2 and 3, with Dyn2 being a ubiq- Actin and microtubule cytoskeletons is required for the uitously expressed form (Cao et al., 1998). GTPase activity internalization of Dyn2 is required for both clathrin-dependent and -independent endocytosis (Lamaze et al., 2001; Le and Nabi, To evaluate the role of the actin cytoskeleton in the bead 2003; Oh et al., 1998; Sabharanjak et al., 2002), and the internalization, the internalization assay was carried out mutant Dyn2-K44A defective in GTP-binding shows a in the presence of the actin polymerization inhibitors, dominant negative effect. As shown in Fig. 2C, the beads cytochalasin D and latrunculin A. As shown in Fig. 3A and were internalized by the cells overexpressing the wild type B, bead internalization was significantly prevented by these Dyn2 fused with GFP. In contrast, overexpression of the reagents. Almost all the beads in the images were white, dominant negative GFP-Dyn2-K44A drastically prevented indicating that they just adhered to the cell surface or were bead internalization (Fig. 2D). Notably, the beads were incompletely internalized. Next, we examined the effect of aggregated on the cell surface, and GFP-Dyn2-K44A was microtubule disorganization on bead internalization, using apparently clustered together with the beads. These results the microtubule assembly inhibitor nocodazole and the indicate that the beads are internalized by the cells in a microtubule-stabilizing drug taxol. These drugs also inhib- clathrin-independent and dynamin-dependent manner. ited the bead internalization by more than 65% (Fig. 3A and

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B). The effect of taxol indicates that not only microtubule assembly but also disassembly is critical to the process. To investigate whether actin filaments and microtubles are remodeled by the MVs-coated bead internalization, the cells were stained with phalloidin for actin and the anti--tubulin antibody for microtubules. These staining images revealed that some beads were surrounded by both F-actin and -tubulin (Fig. 3C, arrowheads). Although it is unclear whether the internalization of the surrounded beads was ongoing or just finished, the vertical optical sections (the x- z plane) indicate that both actin cytoskeleton and microtubules function in the uptake pathway. It is likely that the actin remodeling, which is required for engulfment of the MVs-coated beads, is induced by attachment of the beads to the plasma membrane. Interestingly, when the cells were treated with either nocodazole or taxol, the recruitment of F-actin around the beads was not abolished (Fig. 3D), suggesting that the recruitment of -tubulin follows the engulfment of beads by F-actin. Phosphatidylinositol 3-kinase (PI3K) is involved in rapid actin polymerization during phagocytosis in macrophages (Cox et al., 1999). To elucidate whether the PI3K activity contributes to bead internalization, we examined the effect of PI3K inhibitors, wortmannin and LY294002. As shown in Fig. 4A and B, these drugs strongly prevented bead internalization, which indicates that PI3K is involved in bead internalization.

Lipid rafts play a role in the internalization Recently, accumulating evidence has revealed that lipid rafts serve as entry sites into host cells for some bacteria (Lafont et al., 2004). To examine whether this is the case with P. gingivalis, we perturbed the raft formation by treatment with filipin, nystatin, or methyl--cyclodextrin (MCD). Filipin and nystatin bind to and sequester cholesterol, an essential component of lipid rafts, while MCD Fig. 5. Internalization of the MVs-coated beads is dependent on choles- removes cholesterol from the membrane (Kilsdonk et al., terol. (A) The bead internalization assay described in Fig. 1A and Materials 1995; Smart and Anderson, 2002). As shown in Fig. 5A, the and Methods was carried out in the presence of the indicated drugs. Each number of magenta-colored beads, which were completely image is a projection of 4–5 confocal sections of the middle region. (B) The number of the internalized beads (magenta) per cell was counted, and internalized by the cells, significantly decreased by these presented as percentage of control cells, which were not treated with the drugs, i.e., 40~50% inhibition by filipin and nystatin, and drugs. The mean values from three independent experiments are shown 95% inhibition by MCD. (Fig. 5B). These results indicate with SEs. that cholesterol is required for internalization of the MVs- coated beads. Next, we investigated colocalization of raft markers, gan- Discussion glioside GM1 and caveolin-1, with the MVs-coated beads. After being incubated with the beads, cells were fixed and In this study, we have studied the contribution of several cell surface GM1 was labeled with fluorescent cholera toxin host cell factors to P. gingivalis internalization, using fluo- subunit B (CTxB). As shown in Fig. 6A, GM1 was clearly rescent beads coated with the MVs released by this bacte- recruited around the beads. In addition, the beads were sur- rium. Combined use of the MVs-coated beads and cell rounded by caveolin-1-GFP (Fig. 6B). Thus, lipid rafts were surface-biotinylation provided an easy and reliable assay shown to be indeed localized to the beads entry site. system for the internalization to cells. The MVs-coated beads were efficiently internalized by nonphagocytic HeLa cells, indicating that the bacterial components of the outer

88 Mechanisms of P. gingivalis-Bead Internalization into Cells

Fig. 6. Recruitment of raft markers to the MVs-coated beads. (A) HeLa cells were incubated with the MVs-coated beads (magenta), washed and fixed. The cells were then incubated with Alexa 488-conjugated CTxB to label GM1. (B) HeLa cells transiently expressing caveolin-1-GFP were incubated with the MVs-coated beads (magenta), washed and fixed. Recruitment of caveolin-1 (green) around the beads is indicated in boxed areas. Insets show enlarged images of the boxed areas. Bars, 5 m. membrane are sufficient for the internalization. The present (Sauvonnet et al., 2005). This is probably achieved by internalization mode is apparently distinct from the trigger binding of dynamin to F-actin-interacting proteins (Orth and mechanism, because P. gingivalis does not produce TTSS McNiven, 2003; Schafer, 2004). Therefore, we propose that (Lamont and Yilmaz, 2002). Previous studies of living P. actin dynamics controlled by Dyn2 is critical for internaliza- gingivalis (Deshpande et al., 1998; Lamont et al., 1995; tion of P. gingivalis. In contrast to Listeria monocytogenes Nakagawa et al., 2005; Yilmaz et al., 2002) were reminis- (Veiga and Cossart, 2005), Eps15 was dispensable for inter- cent of the zipper mechanism. Those previous findings are nalization of P. gingivalis. evidenced by the present study showing that actin cyto- We also suggested for the first time that dynamics of skeleton and PI3K are required for the internalization and microtubule assembly and disassembly are required for the that 51-integrin, a receptor for the bacterium, and F- internalization of P. gingivalis. It is known that micro- actin were localized around the beads. tubules are involved in entry of several pathogens into We further demonstrated novel cellular players involved epithelial cells (Yoshida and Sasakawa, 2003). Nocodazole in the internalization of P. gingivalis to cells. We found that inhibited the entry in most of the cases, whereas taxol did the internalization of the MVs-coated beads is dependent on not (Grassme et al., 1996; Kuhn, 1998; Oelschlaeger et Dyn2. Although Dyn2 is required for scission of the clathrin- al., 1993). It is known that taxol does not inhibit motor- coated vesicles, it is also involved in clathrin-independent dependent processes. Internalizations of Campylobacter processes, such as caveolar endocytosis (Nichols, 2003) and jejuni (Hu and Kopecko, 1999) and Chlamydia trachomatis phagocytosis in macrophages (Gold et al., 1999). It has (Clausen et al., 1997), both of which require a dynein been recently suggested that Dyn2 regulates actin poly- motor, were not prevented by taxol. On the contrary, the merization at the site of plasma membrane invagination, MVs-coated bead internalization was inhibited by both in both clathrin-dependent and -independent internalization nocodazole and taxol. This suggests that the internalization

89 K. Tsuda et al. does not require stable microtubules as a rail for the motor- References dependent sliding, and rather that it needs dynamic poly- Benmerah, A., Bayrou, M., Cerf-Bensussan, N., and Dautry-Varsat, A. merization and depolymerization of microtubules. Tubulin 1999. Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J. localized around the beads together with F-actin. Consider- Cell Sci., 112: 1303–1311. ing that neither nocodazole nor taxol inhibited the formation Cao, H., Garcia, F., and McNiven, M.A. 1998. Differential distribution of dynamin isoforms in mammalian cells. Mol. Biol. Cell, 9: 2595–2609. of F-actin-rich region around the beads, microtubule assem- Clausen, J.D., Christiansen, G., Holst, H.U., and Birkelund, S. 1997. bly and disassembly around the beads may follow the step Chlamydia trachomatis utilizes the host cell microtubule network during of actin polymerization to form the membrane-bound com- early events of infection. Mol. Microbiol., 25: 441–449. partment engulfing the beads. Cossart, P. and Sansonetti, P.J. 2004. Bacterial invasion: the paradigms of Recently, it was reported that siRNA-mediated reduction enteroinvasive pathogens. Science, 304: 242–248. of caveolin-1 expression caused inhibition of P. gingivalis Cox, D., Tseng, C.C., Bjekic, G., and Greenberg, S. 1999. A requirement internalization, but the colocalization of intracellular P. for phosphatidylinositol 3-kinase in pseudopod extension. J. Biol. Chem., 274: 1240–1247. gingivalis with caveolin-1 was not clearly shown (Tamai et Deshpande, R.G., Khan, M.B., and Genco, C.A. 1998. Invasion of aortic al., 2005). In addition, the inhibitory effect of MCD for and heart endothelial cells by Porphyromonas gingivalis. Infect. Immun., the internalization was reportedly only 50%. Judging from 66: 5337–5343. those observations, the involvement of lipid rafts in the Dorn, B.R., Dunn, Jr., W.A., and Progulske-Fox, A. 1999. Invasion of internalization cannot be said to be conclusive. However, in human coronary artery cells by periodontal pathogens. Infect. Immun., our experimental system, MCD was found to be a signifi- 67: 5792–5798. Duncan, M.J., Nakao, S., Skobe, Z., and Xie, H. 1993. Interactions of cant inhibitor for the bead internalization. We also showed Porphyromonas gingivalis with epithelial cells. Infect. Immun., 61: an inhibitory effect of nystatin and filipin. Further, the intra- 2260–2265. cellular beads were apparently colocalized with lipid raft Finlay, B.B. and Cossart, P. 1997. Exploitation of mammalian host cell markers (Fig. 6). These results strongly suggest that lipid functions by bacterial pathogens. Science, 276: 718–725. rafts play a pivotal role in the internalization process of P. Garner, M.J., Hayward, R.D., and Koronakis, V. 2002. The Salmonella gingivalis, and that cholesterol is required for formation of pathogenicity island 1 secretion system directs cellular cholesterol redis- the lipid rafts serving as the putative bacterial entry sites. tribution during mammalian cell entry and intracellular trafficking. Cell Microbiol., 4: 153–165. However, more detailed analysis would be required to sub- Gibson, F.C., 3rd, Hong, C., Chou, H.H., Yumoto, H., Chen, J., Lien, E., stantiate the hypothesis. Wong, J., and Genco, C.A. 2004. Innate immune recognition of invasive In summary, we developed a novel assay method to reli- bacteria accelerates atherosclerosis in apolipoprotein E-deficient mice. ably and conveniently examine P. gingivalis internalization Circulation, 109: 2801–2806. to cells, and applied it successfully to obtain important Gold, E.S., Underhill, D.M., Morrissette, N.S., Guo, J., McNiven, M.A., insights into the molecular basis underlying the bacterial and Aderem, A. 1999. Dynamin 2 is required for phagocytosis in mac- entry to host cells. Internalization of the MVs-coated beads rophages. J. Exp. Med., 190: 1849–1856. Grassme, H.U., Ireland, R.M., and van Putten, J.P. 1996. Gonococcal requires host cellular dynamin, actin fibers, microtubules, opacity protein promotes bacterial entry-associated rearrangements of PI3K, and the lipid rafts. Such a combination is so far the epithelial cell actin cytoskeleton. Infect. Immun., 64: 1621–1630. unknown for other bacterial entry, indicating the particular- Grenier, D. and Mayrand, D. 1987. Functional characterization of extra- ity of P. gingivalis internalization to cells and suggesting a cellular vesicles produced by Bacteroides gingivalis. Infect. Immun., 55: diversity of bacterial entry mechanisms. To our surprise, the 111–117. dynamics of microtubule assembly and disassembly is Gruenheid, S. and Finlay, B.B. 2003. Microbial pathogenesis and cytoskeletal function. Nature, 422: 775–781. essential to the process, whereas other bacterial entry mech- Haraszthy, V.I., Zambon, J.J., Trevisan, M., Zeid, M., and Genco, R.J. anisms usually employ stable microtubules. Future studies 2000. Identification of periodontal pathogens in atheromatous plaques. should unravel how the microtubule dynamics is involved J. Periodontol., 71: 1554–1560. in the process, what is the role of the lipid rafts, and what Hu, L. and Kopecko, D.J. 1999. Campylobacter jejuni 81-176 associates relationship exists among each of the components, that is, with microtubules and dynein during invasion of human intestinal cells. dynamin, actin, microtubules, and the lipid rafts. The Infect. Immun., 67: 4171–4182. present bead internalization assay method will no doubt be Inaba, H., Kawai, S., Kato, T., Nakagawa, I., and Amano, A. 2006. Asso- ciation between epithelial cell death and invasion by microsphere con- useful to further investigate these issues. jugated with Porphyromonas gingivalis vesicles with different types of fimbriae. Infect. Immun., 74: 734–739. Acknowledgements. We wish to thank Dr. K. Nakayama (Kyoto Univer- Kilsdonk, E.P., Yancey, P.G., Stoudt, G.W., Bangerter, F.W., Johnson, sity) for the dynamin 2 constructs, Dr. A. Dautry-Varsat (Institut Pasteur) W.J., Phillips, M.C., and Rothblat, G.H. 1995. Cellular cholesterol for the Eps15 constructs, and Dr. A. Helenius (Swiss Federal Institute of efflux mediated by cyclodextrins. J. Biol. Chem., 270: 17250–17256. Technology) for caveolin-1-GFP. We are also very grateful to Dr. N. Shiina Kuhn, M. 1998. The microtubule depolymerizing drugs nocodazole and (National Institute of Genetics) for helpful discussions. 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