Phosphatidylinositol 3-Kinase and Frabin Mediate Cryptosporidium parvum Cellular Invasion via Activation of Cdc42
2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês
10.1074/jbc.m401592200
ISSN1083-351X
AutoresXian‐Ming Chen, Patrick L. Splinter, Pamela S. Tietz, Bing Huang, Daniel D. Billadeau, Nicholas F. LaRusso,
Tópico(s)Ion channel regulation and function
ResumoCryptosporidium parvum invades target epithelia via a mechanism that involves host cell actin reorganization. We previously demonstrated that C. parvum activates the Cdc42/neural Wiskott-Aldrich syndrome protein network in host cells resulting in actin remodeling at the host cell-parasite interface, thus facilitating C. parvum cellular invasion. Here, we tested the role of phosphatidylinositol 3-kinase (PI3K) and frabin, a guanine nucleotide exchange factor specific for Cdc42 in the activation of Cdc42 during C. parvum infection of biliary epithelial cells. We found that C. parvum infection of cultured human biliary epithelial cells induced the accumulation of PI3K at the host cell-parasite interface and resulted in the activation of PI3K in infected cells. Frabin also was recruited to the host cell-parasite interface, a process inhibited by two PI3K inhibitors, wortmannin and LY294002. The cellular expression of either a dominant negative mutant of PI3K (PI3K-Δp85) or functionally deficient mutants of frabin inhibited C. parvum-induced Cdc42 accumulation at the host cell-parasite interface. Moreover, LY294002 abolished C. parvum-induced Cdc42 activation in infected cells. Inhibition of PI3K by cellular overexpression of PI3K-Δp85 or by wortmannin or LY294002, as well as inhibition of frabin by various functionally deficient mutants, decreased C. parvum-induced actin accumulation and inhibited C. parvum cellular invasion. In contrast, the overexpression of the p85 subunit of PI3K promoted C. parvum invasion. Our data suggest that an important component of the complex process of C. parvum invasion of target epithelia results from the ability of the organism to trigger host cell PI3K/frabin signaling to activate the Cdc42 pathway, resulting in host cell actin remodeling at the host cell-parasite interface. Cryptosporidium parvum invades target epithelia via a mechanism that involves host cell actin reorganization. We previously demonstrated that C. parvum activates the Cdc42/neural Wiskott-Aldrich syndrome protein network in host cells resulting in actin remodeling at the host cell-parasite interface, thus facilitating C. parvum cellular invasion. Here, we tested the role of phosphatidylinositol 3-kinase (PI3K) and frabin, a guanine nucleotide exchange factor specific for Cdc42 in the activation of Cdc42 during C. parvum infection of biliary epithelial cells. We found that C. parvum infection of cultured human biliary epithelial cells induced the accumulation of PI3K at the host cell-parasite interface and resulted in the activation of PI3K in infected cells. Frabin also was recruited to the host cell-parasite interface, a process inhibited by two PI3K inhibitors, wortmannin and LY294002. The cellular expression of either a dominant negative mutant of PI3K (PI3K-Δp85) or functionally deficient mutants of frabin inhibited C. parvum-induced Cdc42 accumulation at the host cell-parasite interface. Moreover, LY294002 abolished C. parvum-induced Cdc42 activation in infected cells. Inhibition of PI3K by cellular overexpression of PI3K-Δp85 or by wortmannin or LY294002, as well as inhibition of frabin by various functionally deficient mutants, decreased C. parvum-induced actin accumulation and inhibited C. parvum cellular invasion. In contrast, the overexpression of the p85 subunit of PI3K promoted C. parvum invasion. Our data suggest that an important component of the complex process of C. parvum invasion of target epithelia results from the ability of the organism to trigger host cell PI3K/frabin signaling to activate the Cdc42 pathway, resulting in host cell actin remodeling at the host cell-parasite interface. Cryptosporidium parvum, an intracellular parasite within the protist phylum Apicomplexa, is one of the most commonly reported enteric pathogens worldwide in both immunocompetent and immunocompromised individuals (1O'Donoghue P.J. Int. J. Parasitol. 1995; 25: 139-195Google Scholar). Humans are infected by ingesting C. parvum oocysts. Once ingested, oocysts excyst in the gastrointestinal tract releasing infective sporozoites. Mediated by uncharacterized but specific ligands on the sporozoite surface and unidentified receptors on the host cell, the sporozoite attaches to the apical membrane of the host epithelial cell, inducing host cell actin cytoskeleton remodeling and protrusion of the host cell membrane around the sporozoite to form a parasitophorous vacuole. At the base of each vacuole, the parasite establishes a unique "electron-dense band" of unknown composition, which separates the organism from the host cell cytoplasm. Thus, the parasitophorous vacuole and the dense-band keep the internalized parasite intracellular but extracytoplasmic (2Tzipori S. Griffiths J.K. Adv. Parasitol. 1998; 40: 5-36Google Scholar, 3Chen X.M. Keithly J.S. Paya C.V. LaRusso N.F. N. Engl. J. Med. 2002; 346: 1723-1731Google Scholar, 4Clark D.P. Clin. Microbiol. Rev. 1999; 12: 554-563Google Scholar). Host cell actin is a common molecular target of many pathogenic microbes including viruses and bacteria such as vaccinia virus, Listeria monocytogenes, Escherichia coli, Salmonella enterica, and Shigella flexneri (5Finlay B.B. Cossart P. Science. 1997; 276: 718-725Google Scholar). These microbes utilize host cell actin for multiple reasons including cell attachment and entry, movement within and between cells, vacuole formation and remodeling, and avoidance of phagocytosis (6Gruenheid S. Finlay B.B. Nature. 2003; 422: 775-781Google Scholar). Recent studies by us (7Chen X.M. LaRusso N.F. Gastroenterology. 2000; 118: 368-379Google Scholar) and others (8Forney J.R. DeWald D.B. Yang S. Speer C.A. Healey M.C. Infect. Immun. 1999; 67: 844-852Google Scholar, 9Elliott D.A. Clark D.P. Infect. Immun. 2000; 68: 2315-2322Google Scholar, 10Elliott D.A. Coleman D.J. Lane M.A. May R.C. Machesky L.M. Clark D.P. Infect. Immun. 2001; 69: 5940-5942Google Scholar) have demonstrated that host cell actin remodeling at the host cell-parasite interface is necessary for the cellular invasion of C. parvum. Inhibition of host cell actin polymerization by pharmacological inhibitors, such as cytochalasin B and cytochalasin D (7Chen X.M. LaRusso N.F. Gastroenterology. 2000; 118: 368-379Google Scholar, 8Forney J.R. DeWald D.B. Yang S. Speer C.A. Healey M.C. Infect. Immun. 1999; 67: 844-852Google Scholar), or by cellular expression of specific inhibitory fragments of actin-associated proteins, such as Scar-WA (10Elliott D.A. Coleman D.J. Lane M.A. May R.C. Machesky L.M. Clark D.P. Infect. Immun. 2001; 69: 5940-5942Google Scholar), block C. parvum cellular invasion. Several host cell proteins necessary for the actin-based motility of Listeria and Shigella within the infected host cells (11Pantaloni D. Le Clainche C. Carlier M.F. Science. 2001; 292: 1502-1506Google Scholar) also have been identified at the C. parvum attachment site, including cortactin, Arp2/3 complex, neural Wiskott-Aldrich syndrome protein (N-WASP), 1The abbreviations used are: N-WASP, neural Wiskott-Aldrich syndrome protein; PI3K, phosphatidylinositol 3-kinase; FAB, F-actin binding, GEF, guanine nucleotide exchange factor; GST, glutathione S-transferase; PH, pleckstrin homology; GFP, green fluorescent protein; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; ELISA, enzyme-linked immunosorbent assay; HA, hemagglutinin; DAPI, 4′,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; DH, Dbl homology; PIPES, 1,4-piperazinediethanesulfonic acid. and vasodilator-stimulated phosphoprotein (VASP) (10Elliott D.A. Coleman D.J. Lane M.A. May R.C. Machesky L.M. Clark D.P. Infect. Immun. 2001; 69: 5940-5942Google Scholar). More recent studies have revealed that several intracellular signaling pathways are involved in C. parvum-induced actin remodeling. Whereas the accumulation of cortactin at the C. parvum host cell-parasite interface appears to be dependent upon the activation of the c-Src signaling pathway (12Chen X.M. Huang B.Q. Splinter P.L. Cao H. Zhu G. McNiven M.A. LaRusso N.F. Gastroenterology. 2003; 125: 216-228Google Scholar), the activation of Cdc42, one member of the Rho family of small GTPases, is required for the accumulation of N-WASP and the Arp2/3 complex at the host cell-parasite interface during C. parvum cellular invasion (13Chen X.M. Huang B.Q. Splinter P.L. Orth J.D. Billadeau D.D. McNiven M.A. LaRusso N.F. Infect. Immun. 2004; 72: 3011-3021Google Scholar). Activation of Cdc42 requires guanine nucleotide exchange factors (GEFs). These proteins stimulate the dissociation of GDP from the GDP-bound inactive form, resulting in the binding of GTP to form the GTP-bound active form, the activity of which is often regulated by an upstream signal. One of the GEFs for Cdc42 is frabin, a recently identified protein ubiquitously expressed and implicated in the mechanisms of Cdc42-associated actin remodeling (14Umikawa M. Obaishi H. Nakanishi H. Satoh-Horikawa K. Takahashi K Hotta I Matsuura Y. Takai Y. J. Biol. Chem. 1999; 74: 25197-25200Google Scholar, 15Ikeda W. Nakanishi H. Tanaka Y. Tachibana K. Takai Y. Oncogene. 2001; 20: 3457-3463Google Scholar). Frabin contains an F-actin binding (FAB) domain and two pleckstrin homology (PH) domains (16Obaishi H. Nakanishi H. Mandai K. Satoh K. Satoh A. Takahashi K. Miyahara M. Nishioka H. Takaishi K. Takai Y. J. Biol. Chem. 1998; 273: 18697-18700Google Scholar). Proteins with PH domains can bind to phosphoinositides such as phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) (17Russo C. Gao Y. Mancini P. Vanni C. Porotto M. Falasca M. Torrisi M.R. Zheng Y. J. Biol. Chem. 2001; 276: 19524-19531Google Scholar, 18Han J. Luby-Phelps K. Das B. Shu X. Xia Y. Mosteller R.D. Krishna U.M. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Google Scholar). One important signaling kinase implicated in actin polymerization and activated upon membrane stimulation by a variety of ligands is the class IA phosphatidylinositol 3-kinase (PI3K). Activation of PI3K catalyzes the production of PtdIns(3,4,5)P3 at cell membranes, thus resulting in the recruitment and activation of various signaling components to the cell membrane, some of which have been implicated in the regulation of the cytoskeleton, e.g. growth factor-induced membrane ruffling (19Tolias K.F. Cantley L.C. Carpenter C.L. J. Biol. Chem. 1995; 270: 17656-17659Google Scholar). Using an in vitro model of intestinal cryptosporidiosis, Forney et al. (8Forney J.R. DeWald D.B. Yang S. Speer C.A. Healey M.C. Infect. Immun. 1999; 67: 844-852Google Scholar) have demonstrated previously that a selective PI3K inhibitor, wortmannin, blocked C. parvum infection of cultured bovine fallopian tube epithelial cells. Thus, in this study, we tested the role of PI3K and frabin in C. parvum-induced Cdc42 activation in cellular invasion. We found that C. parvum infection of cultured human biliary epithelial cells induces the accumulation of PI3K at the host cell-parasite interface and results in the activation of PI3K in infected cells. Frabin is also recruited to the host cell-parasite interface, a process that is dependent upon PI3K. Both PI3K and frabin appear to be required for Cdc42 accumulation at the host cell-parasite interface. In addition, the inhibition of PI3K by either selective pharmacologic inhibitors or host cell overexpression of functionally deficient mutants of PI3K and frabin was associated with a reduction of C. parvum-induced actin remodeling and ultimately C. parvum invasion of biliary epithelia. These findings demonstrate that C. parvum invasion of biliary epithelial cells is facilitated by the recruitment and activation of PI3K in host cells, a process that activates the Cdc42 pathway via frabin and is required for C. parvum-induced actin remodeling and cellular invasion. C. parvum and H69 Cells—C. parvum oocysts of the Iowa strain were purchased from a commercial source (Pleasant Hill Farms, Troy, ID). Before infecting cells, oocysts were excysted to release infective sporozoites as described previously (7Chen X.M. LaRusso N.F. Gastroenterology. 2000; 118: 368-379Google Scholar). H69 cells (a gift of Dr. D. Jefferson, Tufts University, Boston, MA) are SV40-transformed human bile duct epithelial cells originally derived from a normal liver harvested for transplant and have been characterized extensively (20Grubman S.A. Perrone R.D. Lee D.W. Murray S.L. Rogers L.C. Wolkoff L.I. Mulberg A.E. Cherington V. Jefferson D.M. Am. J. Physiol. 1994; 266: G1060-G1070Google Scholar). For experiments, H69 cells were used between passages 23 and 30 and maintained for three passages without co-culture cells to ensure that the culture was free of 3T3 fibroblasts. In Vitro Models and Infection Assay—Two in vitro models, an attachment model and an attachment/invasion model, were employed to assay the attachment and invasion of C. parvum to H69 cells as described previously (7Chen X.M. LaRusso N.F. Gastroenterology. 2000; 118: 368-379Google Scholar). H69 cells were seeded into 4-well chamber slides or 6-well Costar tissue culture plates (BD Biosciences Labware, Franklin Lakes, NJ) and grown to 70–80% confluence. For the attachment model, H69 cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) in PBS before exposure to C. parvum sporozoites. In this model, the organism can only attach to the fixed cell surface. For the attachment/invasion model, live cells (without pre-fixation) were exposed directly to C. parvum sporozoites, thus allowing the organism to both attach to and enter into host cells. Infection with C. parvum was done in a culture medium consisting of Dulbecco's modified Eagle's-Ham's F-12 medium, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) and freshly excysted C. parvum sporozoites (1 × 106 sporozoites/slide well or culture plate). Inactivated organisms (treated at 65 °C for 30 min) were used for sham infection experiments (1O'Donoghue P.J. Int. J. Parasitol. 1995; 25: 139-195Google Scholar). Infection assays (attachment rate or attachment/invasion rate) were carried out after a 2-h incubation with the parasite employing an indirect immunofluorescent technique as described previously (7Chen X.M. LaRusso N.F. Gastroenterology. 2000; 118: 368-379Google Scholar). Parasites attaching to pre-fixed cells or invading non-fixed cells were counted, and the results were expressed, respectively, as attachment rate or attachment/invasion rate (the number of parasites/total number of cells × 100). Approximately 2,000 cells were counted for each assay. For the inhibitory experiments, two selective inhibitors of PI3K, LY294002 and wortmannin (21Maeda T. Kawane T. Horiuchi N. Endocrinology. 2003; 144: 681-692Google Scholar), were added in the medium at the time C. parvum was added. A concentration of 10 μm LY294002 or 100 nm wortmannin showed no cytotoxic effects on H69 cells or on C. parvum sporozoites and was selected for the study. Transfection of Cells—H69 cells for transient transfection were grown to 40–60% confluency on 8-well chamber slides and transfected with 1 μg/well plasmid DNA using the LipofectAMINE Plus™ reagent kit according to the manufacturer's recommendations. The plasmid constructs for transient transfection included the following: pEF-BOSΔRI-PI3K-Δp85-HA (a dominant negative mutant of p85 regulatory subunit that cannot interact with the catalytic p110 subunit) (22Sutor S.L. Vroman B.T. Armstrong E.A. Abraham R.T. Karnitz L.M. J. Biol. Chem. 1999; 274: 7002-7010Google Scholar, 23Jackson T.R. Blader I.J. Hammonds-Odie L.P. Burga C.R. Cooke F. Hawkins P.T. Wolf A.G. Heldman K.A. Theibert A.B. J. Cell Sci. 1996; 109: 289-300Google Scholar, 24Rust C. Karnitz L.M. Paya C.V. Moscat J. Simari R.D. Gores G.J. J. Biol. Chem. 2000; 275: 20210-20216Google Scholar) and pEF-BOSΔRI-PI3K-p85-HA (wild type of the p85 subunit of PI3K) (gifts from Dr. L. M. Karnitz, Mayo Medical School, Rochester, MN); pKR5-Cdc42(17N)-Myc (a dominant negative mutant and a gift from Dr. A. Hall, University College London, London, United Kingdom) (25Nobes C.D. Hall A. J. Cell Biol. 1999; 144: 1235-1244Google Scholar); and various constructs of frabin (14Umikawa M. Obaishi H. Nakanishi H. Satoh-Horikawa K. Takahashi K Hotta I Matsuura Y. Takai Y. J. Biol. Chem. 1999; 74: 25197-25200Google Scholar, 15Ikeda W. Nakanishi H. Tanaka Y. Tachibana K. Takai Y. Oncogene. 2001; 20: 3457-3463Google Scholar, 26Ono Y. Nakanishi H. Nishimura M. Kakizaki M. Takahashi K. Miyahara M. Satoh-Horikawa K. Mandai K. Takai Y. Oncogene. 2000; 19: 3050-3058Google Scholar) such as pCMV-Frabin-GFP, pCMV-Frabin-dead-Full-Myc (an internal deletion of amino acids 353–362 in the Dbl homology (DH) domain that abolishes Cdc42-activating activity), and pCMV-Frabin-dead-DH-PH1-Myc (a truncated mutant lacking of the FAB domain but containing the DH and PH1 domains with an internal deletion of amino acids 353–362 in the DH domain) (gifts from Dr. Y. Takai, Osaka University Medical School, Osaka, Japan). Empty vectors were used as controls. Twenty-four h after transfection, cells were exposed to C. parvum sporozoites for the attachment and invasion assays. Immunofluorescent Microscopy—H69 cells were exposed to C. parvum sporozoites as described above. After 2 h of incubation, cells were fixed (0.1 mol/liter PIPES, pH 6.95, 1 mmol/liter EGTA, 3 mmol/liter magnesium sulfate (Sigma-Aldrich), and 2% paraformaldehyde) at 37 °C for 20 min and then permeabilized with 0.2% (v/v) Triton X-100 in PBS. For double immunofluorescent labeling, fixed cells were incubated with primary monoclonal antibodies to associated proteins mixed with a polyclonal antibody against C. parvum sporozoite membrane proteins (a generous gift from Drs. Guan Zhu and Janet Keithly, Wadsworth Center, Albany, NY) followed by rhodamine-labeled anti-mouse and fluorescein-labeled anti-rabbit antibodies (Molecular Probes, Eugene, OR). Some cells were incubated with polyclonal antibodies to associated proteins mixed with a monoclonal antibody against C. parvum (2H2, ImmunuCell, Portland, ME) followed by rhodamine-labeled anti-rabbit and fluorescein-labeled anti-mouse antibodies. To confirm the specificity of the staining, multiple antibodies from different sources were used for some of the proteins including two antibodies against Cdc42 (clone B8, Santa Cruz Biotechnology and a polyclonal antibody from Calbiochem), one monoclonal antibody and one polyclonal antibody to PI3K (Upstate Biotechnology), and a monoclonal antibody to frabin (clone 43, BD Transduction Laboratories). For localization of actin with C. parvum, rhodamine-phalloidin (Sigma-Aldrich) was co-incubated with the secondary antibody. For triple immunofluorescent labeling in experiments with the transient transfected cells, monoclonal antibodies to the c-Myc epitope tag (clone 9B11, Cell Signaling) or HA epitope tag (clone 262K, Cell Signaling) were used to identify the transfected cells. The cells were co-stained with another antibody against the associated protein plus rhodamine-phalloidin to stain actin or 4′,6-diamidino-2-phenylindole (DAPI) (5 μmol/liter) to label the parasite (which stains the nuclei of both the host cells and the parasite in blue) (8Forney J.R. DeWald D.B. Yang S. Speer C.A. Healey M.C. Infect. Immun. 1999; 67: 844-852Google Scholar). Labeled cells were rinsed three times with PBS and once with distilled water, mounted with mounting medium (H-1000, Vector Laboratories), and assessed by confocal laser-scanning microscopy. The numbers of parasites with and without accumulation of associated proteins at the host cell-parasite interface were counted separately for quantitative analysis. Those with the obvious accumulation of each associated protein were counted as positive, and the results were expressed as the accumulation percentage (the number of parasites with an accumulation of the molecules at the host cell-parasite interface/total number of parasites × 100). Between 500 and 1000 C. parvum cells were counted randomly for each assay. Images obtained from the Zeiss 510 confocal microscope (Carl Zeiss, Inc. Oberkochen, Germany) were manipulated uniformly for contrast and intensity using the Adobe Photoshop (Mountain View, CA) software. Immunoprecipitation and Phosphotyrosine of PI3K—H69 cells were grown in 10-cm dishes to 95% confluence and exposed to C. parvum sporozoites at 37 °C for 1 and 2 h. Cells then were rinsed with PBS and scraped into 1 ml of cold lysis buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, and 0.1% SDS) supplemented with 1 mm phenylmethylsulfonyl fluoride, leupeptin, and pepstatin at 20 μg/ml and tyrosine phosphatase inhibitors, sodium orthovanadate and sodium fluoride, at 1 mm. The cell lysates were centrifuged at 13,000 × g for 10 min and were assayed for protein concentration using the Bradford reagent (Sigma-Aldrich). The cell lysates (1 mg of protein) then were incubated with 10 μl of anti-PI3K (Upstate Biotechnology) at 4 °C overnight to immunoprecipitate PI3K. Immune complexes were collected by direct binding to protein A-Sepharose. The immunoprecipitated protein then was released with Western sample buffer (20% glycerol, 4% SDS, 10% β-mercaptoethanol, 0.05% bromphenol blue, and 1.25 m Tris, pH 6.8), incubated at 95 °C for 5 min, separated by SDS-PAGE, and immunoblotted with an antibody to phosphotyrosine (Clone 4G10, Upstate Biotechnology). Immunoreactive bands were developed using a chemiluminescent substrate (ECL Plus, Amersham Biosciences). PI3K and Cdc42 Activity Assay—PI3K activity was detected using an enzyme-linked immunosorbent assay (ELISA). H69 cells were grown on 10-cm dishes to 95% confluency and exposed to C. parvum sporozoites at 37 °C for 1 and 2 h. PI3K protein was immunoprecipitated, and the activity of PI3K was determined using a commercial ELISA kit (Echelon Biosciences Inc.). The activity of PI3K was expressed as the picomole of product phosphatidylinositol 3,4,5-trisphosphate/1 mg protein. A pull-down assay was employed to measure Cdc42 activation in H69 cells after exposure to C. parvum as described previously (13Chen X.M. Huang B.Q. Splinter P.L. Orth J.D. Billadeau D.D. McNiven M.A. LaRusso N.F. Infect. Immun. 2004; 72: 3011-3021Google Scholar, 27Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Google Scholar, 28Ren X.-D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Google Scholar). H69 cells were grown on T75 flasks to 95% confluency and exposed to C. parvum sporozoites at 37 °C for 1 h. Cells then were chilled on ice, washed with ice-cold PBS, and lysed in buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.5% Nonidet P-40, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml pepstatin. The cell lysates then were incubated with GST-Cdc42/Rac-interactive binding domain bound to glutathione-coupled agarose beads (Sigma-Aldrich) for 60 min at 4 °C, washed with washing buffer (50 mm Tris, pH 7.5, 0.5% Triton X-100, 150 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 0.1 mm phenylmethylsulfonyl fluoride), and eluted with SDS sample buffer with 200 mm dithiothreitol. GTP-bound active Cdc42 was analyzed by Western blotting using a monoclonal antibody to Cdc42. PI3K Is Recruited to the Host Cell-Parasite Interface and Is Activated—To test the role of PI3K in the mechanism of host cell actin-dependent invasion of epithelia by C. parvum, we used double immunofluorescent labeling for C. parvum and PI3K. Very strong staining of PI3K was observed at the C. parvum host cell-parasite interface (Fig. 1, A and B). No accumulation of PI3K was detected in sham-infected control cells, and no positive staining of PI3K was found in the parasite itself (data not shown). Quantitative analysis showed 96.7 ± 2.5% of the host cell-parasite interfaces with an accumulation of PI3K. Ligand-stimulated activation of class IA PI3K is dependent upon the phosphorylation of Tyr688 of its p85 subunit (29Cuevas B.D. Lu Y. Mao M. Zhang J. LaPushin R. Siminovitch K. Mills G.B. J. Biol. Chem. 2001; 276: 27455-27461Google Scholar). To test whether C. parvum infection induces the phosphorylation of this p85 subunit in host cells, biliary epithelia were exposed to C. parvum, cell lysates were immunoprecipitated using an antibody to PI3K, and the immunoprecipitates were blotted for phosphotyrosine. As shown in Fig. 1C, phosphotyrosine in the p85 subunit was detected at 1 and 2 h after exposure to C. parvum. Activation of PI3K was determined further by ELISA using the immunoprecipitate as a substrate. As shown in Fig. 1D, cells exposed to C. parvum for 1 and 2 h showed a marked increase in PI3K activity compared with the sham-infected control cells. PI3K-dependent Accumulation of Frabin at the Host Cell-Parasite Interface—To test whether frabin is involved in C. parvum cellular invasion, accumulation of frabin at the host cell-parasite interface was measured first by double immunofluorescent labeling using a monoclonal antibody to frabin and a polyclonal antibody to C. parvum. Strong staining of frabin was observed at the C. parvum host cell-parasite interface (Fig. 2, A1 and A2). No accumulation of frabin in the sham-infected cells or positive staining in the parasite itself was found with the procedure used in the experiments (data not shown). To further confirm the specificity of frabin staining with the antibody, a GFP-frabin construct was used to transfect cells before exposure to C. parvum sporozoites followed by confocal microscopy. As shown in Fig. 2, B1 and B2, GFP-frabin also was found to be recruited to the host cell-parasite interface. To define whether C. parvum-induced frabin accumulation at the host cell-parasite interface is dependent upon PI3K activation, LY294002 and wortmannin, two selective PI3K inhibitors (21Maeda T. Kawane T. Horiuchi N. Endocrinology. 2003; 144: 681-692Google Scholar), and PI3K-Δp85, a dominant negative mutant p85 regulatory subunit of PI3K unable to interact with the catalytic p110 subunit thus blocking PI3K activity (22Sutor S.L. Vroman B.T. Armstrong E.A. Abraham R.T. Karnitz L.M. J. Biol. Chem. 1999; 274: 7002-7010Google Scholar, 23Jackson T.R. Blader I.J. Hammonds-Odie L.P. Burga C.R. Cooke F. Hawkins P.T. Wolf A.G. Heldman K.A. Theibert A.B. J. Cell Sci. 1996; 109: 289-300Google Scholar, 24Rust C. Karnitz L.M. Paya C.V. Moscat J. Simari R.D. Gores G.J. J. Biol. Chem. 2000; 275: 20210-20216Google Scholar), were used. As shown in Fig. 2, C1–D2, a significant decrease in frabin accumulation was found at the host cell-parasite interface in cells treated with LY294002 or transfected with PI3K-Δp85. A similar inhibition of frabin accumulation was detected in cells treated with wortmannin (data not shown). Quantitative analysis showed a significant (p < 0.01) decrease in frabin accumulation in the presence of PI3K inhibitors and in cells transfected with PI3K-Δp85 compared with cells in the absence of inhibitors or cells transfected with the empty vector (Fig. 2E). These data indicate that the recruitment of a Cdc42-specific GEF to the host cell-parasite interface requires the activation of PI3K. To test the possibility that frabin may be required for PI3K accumulation at the host cell-parasite interface, we tested the effects of functionally deficient mutates of frabin on PI3K accumulation. H69 cells were transfected transiently with Frabin-dead-Full or Frabin-DH-PH1, two functionally deficient mutants of frabin (14Umikawa M. Obaishi H. Nakanishi H. Satoh-Horikawa K. Takahashi K Hotta I Matsuura Y. Takai Y. J. Biol. Chem. 1999; 74: 25197-25200Google Scholar, 15Ikeda W. Nakanishi H. Tanaka Y. Tachibana K. Takai Y. Oncogene. 2001; 20: 3457-3463Google Scholar, 26Ono Y. Nakanishi H. Nishimura M. Kakizaki M. Takahashi K. Miyahara M. Satoh-Horikawa K. Mandai K. Takai Y. Oncogene. 2000; 19: 3050-3058Google Scholar), and then exposed to C. parvum followed by co-staining of PI3K. Accumulation of PI3K was found both in cells transfected with Frabin-dead-Full (arrowheads in Fig. 3A2) or Frabin-DH-PH1 (arrowheads in Fig. 3, B2) and in the non-transfected cells (arrows in Fig. 3, A2 and B2). Quantitative analysis showed no significant difference in PI3K accumulation between transfected and non-transfected cells (Fig. 3C), suggesting that frabin is not required for the accumulation of PI3K during C. parvum invasion of biliary epithelial cells. Accumulation/Activation of Cdc42 at the Host Cell-Parasite Interface Requires PI3K and Frabin—Our previous studies indicated that C. parvum cellular invasion activates the Cdc42-Arp/N-WASP network to induce actin remodeling at the attachment site, thus facilitating C. parvum cellular invasion (13Chen X.M. Huang B.Q. Splinter P.L. Orth J.D. Billadeau D.D. McNiven M.A. LaRusso N.F. Infect. Immun. 2004; 72: 3011-3021Google Scholar). To clarify the relationship between PI3K/Frabin signaling pathways and the Cdc42-Arp/N-WASP network during C. parvum cellular invasion, C. parvum-induced Cdc42 accumulation was explored in cells treated with PI3K inhibitors or in cells transfected with PI3K-Δp85 or various mutants of frabin. Consistent with our previous studies (13Chen X.M. Huang B.Q. Splinter P.L. Orth J.D. Billadeau D.D. McNiven M.A. LaRusso N.F. Infect. Immun. 2004; 72: 3011-3021Google Scholar), a strong accumulation of Cdc42 was observed at the host cell-parasite interface in the absence of PI3K inhibitors (Fig. 4, A1 and A2) with 93.7 ± 1.3% of the attachment sites accumulated with Cdc42. A significant decrease in Cdc42 accumulation at the host cell-parasite interface was observed in cells treated with LY294002 (Fig. 4, B1 and B2) or transfected with PI3K-Δp85 (Fig. 4, C1 and C2). A decrease in Cdc42 accumulation also was found in cells transfected with the Frabin-DH-PH1 (Fig. 4, D1 and D2). Quantitative analysis showed a significant decrease in Cdc42 accu
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