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Escherichia coli K-1 Interaction with Human Brain Micro-vascular Endothelial Cells Triggers Phospholipase C-γ1 Activation Downstream of Phosphatidylinositol 3-Kinase*

  • Sunil K. Sukumaran
    Affiliations
    Division of Infectious Diseases, University of Southern California Keck School of Medicine, Los Angeles, California 90027

    University of Southern California Keck School of Medicine, Los Angeles, California 90027
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  • George McNamara
    Affiliations
    Congressman Dixon Image Core, Childrens Hospital Los Angeles, University of Southern California Keck School of Medicine, Los Angeles, California 90027
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  • Nemani V. Prasadarao
    Correspondence
    To whom correspondence should be addressed: Division of Infectious Diseases, MS 51, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027. Tel.: 323-669-5465; Fax: 323-660-2661
    Affiliations
    Division of Infectious Diseases, University of Southern California Keck School of Medicine, Los Angeles, California 90027

    University of Southern California Keck School of Medicine, Los Angeles, California 90027
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grants AI40567 and HD50325 (to N. V. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:September 02, 2003DOI:https://doi.org/10.1074/jbc.M307374200
      Escherichia coli, the most common Gram-negative bacterium that causes meningitis in neonates, invades human brain microvascular endothelial cells (HBMEC) by rearranging host cell actin via the activation of phosphatidylinositol 3-kinase (PI3K) and PKC-α. Here, further, we show that phospholipase (PLC)-γ1 is phosphorylated on tyrosine 783 and condenses at the HBMEC membrane beneath the E. coli entry site. Overexpression of a dominant negative (DN) form of PLC-γ, the PLC-z fragment, in HBMEC inhibits PLC-γ1 activation and significantly blocks E. coli invasion. PI3K activation is not affected in PLC-z/HBMEC upon infection, whereas PKC-α phosphorylation is completely abolished, indicating that PLC-γ1 is downstream of PI3K. Concomitantly, the phosphorylation of PLC-γ1 is blocked in HBMEC overexpressing a dominant negative form of the p85 subunit of PI3K but not in HBMEC overexpressing a dominant negative form of PKC-α. In addition, the recruitment of PLC-γ1 to the cell membrane in both PLC-z/HBMEC and DN-p85/HBMEC is inhibited. Activation of PI3K is associated with the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 1,4,5-trisphosphate (PIP3), which in turn recruits PLC-γ1 to the cell membrane via its interaction with pleckstrin homology domain of PLC-γ1. Utilizing the pleckstrin homology domains of PKC-δ and Btk proteins fused to green fluorescent protein (GFP), which specifically interact with PIP2 and PIP3, respectively, we show herein that E. coli invasion induces the breakdown of PIP2 at the plasma membrane near the site of E. coli interaction. PIP3, on the other hand, recruits the GFPBkt to the cell membrane beneath the sites of E. coli attachment. Our studies further show that E. coli invasion induces the release of Ca2+ from intracellular pools as well as the influx of Ca2+ from the extracellular medium. This elevation in Ca2+ levels is completely blocked both in PLC-z/HBMEC and DN-p85/HBMEC, but not in DN-PKC/HBMEC. Taken together, these results suggest that E. coli infection of HBMEC induces PLC-γ1 activation in a PI3K-dependent manner to increase Ca2+ levels in HBMEC. This is the first report demonstrating the recruitment of activated PLC-γ1 to the sites of bacterial entry.
      To invade host cells, pathogens are known to exploit host-signaling pathways either by direct interaction with their cognate receptors or by introducing molecules that activate host-signaling molecules. One of the pathogens that invades endothelial cells by activating host cell signaling machinery to its advantage is Escherichia coli K1, which causes meningitis in neonates. The incidence of E. coli K1 infections in neonates has recently been reported to be on the rise and has surpassed the incidence of infections by other meningitis-causing pathogens such as Listeria and group B Streptococcus (
      • Stoll B.J.
      • Hansen N.
      • Fanaroff A.A.
      • Wright L.L.
      • Carlo W.A.
      • Ehrenkranz R.A.
      • Lemons J.A.
      • Donovan E.F.
      • Stark A.R.
      • Tyson J.E.
      • Oh W.
      • Bauer C.R.
      • Korones S.B.
      • Shankaran S.
      • Laptook A.R.
      • Stevenson D.K.
      • Papile L.-A.
      • Poole W.K.
      ). One of the most important events in E. coli meningitis is the invasion and transcytosis of the human brain microvascular endothelial cells (HBMEC)
      The abbreviations used are: HBMEC, human brain microvascular endothelial cell; PLC, phospholipase C; PKC, protein kinase C; DN, dominant negative; PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 1,4,5-trisphosphate; IP3, inositol triphosphate; FITC, fluorescein isothiocyanate; DAG, diacylglycerol; cfu, colony-forming unit(s); GFP, green fluorescent protein; SH, Src homology; PH, pleckstrin homology; TBST, Tris-buffered saline plus Tween 20; NA, numeric aperture.
      1The abbreviations used are: HBMEC, human brain microvascular endothelial cell; PLC, phospholipase C; PKC, protein kinase C; DN, dominant negative; PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 1,4,5-trisphosphate; IP3, inositol triphosphate; FITC, fluorescein isothiocyanate; DAG, diacylglycerol; cfu, colony-forming unit(s); GFP, green fluorescent protein; SH, Src homology; PH, pleckstrin homology; TBST, Tris-buffered saline plus Tween 20; NA, numeric aperture.
      by the bacteria to disseminate into the central nervous system.
      Adherence of E. coli to the surface of endothelial cells via S-fimbriae is followed by the interaction of OmpA (an outer membrane protein of E. coli K1) with its receptor, a gp96-like protein, which initiates the invasion process (
      • Stins M.F.
      • Prasadarao N.V.
      • Ibric L.
      • Wass C.A.
      • Luckett P.
      • Kim K.S.
      ,
      • Prasadarao N.V.
      • Wass C.A.
      • Weiser J.N.
      • Stins M.F.
      • Huang S.H.
      • Kim K.S.
      ,
      • Prasadarao N.V.
      ,
      • Prasadarao N.V.
      • Srivastava P.K.
      • Rudrabhatla R.S.
      • Kim K.S.
      • Huang S.H.
      • Sukumaran S.K.
      ). This interaction induces the phosphorylation of focal adhesion kinase and the actin-associated protein, paxillin, which are necessary for cytoskeletal rearrangements in HBMEC (
      • Reddy M.A.
      • Wass C.A.
      • Kim K.S.
      • Schlaepfer D.D.
      • Prasadarao N.V.
      ). Focal adhesion kinase activation requires the physical association of PI3K; thus, overexpression of a dominant negative form of PI3K (either p85 or p110) significantly blocks E. coli invasion of HBMEC (
      • Reddy M.A.
      • Prasadarao N.V.
      • Wass C.A.
      • Kim K.S.
      ). In addition, invasive E. coli induces the phosphorylation and activation of PKC-α, which is recruited to the membrane where it interacts with an actin regulatory molecule, myristoylated alanine-rich C kinase substrate (
      • Sukumaran S.K.
      • Prasadarao N.V.
      ,
      • Sukumaran S.K.
      • Quon M.J.
      • Prasadarao N.V.
      ). Activated PKC-α has been reported to bind to and activate caveolin-1, an integral component of caveolae, which facilitates the transcytosis of bacteria in endothelial cells. The activation of PKC-α is regulated by the mobilization of intracellular calcium by IP3 (inositol triphosphate), a breakdown product of membrane-bound lipid PIP2 (phosphatidylinositol 4,5-bisphosphate) (
      • Rebecchi M.J.
      • Pentyala S.N.
      ). The major enzyme that regulates these events is phospholipase C (PLC).
      Several forms of PLC with different molecular masses, substrate specificity, and Ca2+ requirements have been purified from various mammalian cells (
      • Rebecchi M.J.
      • Pentyala S.N.
      ,
      • Rhee S.G.
      ). Four different families of PLC (β, γ, δ, and ϵ) are present in cells, and all of them possess two conserved X and Y regions, responsible for substrate recognition and catalytic activity, respectively. In addition, γ-type molecules contain a unique structure (Z region) containing two SH2 domains and one SH3 domain (
      • Homma Y.
      • Takenawa T.
      ). Upon stimulation of cells with various growth factors, the SH2 domains of PLC-γ bind the autophosphorylated tyrosine residues of growth factor receptor, leading to tyrosine phosphorylation and activation of PLC-γ (
      • Rebecchi M.J.
      • Pentyala S.N.
      ,
      • Rhee S.G.
      ,
      • Homma Y.
      • Takenawa T.
      ). Except PLC-ϵ, the other three PLC isoforms also contain PH domains. There is now good evidence that PH domains of signaling molecules are involved in targeting to cell membranes (
      • Bae Y.S.
      • Cantley L.G.
      • Chen C.S.
      • Kim S.R.
      • Kwon K.S.
      • Rhee S.G.
      ,
      • Falasca M.
      • Logan S.K.
      • Lehto V.P.
      • Baccante G.
      • Lemmon M.A.
      • Schlessinger J.
      ). The most convincing examples of highly specific, physiologically relevant PH domain targets are observed with PLC-δ PH domain binding to PIP2 as well as IP3 and binding of PH domain of Ser/Thr kinase Akt/PKB/Rac to PI(3,4)P2. The PLC-γ PH domain binds to PIP3, the production of which is regulated by PI3K (
      • Rebecchi M.J.
      • Pentyala S.N.
      ,
      • Rhee S.G.
      ,
      • Falasca M.
      • Logan S.K.
      • Lehto V.P.
      • Baccante G.
      • Lemmon M.A.
      • Schlessinger J.
      ,
      • Varnai P.
      • Balla T.
      ).
      PI3K converts PI(4,5)P2 into PI(3,4,5)P3 at the plasma membrane, which has affinity for PLC-γ and thus anchors it. PIP3 also binds to the c-SH2 of PLC-γ. This process is crucial for the positioning of the enzyme in close proximity to its substrate PI(4,5)P2 (
      • Rameh L.E.
      • Rhee S.G.
      • Spokes K.
      • Kazlauskas A.
      • Cantley L.C.
      • Cantley L.G.
      ). There are two types of PLC-γ: PLC-γ1 and PLC-γ2. It has been demonstrated that the phosphorylation of Tyr-783 of PLC-γ1 is crucial for stimulation of enzymatic activity, whereas phosphorylation of Tyr-753, Tyr-759, and Tyr-1217 is important for PLC-γ2 activity (
      • Kim H.K.
      • Kim J.W.
      • Zilberstein A.
      • Margolis B.
      • Kim J.G.
      • Schlessinger J.
      • Rhee S.G.
      ,
      • Watanabe D.
      • Hashimoto S.
      • Ishiai M.
      • Matsushita M.
      • Baba Y.
      • Kishimoto T.
      • Kurosaki T.
      • Tsukada S.
      ). Because the E. coli invasion of HBMEC is governed by the activation of both PI3K and PKC-α, we hypothesized that PLC-γ would play a role in the invasion process.
      Here we show that PLC-γ1 is activated during E. coli invasion of HBMEC, which is recruited to the membrane beneath the bacterial entry site. Expression of outer membrane protein A (OmpA) on E. coli is necessary for the activation of PLC-γ1. Overexpression of a dominant negative form of PLC-γ in HBMEC significantly abolishes the accumulation of phosphorylated PLC-γ1 beneath the bacteria and subsequent invasion. Our studies also suggest that PLC-γ1 activation is downstream of PI3K but upstream of PKC-α. Furthermore, we show that E. coli invasion of HBMEC mobilizes intracellular calcium as well as calcium influx from extracellular medium in a PLC-γ1-dependent manner.

      EXPERIMENTAL PROCEDURES

      Bacteria—E. coli E44 is a rifampin-resistant mutant of E. coli K1 strain RS 218 (serotype O18:K1:H7), which was isolated from the cerebrospinal fluid of a neonate with meningitis and invades HBMEC in vitro (
      • Prasadarao N.V.
      • Wass C.A.
      • Weiser J.N.
      • Stins M.F.
      • Huang S.H.
      • Kim K.S.
      ). E91 is a non-invasive derivative of E44 in which the ompA gene has been disrupted, resulting in lack of OmpA expression. Bacteria were grown in brain heart infusion broth (Difco Labs) with appropriate antibiotics.
      Materials—Tissue culture dishes containing a glass coverslip in the middle were obtained from Mattek Corp. (Ashland, MA). Antibodies to PLC-γ, phospho-PLC-γ1 (Tyr-783), and phospho-PLC-γ2 (Tyr-1217) were obtained from Cell Signaling Technologies (Beverly, MA). Fura 2/AM and fluorescein isothiocyanate-conjugated secondary antibodies and rhodamine phalloidin were obtained from Molecular Probes, Inc. (Eugene, OR). Cy3-conjugated secondary antibody was obtained from Rockland Immunochemicals (Gilbertsville, PA). Normal goat serum and the Vectashield mounting medium with 4′,6-diamidino-2-phenylindole were obtained from Vector Laboratories Inc. (Burlingame, CA). The PepTag nonradioactive PKC assay was obtained from Promega (Madison, WI). SuperSignal chemiluminescence reagent was obtained from Pierce. The mammalian expression vectors for pXf-PLC-z and pXf-control have been described previously, which provide methotrexate resistance to transfected cells (
      • Kassis J.
      • Moellinger J.
      • Lo H.
      • Greenberg N.M.
      • Kim H.G.
      • Wells A.
      ). Mammalian expression vectors containing PHPLC-δ-GFP and PHBtk-GFP were kindly provided by Dr. Tamas Ballas (
      • Varnai P.
      • Balla T.
      ). Dominant negative (DN) constructs of p85 and PKC-α have been described previously (
      • Reddy M.A.
      • Prasadarao N.V.
      • Wass C.A.
      • Kim K.S.
      ,
      • Sukumaran S.K.
      • Prasadarao N.V.
      ). LipofectAMINE-plus was obtained from Invitrogen. All other reagents were obtained from Sigma.
      HBMEC Culture Maintenance and Transfections—HBMEC were isolated and cultured as described previously (
      • Stins M.F.
      • Prasadarao N.V.
      • Wass C.A.
      • Kim K.S.
      ). HBMEC cultures were maintained in RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 10% Nu-serum, 2 mm glutamine, 1 mm sodium pyruvate, streptomycin (100 μg/ml), penicillin (100 units/ml), essential amino acids, and vitamins. HBMEC were transfected with mammalian expression vectors using LipofectAMINE-plus. Briefly, DNA-LipofectAMINE-plus in RPMI 1640 was added to 50% confluent HBMEC monolayers. After 6 h of incubation at 37 °C, the cells were washed with RPMI 1640, and complete medium was added. After 48 h, the complete medium was replaced with medium containing either G418 (400 μg/ml) or methotrexate (1200 nm/ml) and maintained at least for 4 weeks before performing the invasion experiments.
      E. coli Invasion Assays—Confluent HBMEC in 24-well plates were incubated with 1 × 107 E. coli in experimental medium (1:1 mixture of Ham's F-12 and M-199 containing 5% heat-inactivated fetal bovine serum) for 90 min at 37 °C. The monolayers were washed three times with RPMI 1640 and incubated in experimental medium containing gentamicin (100 μg/ml) for 1 h to kill extracellular bacteria. The monolayers were washed again and lysed with 0.5% Triton X-100. The intracellular bacteria were enumerated by plating on sheep blood agar plates. In parallel experiments, total cell associated bacteria was determined as described above except that the gentamicin step was omitted. In some experiments, HBMEC were pretreated with various inhibitors for 30 min prior to the addition of bacteria. The effects of these inhibitors on HBMEC viability were assessed using trypan blue exclusion method, and effects on bacterial viability were tested by quantitative culture (
      • Prasadarao N.V.
      • Wass C.A.
      • Weiser J.N.
      • Stins M.F.
      • Huang S.H.
      • Kim K.S.
      ).
      Preparation of HBMEC Membranes—Confluent monolayers of HBMEC grown on collagen-coated dishes (60-mm diameter) were washed with RPMI 1640, and E. coli suspended in experimental medium was added. Following stimulation for different time periods (0, 5, 10, 15, and 30 min), the cells were rinsed twice in ice-cold phosphate-buffered saline and placed on ice. The cells in each 60-mm dish were harvested by scraping into 2 ml of ice-cold cell homogenization buffer (buffer A) consisting of 20 mm Tris, pH 7.5, 0.25 m sucrose, 10 mm EGTA, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 2 mm dithiothreitol. The cells were subjected to mild sonication, and a portion of this cell lysate was centrifuged at low speed (5,000 × g) to remove cell debris. The supernatant was the subjected to ultracentrifugation (100,000 × g) to pellet the membrane fraction, which was then solubilized in Tris buffer containing 0.5% Triton X-100. Both the membrane fractions and the cell lysates were used for the PepTag assay and immunoprecipitation studies.
      PepTag Assay for Non-radioactive Detection of PKC Activity—The PepTag (Promega) assay utilizes a florescent peptide substrate that is highly specific to PKC according to the instructions from the manufacturer. Phosphorylation by PKC changes the net charge of the peptide substrate from +1 to -1, thereby allowing the phosphorylated and non-phosphorylated versions of the substrate to be separated using agarose (0.8%) gel electrophoresis. The phosphorylated species migrates toward the positive electrode, whereas the non-phosphorylated substrate migrates toward the negative electrode. HBMEC total lysates or membrane proteins (10-25 μgin10 μl) were incubated with the PKC reaction mixture (25 μl) according to the protocol from the manufacturer at 30 °C for 30 min. The reactions were stopped by placing the tubes in a boiling water bath. After adding 80% glycerol (1 μl), the samples were loaded onto an agarose gel (0.8% agarose in 50 mm Tris-HCl, pH 8.0) and separated at 100 V for 15 min. The peptide bands were visualized under UV light.
      Immunoprecipitations and Western Blotting—HBMEC in 60-mm dishes were exposed to bacteria for varying time periods in infection medium and the harvested by scraping on ice into 2 ml of cell homogenization buffer A. The cells were subjected to mild sonication, and the total cell lysates were centrifuged at 16,000 rpm in a microcentrifuge for 20 min at 4 °C. The supernatant was collected, and protein content was determined. For immunoprecipitation, 300-500 μg of protein was incubated with the appropriate antibody overnight at 4 °C and then incubated for 1 h with protein A-agarose. The immune complexes were washed four times with the cell lysate buffer, and the proteins bound to agarose were eluted in SDS sample buffer and separated by 10% SDS-polyacrylamide gel electrophoresis. The proteins were transferred to a nitrocellulose membrane, which was then blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.05% Tween 20 (TBST) for 2 h at room temperature. The blot was incubated with the primary antibody overnight at 4 °C in 5% bovine serum albumin/TBST. The blot was washed with TBST and further incubated with the horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Subsequently, the blot was washed four times with TBST for a total time of 1 h, developed with SuperSignal chemiluminescence reagent, and exposed to x-ray film to visualize the proteins. The protein bands on the x-ray films were quantitated on a Bio-Rad densitometer using Alpha-imager software (Alpha Innotech Corp., San Francisco, CA).
      Immunofluorescence Staining—HBMEC were grown in 8-well chamber slides coated with collagen and infected with E. coli as described above. The monolayers were then washed with phosphate-buffered saline and fixed in 2% paraformaldehyde for 15 min at room temperature. Subsequently, the monolayers were incubated with 5% normal goat serum in phosphate-buffered saline containing 1% Triton X-100 for 30 min and further incubated with the primary antibody in 5% normal goat serum in phosphate-buffered saline containing 1% Triton X-100 for 1 h at room temperature. The cells were then washed with phosphate-buffered saline and incubated with secondary antibodies conjugated to the fluorochromes Cy3 or FITC for 30 min at room temperature. F-actin was detected by incubating the cells with rhodamine phalloidin for 30 min at room temperature. The cells were washed again, the chambers were removed, and the slides were mounted in Vectashield (Vector Laboratories) anti-fade solution containing 4′,6-diamidino-2-phenylindole. Cells were viewed with a Leica (Wetzlar, Germany) DMRA microscope with Plan-apochromat 40×/1.25 NA and 63×/1.40 NA oil immersion objective lenses. Image acquisition was with a SkyVision-2/VDS digital CCD (12-bit, 1280 × 1024 pixel) camera in unbinned or 2 × 2-binned models into EasyFISH software, saved as 16-bit monochrome, and merged as 24-bit RGB TIFF images (Applied Spectral Imaging Inc., Carlsbad, CA).
      Estimation of Intracellular Calcium Levels Using Fura-2/AM—HBMEC plated in a dish containing a glass coverslip at the center were washed with Ringer's solution (10 mm HEPES, 1 mm MgSO4,5mm KCl, 12 mm NaCl, 2 mm CaCl2, and 10 mm glucose). Fura-2/AM and Pluronic (Molecular Probes) were added to the HBMEC monolayer in equal proportions at 37 °C for active uptake of Fura-2/AM by the cells (
      • Pallone T.L.
      • Silldorff E.P.
      • Cheung J.Y.
      ). The cells were then rinsed with Ringer's solution containing 1 mm calcium and maintained in the same medium. The cells were infected with 1 × 107 E. coli K1, and the change in the ratio of the intensities of fluorescence at 340 and 380 nm was monitored over a period of 40 min. After 40 min, ionomycin (1 μm) was added and cells were monitored over a period of 1 h. Fura-2 experiments were performed on a Nikon Instruments (Melville, NY) Diaphot TMD 300 inverted microscope, using a Nikon Fluor 40×/1.3 NA Ph4DL oil immersion objective lens. A Hamamatsu Corp. (Bridgewater, NJ) ORCA-100 (C4742-95-12NR) 12-bit digital camera was operated in 4 × 4 binning mode, with typical exposure times of 100 ms/channel. The microscope was equipped with a Ludl Electronics Products Ltd. (Hawthorne, NY) Mac2000 XYZ stage and focus controller. The imaging rig was controlled by MetaMorph 4.5 (Universal Imaging Corp., Downingtown, PA).

      RESULTS

      Inhibition of PLC-γ Blocks OmpA+ E. coli Invasion of HBMEC—Our previous studies demonstrated that PKC-α plays an important role in the cytoskeletal rearrangements observed during OmpA+ E. coli invasion of HBMEC (
      • Sukumaran S.K.
      • Prasadarao N.V.
      ). It has been well established that the activation of PKC-α is regulated by PLC-γ via mobilization of calcium and production of DAG. In this context, we decided to test the role of PLC-γ in bacterial invasion of HBMEC using specific inhibitors of PLC (
      • Xie Z.Z.
      • Bikle D.D.
      ). OmpA+ E. coli invasion of HBMEC was significantly blocked in a dose-dependent manner when the cells were pretreated with a cell permeable PLC inhibitory compound, U-73122. This result was in direct contrast to that obtained using a control inactive analogue compound, U-73343. At 2 μm, U-73122 blocked the invasion by more than 50% and a concentration of 4 μm caused >80% inhibition of invasion when compared with the invasion of U-73343-treated cells (10,535 + 1,200 cfu/well with U-73343 versus 2,100 + 650 cfu/well with U-73122, p < 0.001) (Fig. 1). The extent of binding of the bacteria to HBMEC treated with either inhibitor remained the same. The inhibitors were neither cytotoxic nor exerted any anti-bacterial effect. These results suggest that E. coli invasion into HBMEC requires the activity of PLC.
      Figure thumbnail gr1
      Fig. 1Inhibition of E. coli invasion by PLC inhibitors. HBMEC monolayers were treated with various concentrations of either U-73122 or U-73343 for 30 min prior to infection with OmpA+ E. coli. Total cell associated (binding) and intracellular (invasion) bacteria were determined as described under “Experimental Procedures.” OmpA- E. coli was used as a negative control for invasion. The data represent the mean ± S.D. values from three different experiments carried out in triplicate and expressed as relative invasion, taking OmpA+ E. coli binding or invasion of HBMEC as 100%.
      E. coli Induces the Phosphorylation on Tyr-783 and Hence the Activation of PLC-γ1—Having demonstrated the importance of PLC in E. coli invasion of HBMEC, we proceeded to examine the activation of PLC-γ and which isoform of PLC-γ was involved. It is widely reported that phosphorylation at tyrosine 783 of PLC-γ1 and at tyrosines 753, 759, and 1217 of PLC-γ2 bring about their activation (
      • Rhee S.G.
      ). Thus, we used antibodies that specifically recognize the phosphorylated form of tyrosine 783 of PLC-γ1 and tyrosine 1217 of PLC-γ2 for immunoblotting. HBMEC were infected with either OmpA+ or OmpA- E. coli for varying periods of time, and the total cell lysates were immunoprecipitated with an anti-PLC-γ antibody (recognizes both PLC-γ isoforms). The immune complexes were subjected to Western blotting with phospho-PLC-γ1 antibodies. An increase in phosphorylation on Tyr-783 of PLC-γ1, but not PLC-γ2, was observed in the cell lysates of HBMEC infected with OmpA+ E. coli. This change was observed at 5 min of infection with peak activation at 15 min, followed by a slight decline at 30 min after infection (Fig. 2A). In contrast, no phosphorylation at tyrosine 1217 of PLC-γ2 (only antibody available to phospho-PLC-γ2 is to tyrosine 1217) was detected under the same conditions. To ensure no other change in phosphorylation on PLC-γ2 had been missed, total cell lysates were immunoprecipitated with anti-PLC-γ2 antibody followed by immunoblotting with anti-phosphotyrosine antibody (4G10). Despite the presence of significant amounts of PLC-γ2, no change in phosphorylation was observed after infecting with either the bacteria (Fig. 2B). In addition, no significant levels of phospho-PLC-γ1 were observed in OmpA- E. coli-treated HBMEC at any time point. These blots, when stripped and reprobed with anti-PLC-γ, indicated that equal amounts of proteins were loaded in each lane. Densitometric scanning of the bands in these blots (from Fig. 2A) showed that a 10-fold increase in phosphorylation of PLC-γ1 by 15 min after infection (Fig. 2C). The densities of the bands were linear up to 100,000 units according to the calibration curve obtained by scanning an immunoblot containing increasing concentrations of actin. These results suggest that E. coli interaction with HBMEC induces the phosphorylation of PLC-γ1 at Tyr-783, thereby activating it.
      Figure thumbnail gr2
      Fig. 2Phosphorylation of PLC-γ1 in HBMEC infected with OmpA+ E. coli. HBMEC monolayers were infected either with OmpA+ E. coli or OmpA- E. coli for varying periods of time. Either total cell lysates (A, B, and D) or membrane fractions (E) were prepared as described under “Experimental Procedures.” In some experiments, the monolayers were pretreated with U-73343 or U-73122 prior to adding bacteria. Approximately 40 μg of protein was separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose. Immunoblotting was carried out with antibodies to PLC-γ, phospho-PLC-γ1, or phospho-PLC-γ2. The bands in the blot containing the total cell lysates were scanned on a densitometer, and the intensity of the bands was presented (C). In some experiments, 300 μg of protein was immunoprecipitated with anti-PLC-γ2 antibody followed by Western blotting with anti-phosphotyrosine and anti-PLC-γ2 antibodies. The blot was stripped and reprobed with anti-PLC-γ2 antibody (D). IP, immunoprecipitation; WB, Western blotting.
      To further confirm that the inhibition of E. coli invasion into U-73122-treated HBMEC was the result of inhibition of PLC-γ1 activity, cell lysates of OmpA+ E. coli-infected HBMEC pretreated with either U-73122 or its inactive analog U-73343 were analyzed by Western blotting. The results demonstrate that the pattern of phosphorylation of PLC-γ1 in the U-73343-treated cells is similar to that of the pattern observed in untreated control cells, but not in the U-73122-treated HBMEC (Fig. 2D).
      As mentioned, the activation of PLC-γ1 includes its recruitment to the cell membrane for PIP2 hydrolyzing activity. Therefore, membrane fractions of HBMEC infected with either OmpA+ or OmpA- E. coli were prepared and subjected to Western blot analyses with anti-PLC-γ and anti-phospho-PLC-γ1 antibodies. As shown in Fig. 2E, HBMEC membranes infected with OmpA+ E. coli exhibited significant amounts of PLC-γ with a concomitant increase in the levels of phosphorylation of PLC-γ1. In contrast, membrane proteins from HBMEC infected with OmpA- E. coli contained neither PLC-γ nor phospho-PLC-γ1. To further confirm the recruitment of phospho-PLC-γ1 to the HBMEC membrane, we analyzed the OmpA+ and OmpA- E. coli-infected HBMEC by immunocyto-chemistry using anti-phospho-PLC-γ1 antibody. In addition, the cells were stained for actin with rhodamine phalloidin to examine the spatial relationship of actin rearrangements with phospho-PLC-γ1. As shown in Fig. 3, control uninfected HBMEC showed actin stress fibers throughout the cell, whereas the staining of phospho-PLC-γ1 was punctate and very weak (panels A-D). After infection with OmpA+ E. coli, actin was rearranged to condense beneath few groups of bacteria (Fig. 3, E-H). The bacteria, which did not induce any condensation of actin, might be in the process of adherence. These results are in agreement with our previous studies, which showed that OmpA+ E. coli induces actin nucleation beneath the bacterial entry site. The same monolayers, when stained for phospho-PLC-γ1, showed intense staining beneath groups of bacteria in OmpA+ E. coli-infected HBMEC (Fig. 3G). In addition, the phospho-PLC-γ1 co-localized at the actin condensation sites. In contrast, neither actin condensation nor phospho-PLC-γ1 staining was observed in HBMEC infected with OmpA- E. coli (Fig. 3, I-L). Interestingly, OmpA+ E. coli infection also mobilized some phospho-PLC-γ1 to the nucleus (Fig. 3G). Taken together, these data indicate that, during E. coli invasion, activated PLC-γ1 is recruited to the membrane at bacterial attachment sites.
      Figure thumbnail gr3
      Fig. 3Condensation of phospho-PLC-γ1 at the sites of OmpA+ E. coli attachment in infected HBMEC. HBMEC were seeded in 8-well chamber slides and cultured until confluent. The monolayers were then infected with either OmpA+ or OmpA- E. coli for 15 min, followed by washing three times with RPMI 1640. The monolayers were then fixed and incubated with rhodamine phalloidin (B, F, and J) and anti-phospho-PLC-γ1 antibody (C, G, and K). The antibody was probed with FITC-conjugated secondary antibody. Cell morphology, along with bacteria, was viewed under transmitted light using a blue filter (A, E, and I). Actin and phospho-PLC-γ1 were observed using a fluorescent light source. Overlay images were presented to show the co-localization of actin and phospho-PLC-γ1 (D, H, and L). Arrows indicate the positions of the bacteria and the corresponding accumulation of actin and phospho-PLC-γ1.
      Overexpression of a Dominant Negative Form of PLC-γ Inhibits E. coli Invasion of HBMEC—To further confirm the role of PLC-γ1 in E. coli invasion, HBMEC were transfected with a mammalian expression vector encoding the PLC-z fragment, which is a dominant negative form of PLC-γ consisting of the SH2 and SH3 domains and an inhibitory domain. PLC-z/HBMEC transfectants showed significant expression of PLC-z, a 46-kDa protein by Western blot analysis of the cell lysates of the transfectants when compared with either control or vector alone transfected cells (Fig. 4A). Invasion assays performed using PLC-z/HBMEC indicated that inhibition of PLC-γ activity significantly abolished the invasion of E. coli into the endothelial cells (1,575 + 250 cfu/well for PLC-z/HBMEC versus 9,300 + 1,050 cfu/well for vector/HBMEC, p < 0.001) (Fig. 4B). This difference in the invasion was not the result of inefficient binding of the bacteria to the transfectants, as both the transfectants showed similar levels of total cell-associated bacteria. These results confirm our inhibitor studies and demonstrate that PLC-γ plays an essential role in E. coli invasion of HBMEC.
      Figure thumbnail gr4
      Fig. 4Overexpression of a dominant negative form of PLC-γ blocks the invasion of E. coli into HBMEC because of inhibition of phosphorylation of PLC-γ1. A, total cell lysates of non-transfected HBMEC, HBMEC transfected with vector alone (V/HBMEC), or HBMEC transfected with PLC-z-containing plasmid (PLC-z/H) were separated on a SDS-polyacrylamide gel and subjected to immunoblotting with anti-PLC-γ antibody. The dominant negative form of PLC-γ, PLC-z, is a 46-kDa protein. B, confluent monolayers of control HBMEC, vector/HBMEC, and PLC-z/HBMEC were used for E. coli invasion assays. Intracellular bacteria (invasion) and total cell-associated bacteria (binding) were assayed as described under “Experimental Procedures.” Experiments were carried out at least three times in triplicate, and the data are presented as percentage of invasion, taking invasion of control HBMEC as 100%. The bars represent mean ± S.D. C, total cell lysates of vector/HBMEC and PLC-z/HBMEC infected with OmpA+ E. coli for varying periods of time were subjected to Western blotting with anti-PLC-γ antibody. The blot was stripped and reprobed with anti-phospho-PLC-γ1 antibody. D, membrane fractions were prepared from the total cell lysates of vector/HBMEC and PLC-z/HBMEC infected with OmpA+ E. coli for varying periods of time and subjected to Western blotting as described in C.
      The activation of PLC-γ1 was assessed in PLC-z/HBMEC after infecting with OmpA+ E. coli. No phospho-PLC-γ1 was detected in the total cell lysates of PLC-z/HBMEC infected with OmpA+ E. coli, whereas the vector/HBMEC showed the presence of phosphorylated PLC-γ1 from 5 to 30 min after infection (Fig. 4C). The blot, when reprobed with PLC-γ antibody, showed the presence of equal quantities of protein in each lane. The PLC-z/HBMEC lysates revealed both wild type PLC-γ and PLC-z fragment. The membrane fractions of the same PLC-z/HBMEC, when probed with anti-PLC-γ1 antibody, revealed the presence of PLC-z fragment, which did not react to anti-phospho-PLC-γ1 antibody, indicating that the dominant negative fragment is not phosphorylated at the membrane. In contrast, control cell membranes showed PLC-γ1 in phosphorylated form. These results suggest that PLC-z fragment probably recruits to the membrane because of the SH2 interaction with PIP3 but could serve as a substrate for phosphorylation. In addition, the overexpression of PLC-z fragment blocks the recruitment of native PLC-γ1 and thereby its activation.
      To investigate whether the overexpression of PLC-z in HBMEC had any effect on the accumulation of phospho-PLC-γ1 and actin in E. coli invasion, we also examined the PLC-z/HBMEC monolayers infected with OmpA+ E. coli by immunocytochemistry. Despite adherence of OmpA+ E. coli, these cells showed no accumulation of either actin or phospho-PLC-γ1 at the bacterial engulfment site (Fig. 5, E-H) even after longer times of incubation (data not shown). In contrast, the vector/HBMEC showed both actin and phospho-PLC-γ1 accumulation beneath the bacteria (Fig. 5, A-D). These results suggest that PLC-z overexpression blocks the recruitment of native PLC-γ1 to membranes and are in good agreement with the immunoblotting studies.
      Figure thumbnail gr5
      Fig. 5Inhibition of phospho-PLC-γ1 and actin condensation beneath OmpA+ E. coli in HBMEC overexpressing dominant negative forms of PLC-γ and PI3K. HBMEC monolayers transfected with either vector alone (A-D), PLC-z (E-H), a dominant negative p85 subunit (I-L), or a dominant negative form of PKC-α (M-P) were infected with OmpA+ E. coli for 15-20 min. The monolayers were then washed and stained with rhodamine phalloidin (B, F, J, and N) or incubated with anti-phospho-PLC-γ1 antibody (C, G, K, and O). The phospho-PLC-γ1 antibody was probed with FITC-conjugated secondary antibody. The morphology of the monolayers along with the bacteria was viewed under transmitted light using a blue filter (A, E, I, and M) and the actin and phospho-PLC-γ1 with a fluorescence light. Overlay pictures were also included to show the co-localization of actin and phospho-PLC-γ1. Arrows indicate the positions of the bacteria and the corresponding presence or absence of either actin or phospho-PLC-γ1.
      Activation of PLC-γ1 in HBMEC Overexpressing a Dominant Negative Form of PKC-α, but Not in Cells Overexpressing a Dominant Negative Form of PI3K—Because our earlier studies showed that both PI3K and PKC-α activation are required for E. coli invasion (
      • Reddy M.A.
      • Prasadarao N.V.
      • Wass C.A.
      • Kim K.S.
      ,
      • Sukumaran S.K.
      • Prasadarao N.V.
      ), the activation of these molecules was assessed in PLC-z/HBMEC. PI3K activation was monitored by assessing the phosphorylation of Akt, a well established downstream substrate, by Western blot analysis of total cell lysates. As shown in Fig. 6A, the phosphorylation of Akt was not affected in PLC-z/HBMEC, when compared with vector/HBMEC in which the phosphorylation of Akt peaked between 10 and 15 min after infection. The phosphorylation pattern of Akt was similar to that of the phospho-Akt pattern observed in HBMEC infected with OmpA+ E. coli as previously reported. The activity of PKC-α was assessed using a non-radioactive PepTag assay. Interestingly, the PKC-α activation was completely abolished in PLC-z/HBMEC at all time points, whereas vector/HBMEC showed significant activation by 15 min (Fig. 6B). These results suggest that PLC-γ1 activation is downstream of PI3K and upstream of PKC-α.
      Figure thumbnail gr6
      Fig. 6Inhibition of PKC-α and PLC-γ1 activities in HBMEC overexpressing dominant negative PLC-z fragment and p85 subunit of PI3K, respectively upon infection with OmpA+ E. coli. The PLC-z/HBMEC were infected with OmpA+ E. coli for varying periods of time, and total cell lysates were prepared for either immunoblotting with anti-Akt antibody (A) or the PepTag assay (B). The blot was stripped and reprobed with anti-phospho-Akt antibody. Total cell lysates of HBMEC transfected with dominant negative forms of either p85 or PKC-α were subjected to immunoblotting with anti-PLC-γ antibody (C). The blot was stripped and reprobed with anti-phospho-PLC-γ1 antibody. HBMEC transfected with vector alone were used as a control.
      Previous studies have shown that overexpression of either a mutated form of the p85 subunit of PI3K or PKC/CAT-KR for PKC-α, which both act as dominant negative (DN) forms, significantly blocked the OmpA+ E. coli invasion of HBMEC (
      • Reddy M.A.
      • Prasadarao N.V.
      • Wass C.A.
      • Kim K.S.
      ,
      • Sukumaran S.K.
      • Prasadarao N.V.
      ). Therefore, we further investigated to confirm these results by examining the PLC-γ1 activation in HBMEC overexpressing these dominant negative forms. The total cell lysates of DN-p85/HBMEC and DN-PKC/HBMEC infected with OmpA+ E. coli were subjected to Western analysis with both anti-PLC-γ1 and anti-phospho-PLC-γ1 antibody (Fig. 6C). The anti-PLC-γ antibody immunoblot showed the presence of equal amounts of PLC-γ in all cell lysates, whereas phospho-PLC-γ1 was observed only DN-PKC/HBMEC, whereas no phosphorylation of PLC-γ1 was observed in DN-p85/HBMEC. These data support our findings with the PLC-z fragment overexpression that PKC-α activation induced by OmpA+ E. coli is downstream of PLC-γ1 activation.
      To further analyze the distribution of phospho-PLC-γ1 in these transfectants infected with OmpA+ E. coli, immunocytochemistry was carried out. As shown in Fig. 5, the vector/HBMEC, when infected with OmpA+ E. coli, showed accumulation of phospho-PLC-γ1 at the site of bacterial engulfment. Staining with rhodamine phalloidin of this same monolayer indicated that actin was present along with phospho-PLC-γ1, as seen in the non-transected HBMEC (Fig. 5, A-D). In contrast, DN-p85/HBMEC revealed accumulation neither of actin nor phospho-PLC-γ1 beneath the bacteria (Fig. 5, I-L), further supporting our conclusion that PI3K activity is an event upstream of PLC-γ1 activation. Interestingly, DN-PKC/HBMEC infected with OmpA+ E. coli showed localization of phospho-PLC-γ1 similar to that observed in control cells, beneath the E. coli entry point (Fig. 5, M-P). However, no signs of actin condensation were observed in these cells. These experiments suggest that PI3K activity may be necessary for the migration of PLC-γ1 to the membrane, but that other signaling events downstream of PKC-α are needed for actin rearrangements, which are important for E. coli invasion.
      Phosphatidylinositol 1,4,5-Trisphosphate (PIP3) Condensation Is Associated with OmpA+ E. coli Invasion—The activation of PLC-γ1 results in the generation of two intracellular messengers, DAG and IP3 from PIP2, which promote the activation of PKC and the release of Ca2+ from intracellular stores, respectively. PIP2 is a precursor not only of IP3 and DAG but also of PIP3, which is produced by the action of PI3K. The PH domains of PLC-γ1 and Btk have great affinity for PIP3, which recruits or firmly docks PLC-γ1 to the membrane close to its substrate, PIP2. Therefore, to examine the distribution of these two phosphoinositides (PIP2 and PIP3), we transfected HBMEC with PH-GFP fusion constructs that specifically bind these phosphoinositides. PH-GFP derived from PKC-δ binds PIP2 and IP3, whereas PH-GFP based on Btk specifically interacts with PIP3. PHPLC-δ-GFP expression was mostly observed in plasma membrane in uninfected HBMEC (Fig. 7, A and B), although a small amount of fluorescence was always present in the cytosol and nucleus. Upon infection with OmpA+ E. coli, the fluorescence at the cell membrane was reduced (E and F). However, fluorescence intensity was increased in the cytoplasm, possibly because of increased binding of PHPLC-δ-GFP to IP3. Interestingly, the plasma membrane localization of PHPLC-δ-GFP was decreased only near the areas of bacterial adherence, indicating a spatial relationship. In contrast, these cells when infected with OmpA- E. coli revealed no such redistribution (I and J). The PHBtk-GFP-transfected cells on the other hand showed the presence of fluorescence in the cytoplasm but not on the membranes (C and D). These PHBtk-GFP/HBMEC, upon infection with OmpA+ E. coli, showed the recruitment of fluorescence to the sites of E. coli attachment (G and H). No redistribution of the fluorescence was observed in OmpA- E. coli-infected cells.
      Figure thumbnail gr7
      Fig. 7Distribution of PIP2 and PIP3 in HBMEC infected with OmpA+ E. coli. HBMEC were transfected with mammalian expression vectors containing either PHPLC-δ-GFP (A, E, I, and M) or PHBtk-GFP (C, G, K, and O), and the monolayers were infected with either OmpA+ E. coli (E-H) or OmpA- E. coli (I-L). In some experiments, the transfected cells were pre-treated with U-73122 prior to infection with OmpA+ E. coli (M-P). The arrows indicate the location of bacteria and either loss or condensation of fluorescence.
      To further investigate whether PLC-γ1 is downstream of PI3K, the PH-GFP-transfected cells were pretreated with U-73122, U-73343, or wortmannin prior to E. coli infection. The PHPLC-δ-GFP-expressing cells showed no decrease in fluorescence in the HBMEC membrane (Fig. 7, M and N), whereas PHBtk-GFP fluorescence did condense beneath the E. coli entry site in HBMEC pretreated with U-73122 (O and P). However, U-73343-treated cells behaved similarly to untreated cells, suggesting that PIP3 accumulation was not affected by inhibition of PLC-γ1 activity (data not shown). In contrast, neither PIP3 accumulation beneath the bacteria nor redistribution of membrane PIP2 was observed in PHBtk-GFP/HBMEC or PHPLC-δ-GFP/HBMEC, respectively when pretreated with wortmannin (similar to Fig. 7, I-L). These experiments suggest that blocking of PI3K activity abolished the conversion of PIP2 to PIP3 and the hydrolysis of PIP2 to its product by PLC-γ1. Thus, PI3K activity is upstream of PLC-γ1 and the lipid products of PI3K most likely serve as targets of the PH domain of PLC-γ1 in infected cells.
      Ca2+ Mobilization in HBMEC during E. coli Invasion Is PLC-γ-dependent—It is well established that PLC-γ hydrolyzes PIP2 to generated IP3 and DAG, which mediate the release of Ca2+ from intracellular stores and PKC activation, respectively. Our previous studies showed that E. coli induces PKC-α activation during the invasion of HBMEC. Therefore, we analyzed the changes in intracellular calcium during the E. coli invasion process. For these experiments HBMEC were loaded with the Fura-2/AM ester for 30 min, then washed with Ringer's solution, and treated with the bacteria. Changes in calcium levels were observed continuously using a fluorescence microscope equipped with image acquisition capacity at 15-s time intervals. The monolayers were imaged for 5 min, after which time either OmpA+ or OmpA- E. coli were added to the monolayers. In most experiments, the bacteria settled onto the monolayer within 15-20 min after infection. Representative pictures are shown for each experiment. The control cells showed a constant basal level fluorescence up to 40 min after infection at which time ionomycin (1 μm) was added (Fig. 8A). Ionomycin caused a sharp transient increase in Ca2+ mobilization, which is at ∼3 μm in concentration as calculated from a standard curve obtained using a calibration kit. In Fig. 8, an image of the monolayer at the time we calculated the Ca2+ intensity (peak) prior to adding ionomycin is included as an inset. The monolayers when infected with OmpA+ E. coli showed an increase in Ca2+ that lasted for at least 10 min before beginning to decline (Fig. 8B). The concentration of this elevated Ca2+ was ∼1-1.5 μm. In contrast, OmpA- E. coli infection showed no such elevation in Ca2+ levels (Fig. 8C), suggesting that OmpA expression in E. coli is necessary for the induction of Ca2+ mobilization. To further distinguish whether the increase in calcium levels is the result of intracellular Ca2+ release or the influx of Ca2+ from the medium, calcium measurements were also carried out in the absence of calcium in external medium (either no calcium or chelated with EGTA). The results showed that OmpA+ E. coli infection induced an increase of Ca2+ levels, up to 0.8-1.0 μm (data not shown). Ionomycin induced a very small rise in calcium in these cells.
      Figure thumbnail gr8
      Fig. 8Elevation of intracellular calcium in HBMEC infected with OmpA+ E. coli. HBMEC monolayers either non-transfected (A, B, and C), transfected with PLC-z (D), dominant negative p85 (E), or dominant negative PKC-α (F) were loaded with Fura 2/AM as described under “Experimental Procedures.” The monolayers were then infected with either OmpA+ E. coli (B, D, E, and F) or OmpA- E. coli (C) and monitored for the increase in intracellular calcium every 15 s with an automated fluorescence microscope. At 40 min after infection, ionomycin (1 μm) was added. An image of the monolayer at the time the peak intensity of Ca2+ was measured prior to adding ionomycin is included as an inset. The y axis represents the ratio of intensity of fluorescence at 340 and 380 nm.
      HBMEC transfected with PLC-z fragment did not show increased Ca2+ levels even after OmpA+ E. coli infection confirming that PLC-γ1 activity is required for calcium mobilization (Fig. 8D). In addition, we also examined the DN-p85/HBMEC and DN-PKC/HBMEC monolayers for their response to OmpA+ E. coli induced Ca2+ increase. The DN-p85/HBMEC exhibited no elevation in Ca2+ levels inside the cell (Fig. 8F), whereas Ca2+ levels in DN-PKC/HBMEC increased to 0.5-μm in concentration (Fig. 8G). The number of DN-PKC/HBMEC responding to OmpA+ E. coli infection was similar to that of non-transfected HBMEC, although the fluorescence intensity of the cells was slightly lower. These transfectants responded to ionomycin in a manner equal to that of control cells. Taken together, these studies indicate that PLC-γ1 is important for the elevation of Ca2+ in HBMEC in response to OmpA+ E. coli infection.

      DISCUSSION

      E. coli invasion of HBMEC induces gross cytoskeletal rearrangements involving accumulation of actin and myosin at the site of bacterial entry (
      • Prasadarao N.V.
      • Wass C.A.
      • Stins M.F.
      • Shimada H.
      • Kim K.S.
      ,
      • Rudrabhatla R.S.
      • Sukumaran S.K.
      • Bokoch G.M.
      • Prasadarao N.V.
      ). This mechanism of condensation of cytoskeletal elements is regulated by a series of signaling events triggered in host cell upon bacterial contact. The invasion E. coli is then transported in an endosome, probably formed by the fusion of several caveolae across the endothelial cell, thereby avoiding fusion with host cell lysosomes. We have previously shown that OmpA interaction with a gp96-like receptor, a surface glycoprotein on HBMEC, is crucial for transducing these signals from the cell surface to the cell signaling machinery (
      • Prasadarao N.V.
      ). Our studies also showed that both PI3K and PKC-α become activated and are recruited to the sites of bacterial attachment (
      • Reddy M.A.
      • Prasadarao N.V.
      • Wass C.A.
      • Kim K.S.
      ,
      • Sukumaran S.K.
      • Prasadarao N.V.
      ); however, the signaling cascades leading to activation of PI3K and PKC-α under these conditions have not been identified.
      In the present study, we have demonstrated that OmpA+ E. coli stimulates PLC-γ1 activity, resulting in the release of Ca2+ from intracellular stores, probably by generation of IP3. OmpA+ E. coli induced activation of PLC-γ1 involves phosphorylation on Tyr-783 of PLC-γ1, which results in 10-fold greater activity of the enzyme compared with the activity detected in HBMEC infected with OmpA- E. coli. Inhibition of PLC-γ1 phosphorylation by a PLC-γ-specific inhibitor, as shown by Western blot analysis, indicates that PLC-γ1 but not PLC-γ2 activation is necessary for E. coli invasion. Another meningitis causing bacterium, Listeria monocytogenes, has also been shown to activate PLC-γ1 via a bacterial protein, InlB (
      • Bierne H.
      • Dramsi S.
      • Gratacap M.P.
      • Randriamampita C.
      • Carpenter G.
      • Payrastre B.
      • Cossart P.
      ). Interestingly, these studies with InlB could not detect any tyrosine phosphorylation of PLC-γ1 but showed the transient formation of IP3 and the association of PLC-γ1 with other phosphotyrosyl proteins. Similar to E. coli K1, enteropathogenic E. coli also triggers PLC-γ1 activation by increasing its tyrosine phosphorylation, which requires the protein intimin and its interaction with a receptor on epithelial cells (
      • Kenny B.
      • Finlay B.B.
      ). Of note, our studies for the first time showed the accumulation of activated PLC-γ1 at the sites of bacterial attachment by immunocytochemistry. The presence of phospho-PLC-γ1 in the membranes of HBMEC infected with the OmpA+ E. coli further supported the notion that activated PLC-γ1 is recruited to the host cell membrane. OmpA- E. coli could not induce the activation of PLC-γ1 even after longer periods of infection, indicating that OmpA interaction with HBMEC is critical for downstream signaling.
      The requirement for PLC-γ1 in E. coli invasion of HBMEC was further demonstrated by blocking the PLC-γ1 activation by overexpressing a PLC-z fragment, which acts as a dominant negative molecule. The PLC-z fragment has been shown to block both PLC-γ1 and -γ2; however, lack of PLC-γ2 activation in E. coli-infected cells indicates that down-regulation of PLC-γ1 activity may be responsible for the inhibition of invasion. Results from immunoprecipitation studies confirm that no phosphorylation of endogenous PLC-γ1 occurs in PLC-z/HBMEC. It is assumed that the PLC-z fragment is recruited to the cell membrane because of its interaction with PIP3 via cSH2 domain; however, no previous study has shown its presence in the cell membrane. Thus, our observations are the first to report the presence of PLC-z in the HBMEC membrane. Recruitment of PLC-z also served to prevent the translocation of native PLC-γ1 to the membrane, thus inhibiting the production of IP3 as assessed by measurement of Ca2+ levels. PLC-γ activation has been proposed to be involved in actin rearrangements, as this enzyme modulates the levels of IP3 and Ca2+, two well known regulators of actin binding proteins. Furthermore, our previous studies have shown that actin accumulation at the site of bacterial adherence is necessary for E. coli invasion (
      • Prasadarao N.V.
      • Wass C.A.
      • Stins M.F.
      • Shimada H.
      • Kim K.S.
      ). Overexpression of the PLC-z fragment not only significantly reduced E. coli invasion, but completely abolished the actin condensation associated with E. coli, indicating that activation of PLC-γ1 is indeed required for bacterial uptake and is upstream of actin rearrangements. These results are in sharp contrast with the invasion process of Listeria, in which blocking of PLC-γ activity had no effect on invasion but appeared to be involved in post-internalization events (
      • Bierne H.
      • Dramsi S.
      • Gratacap M.P.
      • Randriamampita C.
      • Carpenter G.
      • Payrastre B.
      • Cossart P.
      ).
      Our previous studies have demonstrated that OmpA+ E. coli invasion of HBMEC is dependent on both PI3K and PKC-α activation. Inhibiting PLC-γ1 activity by PLC-z did not alter PI3K activity but abolished PKC-α activity to a significant extent, suggesting that activation of PLC-γ1 is required for PKC-α but not for PI3K activation. Demonstrating that the PLC-γ1 activation is not affected in DN-PKC-α/HBMEC, whereas no activation was observed in DN-p85/HBMEC upon infection, further supports these results. Interestingly, we still observed the accumulation of phospho-PLC-γ1 at the sites of E. coli entry in DN-PKC-α/HBMEC, although no actin condensation was observed. Thus, the lack of PKC-α activation in DN-PKC/HBMEC did not alter the recruitment of PLC-γ1 to the membrane but prevented actin rearrangements.
      The activation of PI3K leads to the formation of PIP3, which in turn modulates the PLC-γ1 activity. The PH domain and SH2 domain of PLC-γ1 that have high affinity to PIP3 presumably target the enzyme to its membrane substrates, leading to PLC-γ1-catalyzed hydrolysis of PIP2 to IP3 and DAG. We used the PH domains of PLC-δ specific to PIP2 and IP3, and Btk specific to PIP3, which are fused to GFP to visualize the distribution of these two phosphoinositides during E. coli invasion. Because PHPLCδ-GFP interacts with the inositol head group of PIP2, hydrolysis of lipid by PLC-γ1 is reflected by the release of the fluorescent probe from the plasma membrane. Such changes in localization were dramatically demonstrated when HBMEC overexpressing PHPLCδ-GFP protein was infected with OmpA+ E. coli, but not with OmpA- E. coli. In contrast, the PHBtk-GFP protein, which is generally present in the cytosol relocated to the sites of E. coli entry. The PIP2 localization in the cytosol of PHPLCδ-GFP expressing HBMEC could be the result of generation of significant quantities of IP3 by PLC-γ1 action, which also binds to PHPLC-δ-GFP. It is also possible that the resynthesis of PIP2 may be increased in the cell during the E. coli invasion process. Similar resynthesis of PIP2 was observed previously in NIH 3T3 cells expressing PHPLCδ-GFP in response to angiotensin II treatment (
      • Varnai P.
      • Balla T.
      ). Several other pathogens such as EPEC and Salmonella have been shown to require phosphoinositide metabolism for invasion into epithelial cells using biochemical methods, but not by direct visualization (
      • Foubister V.
      • Rosenshine I.
      • Finlay B.B.
      ,
      • Ruschkowski S.
      • Rosenshine I.
      • Finlay B.B.
      ).
      Activation of PLC-γ1 induces the production of IP3, which subsequently causes release of Ca2+ from intracellular pools. This event is reflected by the increase in intracellular Ca2+ levels in HBMEC infected with OmpA+ E. coli. The Ca2+ increase in infected cells was mostly caused by the mobilization of Ca2+ from intracellular stores, although the influx of Ca2+ from extracellular medium is also necessary. In agreement with this observation, we have previously shown that EGTA chelation of extracellular calcium significantly blocked the E. coli invasion. Similarly, Listeria infection of Hep2 cells induced the release of intracellular Ca2+ and influx of extracellular Ca2+. However, blocking of PLC-γ1 with the PLC inhibitor U-73122 had no effect on Listeria invasion (
      • Bierne H.
      • Dramsi S.
      • Gratacap M.P.
      • Randriamampita C.
      • Carpenter G.
      • Payrastre B.
      • Cossart P.
      ). In contrast, PLC-γ1 inhibition significantly blocked both E. coli invasion as well as the elevation of Ca2+ levels in HBMEC. Similarly, inhibition of PI3K activity also blocked the elevation of calcium levels, whereas blocking of PKC-α caused a 50% reduction in the rise of Ca2+ levels. Despite the presence of calcium elevation in DN-PKC/HBMEC, no invasion of E. coli was observed, suggesting that intact PKC-α activity is necessary to trigger the actin rearrangements for invasion. It is not clear at this point why DN-PKC/HBMEC show only a 50% rise in calcium when compared with controls despite normal activation of PLC-γ1. It is possible that overexpression of DN-PKC, which blocks the native PKC-α activity, may initiate feedback inhibition signals to PLC-γ1. It has been reported that the magnitude and/or the duration of intracellular calcium elevations differentially activate transcriptions factors (
      • Dolmetsch R.E.
      • Lewis R.S.
      • Goodnow C.C.
      • Healy J.I.
      ). Thus, the calcium elevation in DN-PKC/HBMEC may not be sufficient to modulate other factors required for E. coli invasion.
      In summary, we have demonstrated that PLC-γ1 is activated by E. coli invasion of HBMEC in an OmpA-dependent manner and is downstream from PI3K. The activated enzyme is condensed at the site of bacterial adherence probably to hydrolyze PIP2 to IP3 and DAG, resulting in elevation of Ca2+ levels and activation of PKC-α in HBMEC. Further studies are in progress to delineate how OmpA receptor, a gp96-like molecule, transduces signals to induce the tyrosine phosphorylation and activation of PLC-γ1.

      Acknowledgments

      We sincerely thank Drs. Allan Wells, Tamas Ballas, L. C. Cantley, and I. B. Weistein for providing constructs expressing PLC-z, PH-GFP, dominant negative p85, and dominant negative PKC-α, respectively. We also thank Drs. Scott Filler, Martine Torres, and Barbara Driscoll for critical reading of this manuscript. We thank Dr. Tom Coates for allowing us to use the time-lapsed microscope and Image Core facility at Childrens Hospital Los Angeles.

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