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Phosphatidylinositol 3-Kinase Activation and Interaction with Focal Adhesion Kinase in Escherichia coli K1 Invasion of Human Brain Microvascular Endothelial Cells*

Open AccessPublished:November 24, 2000DOI:https://doi.org/10.1074/jbc.M007382200
      Invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for successful crossing of the blood-brain barrier by Escherichia coli K1. We have previously demonstrated the requirement of cytoskeletal rearrangements and activation of focal adhesion kinase (FAK) in E. coli K1 invasion of human BMEC (HBMEC). The current study investigated the role of phosphatidylinositol 3-kinase (PI3K) activation and PI3K interaction with FAK in E. coli invasion of HBMEC. PI3K inhibitor LY294002 blocked E. coli K1 invasion of HBMEC in a dose-dependent manner, whereas an inactive analogue LY303511 had no such effect. In HBMEC, E. coli K1 increased phosphorylation of Akt, a downstream effector of PI3K, which was completely blocked by LY294002. In contrast, non-invasive E. coli failed to activate PI3K. Overexpression of PI3K mutants Δp85 and catalytically inactive p110 in HBMEC significantly inhibited both PI3K/Akt activation and E. coli K1 invasion of HBMEC. Stimulation of HBMEC with E. coli K1 increased PI3K association with FAK. Furthermore, PI3K/Akt activation was blocked in HBMEC-overexpressing FAK dominant-negative mutants (FRNK and Phe397FAK). These results demonstrated the involvement of PI3K signaling in E. coli K1 invasion of HBMEC and identified a novel role for PI3K interaction with FAK in the pathogenesis ofE. coli meningitis.
      BMEC
      brain microvascular endothelial cells
      HBMEC
      human BMEC
      FAK
      focal adhesion kinase
      FRNK
      FAK dominant-negative mutant
      PI3K
      phosphatidylinositol 3-kinase
      PH
      pleckstrin homology
      Akt
      protein kinase B
      cfu
      colony-forming units
      EGF
      epidermal growth factor
      E44
      spontaneous rifampin-resistant mutant of strain RS218 (018:K1:H7)
      GEF
      guanine nucleotide exchange factor
      In neonates, Escherichia coli is the most common Gram-negative bacterium that causes meningitis, a serious disease affecting the central nervous system. The most distressing aspect of neonatal Gram-negative meningitis is high morbidity and mortality despite advances in antimicrobial chemotherapy and supportive care. Increased understanding of the pathogenesis and pathophysiology of this disease can lead to improved outcome. Intravascular survival and penetration of the blood-brain barrier by circulating bacteria represent the most critical events in the development of bacterial meningitis (
      • Leib L.S.
      • Tauber M.G.
      ).
      Invasion of brain microvascular endothelial cells (BMEC)1 is a requirement forE. coli crossing of the blood-brain barrier, and it involves attachment of E. coli to BMEC through interaction of bacterial ligands with corresponding receptors present on BMEC cell surface (
      • Kim K.S.
      ). Several E. coli determinants (OmpA, IbeA, Ibe B, and YijP) involved in the invasion of BMEC have been identified (
      • Prasadarao N.V.
      • Wass C.A.
      • Weiser J.N.
      • Stins M.F.
      • Huang S.H.
      • Kim K.S.
      ,
      • Huang S.H.
      • Wass C.A.
      • Fu Q.
      • Prasadarao N.V.
      • Stins M.F.
      • Kim K.S.
      ,
      • Huang S.H.
      • Chen Y.H.
      • Fu Q.
      • Stins M.
      • Wang Y.
      • Wass C.
      • Kim K.S.
      ,
      • Wang Y.
      • Huang S.H.
      • Wass C.A.
      • Stins M.F.
      • Kim K.S.
      ). Receptors on the surface of BMEC for two of these E. coli determinants (OmpA and IbeA) have been characterized biochemically (
      • Prasadarao N.V.
      • Wass C.A.
      • Kim K.S.
      ,
      • Prasadarao N.V.
      • Wass C.A.
      • Huang S.H.
      • Kim K.S.
      ). We have recently demonstrated that E. coli invasion of BMEC occurs via a zipper-like mechanism and requires cytoskeletal rearrangements in the host cell (
      • Prasadarao N.V.
      • Wass C.A.
      • Stins M.F.
      • Shimada H.
      • Kim K.S.
      ). We have further characterized in BMEC that E. coli K1 induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, a cytoskeletal protein known to associate with FAK (
      • Reddy M.A.
      • Wass C.A.
      • Kim K.S.
      • Schlaepfer D.D.
      • Prasadarao N.V.
      ). Furthermore, using FAK dominant-negative mutants we have shown that FAK kinase activity and its autophosphorylation site tyrosine 397 (Tyr-397) are critical for E. coli K1 invasion of HBMEC (
      • Reddy M.A.
      • Wass C.A.
      • Kim K.S.
      • Schlaepfer D.D.
      • Prasadarao N.V.
      ). These results establish that FAK signaling is essential in E. coliK1 invasion of HBMEC.
      FAK is a 125-kDa non-receptor tyrosine kinase and plays an important role in the assembly of signaling complexes that regulate the organization of the cytoskeleton and modulate the function of growth factors. An amino- and a carboxyl-terminal domain of about 400 amino acids each flank the kinase domain of FAK. The amino-terminal domain contains an autophosphorylation site (Tyr-397), and the carboxyl-terminal domain contains focal adhesion targeting sequences needed for targeting to focal adhesions and binding sites for intracellular signaling molecules and cytoskeletal proteins (
      • Schlaepfer D.D.
      • Hauck C.R.
      • Sieg D.J.
      ). However, the signaling molecules that interact with FAK in E. coli K1 invasion of BMEC have not been identified. The autophosphorylation site Tyr-397 of FAK has been shown to bind to Src kinases and phosphatidylinositol 3-kinase (PI3K), and binding to one or both is required for FAK-mediated functions (
      • Xing Z.
      • Chen H.-C.
      • Nowlen J.K.
      • Taylor S.
      • Shalloway D.
      • Guan J.-L.
      ,
      • Cobb B.S.
      • Schaller M.D.
      • Leu T.-H.
      • Parsons J.T.
      ,
      • Chen H.-C.
      • Guan J.-L.
      ). Interestingly, pretreatment of HBMEC with Src kinase-specific inhibitor, PP1, did not affect E. coli K1 invasion of HBMEC (
      • Reddy M.A.
      • Wass C.A.
      • Kim K.S.
      • Schlaepfer D.D.
      • Prasadarao N.V.
      ). Furthermore, overexpression of Src kinase dominant-negative mutants did not blockE. coli K1 invasion of HBMEC,
      M. A. Reddy, N. V. Prasadarao, and K. S. Kim, unpublished results.
      2M. A. Reddy, N. V. Prasadarao, and K. S. Kim, unpublished results.
      suggesting an insignificant role for Src kinases.
      In the present study, we examined the role of PI3K in E. coli invasion of HBMEC. PI3K proteins are subdivided into three major groups, and group I PI3K has been identified as a downstream effector of both receptor and non-receptor tyrosine kinases (
      • Wymann M.P.
      • Pirola L.
      ). Group I PI3K is a heterodimer consisting of a regulatory subunit (p85) and a 110-kDa catalytic subunit (p110). Regulatory subunit p85 contains an SH3 domain and two SH2 domains that interact with various intracellular signaling molecules. Interaction of the SH2 domains of p85 subunit with phosphotyrosine residues on tyrosine kinases, including Src and FAK, results in PI3K activation. PI3K catalytic subunit p110 phosphorylates the d-3 position of the inositide ring of phosphatidylinositol, and its derivatives form the second messenger phosphatidylinositol 3,4,5-trisphosphate. Phosphatidylinositol 3,4,5-trisphosphate binding to pleckstrin homology (PH) domains of various cellular and cytoskeletal proteins mediates membrane recruitment of several kinases including, Akt (protein kinase B), PDK1, and PDK2. Akt is then activated by sequential phosphorylation on Thr-308 and Ser-473 by phosphoinositide-dependent protein kinases PDK1 and PDK2, respectively (
      • Downward J.
      ). PI3K signaling has been implicated in a variety of cellular processes, including survival, proliferation, migration, metabolic changes (18 and 19) andListeria monocytogenes invasion of epithelial cells (
      • Ireton K.
      • Payrastre B.
      • Chap H.
      • Ogawa W.
      • Sakaue H.
      • Kasuga M.
      • Cossart P.
      ). PI3K has been shown to act downstream of FAK in cell migration and survival (
      • Reiske H.R.
      • Kao S.-C.
      • Cary L.A.
      • Guan J.-L.
      • Lai J.-F.
      • Chen H.-C.
      ,
      • Sonoda Y.
      • Watanabe S.
      • Matsumoto Y.
      • Aizu-Yokota E.
      • Kasahara T.
      ,
      • Chan P.-C.
      • Lai J.-F.
      • Cheng C.-H.
      • Tang M.-J.
      • Cjiu C.-C.
      • Chen H.-C.
      ). In this paper, we report the activation of PI3K/Akt signaling in E. coli K1 invasion of HBMECs and demonstrate that PI3K interaction with FAK is required for the activation of PI3K/Akt signaling in this process.

      DISCUSSION

      Bacterial entry of mammalian cells involves a complex interplay of bacterial determinants with host cell receptors and is associated with activation of specific signaling pathways (
      • Kim K.S.
      ). Our previous studies have demonstrated that FAK plays a central role in E. coliK1 invasion of HBMEC (
      • Reddy M.A.
      • Wass C.A.
      • Kim K.S.
      • Schlaepfer D.D.
      • Prasadarao N.V.
      ). However, the mechanisms of FAK activation and signaling molecules that interact with FAK in this process have been not identified. In this paper, we report that PI3K activation and its interaction with FAK are required for E. coli K1 invasion of HBMEC. This was shown by blockade of both PI3K activation and E. coli invasion of HBMEC with specific PI3K inhibitor (LY294002) as well as using dominant-negative mutants of PI3K (Δp85 and p110ΔK) and FAK (FRNK and FAKY397F).
      The regulatory subunit p85 of PI3K contains an amino-terminal SH3 domain, a breakpoint cluster homology domain and two SH2 domains. These domains allow p85 to simultaneously interact with multiple intracellular signaling molecules (
      • Wymann M.P.
      • Pirola L.
      ). Thus, PI3K can recruit a variety of signaling molecules to the site of bacterial entry. There are several phosphoinositide binding proteins, which can be potential downstream effectors of PI3K, that can participate at several steps inE. coli invasion of HBMEC, including cytoskeletal rearrangements or the endosome formation. One of them is the Rho family of GTP binding proteins that regulates actin rearrangement. Rho family members cycle between the inactive GDP binding form and the active GTP binding form. Guanine nucleotide exchange factors (GEF) and GTPases regulate the cycling of Rho-like GTPases. GEFs mediate conversion of inactive GDP-bound form to active GTP-bound form. PI3K products can bind to pleckstrin homology domains in GEF and target them to plasma membrane to activate Rho family of GTPases (
      • Carpenter C.L.
      • Tolias K.F.
      • Couvillion A.C.
      • Hartwig J.H.
      ). We showed that the E. coli K1 mutant lacking cytotoxic necrotizing factor-1 failed to invade HBMEC (

      Wang, Y., Wass, C., and Kim, K. S. (2000) in Abstracts of the 100th American Society for Microbiology General Meeting, Los Angeles, May 21–25; Abstr. B108, p. 65.

      ), suggesting the involvement of Rho GTPases. Cytotoxic necrotizing factor-1 is known to activate Rho GTPases in mammalian cells (
      • Schmidt G.
      • Sehr P.
      • Wilm M.
      • Selzer J.
      • Mann M.
      • Aktories K.
      ,
      • Flatau G.
      • Lemichez E.
      • Gauthier M.
      • Chardin P.
      • Fiorentini C.
      • Boquet P.
      ). PI3K may be required for the regulation of Rho-like GTPases in HBMEC. Many cytoskeletal proteins involved in the regulation of actin polymerization bind to phosphoinositides through their pleckstrin homology (PH) domains,e.g. profilin and capping proteins bind to monomeric actin and prevent its polymerization. Binding of phosphoinositides to the PH domains of these proteins releases actin and promotes polymerization (
      • Schmidt A.
      • Hall M.N.
      ). Phosphoinositide-induced changes in the conformation of another cytoskeletal protein vinculin may also play a role in focal adhesion assembly by promoting cross-linking of talin to actin. Another mechanism for the PI3K involvement could be the activation of Akt, a Ser/Thr kinase, which is recruited to plasma membrane by binding to phosphoinositides through its PH domain. Evidence exists for the involvement of Akt in actin reorganization and migration of microvascular endothelial cells (
      • Morales-Ruiz M.
      • Fulton D.
      • Sowa G.
      • Languino L.R.
      • Fujio Y.
      • Wlash K.
      ). Upon entry into HBMEC, E. coli have been shown to be located in vacuoles and cross the HBMEC by transcytosis (
      • Prasadarao N.V.
      • Wass C.A.
      • Stins M.F.
      • Shimada H.
      • Kim K.S.
      ). This is another level where PI3K may be involved, because PI3K has been shown to participate in the recruitment of early endosome proteins as well as movement of the endosomes along the microtubules (
      • Nielsen E.
      • Severin F.
      • Backer J.M.
      • Hyman A.A.
      • Zerial M.
      ,
      • Gaullier J.M.
      • Simonsen A.
      • D'Arrigo A.
      • Bremnes B.
      • Stenmark H.
      • Aasland R.
      ). Studies are in progress to examine the role of PI3K in cytoskeleton rearrangements and trafficking of E. coli-containing vacuoles in HBMEC.
      Our recent results have demonstrated the cytoskeletal changes in HBMEC at the site of bacterial entry (
      • Prasadarao N.V.
      • Wass C.A.
      • Stins M.F.
      • Shimada H.
      • Kim K.S.
      ). These sites were similar to focal adhesions that are intracellularly associated with protein complexes consisting of cytoskeletal proteins and tyrosine kinase FAK (
      • Brigitte M.J.
      • Bubeck P.
      • Giegl K.
      • Kroemker M.
      • Moschner J.
      • Rothkegel M.
      • Rudiger M.
      • Schluter K.
      • Stanke G.
      • Winkler J.
      ). The role of FAK has been studied in many cellular responses associated with cytoskeleton rearrangement, including adhesion and migration (
      • Guan J.-L.
      • Chen H.-C.
      ). However, the mechanism of FAK activation and the proteins recruited by FAK in E. coli invasion of HBMEC have not been identified. The present study identified that PI3K interacts with FAK in HBMEC and its interaction is enhanced in HBMEC stimulated with E. coliK1, suggesting that FAK recruits PI3K to the site of bacterial entry. PI3K activation was abolished by FAK dominant-negative mutants FRNK and FAKY397F, indicating that FAK may be upstream of PI3K in E. coli K1 invasion of HBMEC. In contrast, tyrosine phosphorylation of FAK was not affected by PI3K inhibitors (data not shown), providing further support for this mechanism.
      Several E. coli K1 determinants involved in the HBMEC invasion have been identified, and some of these bacterial proteins induced cytoskeleton rearrangements in HBMEC (
      • Kim K.S.
      ). However, at present, the identity of E. coli K1 structures responsible for the activation of FAK and/or PI3K and the mechanism of activation in HBMEC are not yet defined. Of interest, uropathogenic E. coliinvasion of bladder epithelial cells was found to require host actin reorganization, FAK phosphorylation at Tyr-397 and PI3K activation, but not activation of Src-family tyrosine kinases (
      • Martinez J.J.
      • Mulvey M.A.
      • Schilling J.D.
      • Pinkner J.S.
      • Hultgren S.J.
      ). These events were mediated by FimH and correlated with the formation of complexes between FAK and PI3K. Thus, our findings of PI3K activation and PI3K/FAK interactions reported here with E. coli K1 invasion of HBMEC are similar to those observed with type 1 pilus E. coliinvasion of uroepithelial cells.
      We have previously demonstrated that other meningitis-causing bacteria,e.g. group B streptococcus (
      • Nizet V.
      • Kim K.S.
      • Stins M.
      • Jonas M.
      • Chi E.Y.
      • Nguyen D.
      • Rubens C.E.
      ), Citrobacter freundii (
      • Badger J.L.
      • Stins M.F.
      • Kim K.S.
      ), and L. monocytogenes (
      • Greiffenberg L.
      • Goebel W.
      • Kim K.S.
      • Weiglein I.
      • Rubert A.
      • Engelbrecht R.
      • Stins M.
      • Kuhn M.
      ), are able to invade HBMEC. PI3K involvement has been demonstrated in L. monocytogenes invasion of epithelial cells. However, PI3K involvement is independent of FAK in L. monocytogenesinvasion of epithelial cells (
      • Ireton K.
      • Payrastre B.
      • Cossart P.
      ). In contrast, our findings indicate the requirement of PI3K interaction with FAK in E. coli K1 invasion of HBMEC. Thus, the mechanism of E. coli K1 invasion of HBMEC is strikingly different from that of L. monocytogenes. This may reflect the differences in cell types, bacterial ligands, and mechanisms of diseases. It would be interesting to see whether PI3K/FAK interactions are also involved in the invasion of HBMEC by other meningitis-causing bacteria.

      Acknowledgments

      We thank Dr. M. F. Stins for help with culturing human brain microvascular endothelial cells.

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