Campellone KG, Rankin S, Pawson T, Kirschner MW, Tipper DJ, Leong JM.

R01-AI46454 NIAID NIH HHS , R01 AI046454 NIAID NIH HHS

	Figure 1. Intimin-expressing bacteria attach efficiently to mammalian cells expressing membrane-targeted Tir. (A) Full-length Tir (TirFL) and TirMC, a Tir derivative lacking the NH2-terminal cytoplasmic domain, are depicted. TirMC was directed to the plasma membrane by an NH2-terminal targeting sequence derived from a viral HN-protein. Arrows indicate the positions of HA-epitope tags and Y474. (B) HEp-2 cells transfected with plasmids encoding TirFL or TirMC were stained with an mAb to the HA epitope to identify Tir-expressing cells, with an mAb to visualize phosphotyrosine (Y-PO4), and with phalloidin to visualize F-actin. The inset shows several TirFL-expressing cells. (C) Mock-transfected HeLa cells, or cells expressing TirFL, TirMC, or TirMC(Y474F) (identified by anti-HA fluorescence) were challenged with a laboratory strain of E. coli carrying a plasmid-encoding intimin or a pUC19 vector control. Cell binding index, defined as the percentage of cells with at least five bacteria bound, was measured microscopically, and data represent the mean of two experiments.
	Figure 2. Tir expressed in mammalian cells allows a tir-deficient EPEC strain to form actin pedestals in the absence of the NH2-terminal cytoplasmic domain. (A) HEp-2 cells expressing TirFL were infected with either EPECΔtir (top panels) or EPECΔtirΔeae (bottom panels) and stained as described in the Fig. 1 legend. The arrow indicates positions of several bound bacteria. (B) HEp-2 cells expressing either TirMC (top panels) or a Y474F mutant of TirMC (bottom panels) were infected with EPECΔtir and stained as described in the Fig. 1 legend. The arrow indicates positions of several bound bacteria. (C) HeLa cells transfected with plasmids encoding TirFL or TirMC were infected with wild-type EPEC, EPECΔtir + pTir, or EPECΔtir. Cells with at least five bound bacteria were examined, and the percentage of Tir-expressing cells (identified by anti-HA fluorescence) or Tir-nonexpressing cells generating actin pedestals was quantitated. Data represent the means of triplicate samples of 200 cells each. Similar results were observed in independent experiments.
	Figure 3. Clustering of the COOH terminus of EPEC Tir beneath the plasma membrane is sufficient to trigger actin pedestal formation. (A) HEp-2 cells expressing TirMC were challenged with a laboratory strain of E. coli harboring either an intimin-expressing plasmid (top panels) or a vector control (bottom panels). Tir and F-actin were visualized with an anti-HA antibody and phalloidin, respectively. The arrow indicates positions of bound bacteria. (B) HeLa cells expressing TirMC were treated with antibodies to either TirM, the extracellular domain of Tir (top panels), to the HA epitope (middle panels), or left untreated (bottom panels). Then, they were challenged with S. aureus particles that carry protein A on their surface (top panels) or anti–mouse IgG-coated S. aureus (middle and bottom panels). Tir and F-actin were visualized as described in A. S. aureus particles are visible when staining for TirMC because they bind the fluorescently labeled secondary antibody. The inset shows a magnification of several pedestals.
	Figure 4. Actin pedestal formation resulting from clustering of TirMC is quantitatively, kinetically, and morphologically similar to pedestal formation after EPEC infections. (A) TirMC-expressing HeLa cells were challenged with particles of the indicated type, and the percentage of bound particles generating actin pedestals was quantitated. Data represent the means ± SD of three experiments in which 50 cells were examined for each type of particle in each experiment. A range of 330–635 particles was counted per 50 cells. (B) HeLa cells were either transfected with a plasmid encoding TirMC or were primed with an EPEC strain that delivers TirFL and other Esps to host cells but does not express intimin. Cells were subsequently challenged with E. coli expressing intimin, fixed at various time points, and quantitated for pedestal formation as described in the Fig. 2 legend. Each data point represents the mean of two independent experiments in which 200 cells were examined. (C) The transfected or EPEC-primed HeLa cells described in B were examined at 10 or 60 min after challenge. Tir and F-actin were visualized with an anti-HA antibody and phalloidin, respectively. Arrows indicate examples of Tir localizations at the tips of pedestals.
	Figure 5. Clustering results in tyrosine phosphorylation of TirMC and recruitment of Nck, N-WASP, Arp2/3 complex, and α-actinin. (A) HeLa cells were either mock transfected and infected with wild-type EPEC (top panels) or were transfected with plasmids encoding TirMC or TirMC(Y474F) and challenged with E. coli expressing intimin (middle and bottom panels). Cells were stained with antibodies to detect phosphotyrosine (Y-PO4), Nck, N-WASP, Arp3, or α-actinin (all shown in green). Bacterial DNA was visualized by DAPI staining (blue). (B) HeLa cells were mock transfected and infected with EPECΔtir + pTir (left lane), or were transfected with a plasmid encoding TirMC and treated with particles coated with anti-TirM antibodies (right lane). HA-tagged Tir was immunoprecipitated from cell lysates and Western blotted for HA-Tir and phosphotyrosine.
	Figure 6. Clustering of TirMC does not effectively trigger actin pedestal formation in cells that lack Nck. (A) TirMC was expressed in MEFs that express Nck1 (Nck +/− cells; top panels) and MEFs that do not express either Nck1 or Nck2 (Nck −/− cells; bottom panels). Cells were treated with anti-TirM antibodies and S. aureus particles and visualized as described in the Fig. 3 legend. (B) Nck-proficient MEFs (top panels) and Nck-deficient MEFs (bottom panels) were either mock transfected and infected with EPECΔtir + pTir (left panels) or were transfected with a plasmid encoding TirMC and challenged with E. coli expressing intimin (right panels). Cells were stained with antibodies to detect phosphotyrosine (green) and with DAPI to visualize bacterial DNA (blue).
	Figure 7. A clustered Nck-binding peptide from EPEC Tir is sufficient to trigger actin assembly in vitro. (A) Latex beads were coated with 12aa(+PO4), a peptide that binds human Nck; 12aa, a nonphosphorylated peptide that lacks Nck-binding activity; or 7aa(+PO4), a peptide that binds Nck poorly (Campellone et al., 2002). EPEC Tir residues present within the depicted peptides are underlined. Peptide-coated beads were added to Xenopus egg extracts supplemented with rhodamine-actin and examined microscopically. (B) Xenopus egg extracts were supplemented with the peptides described in A. Biotinylated peptides and associated proteins were collected from extracts using streptavidin-labeled particles and subjected to anti-Nck immunoblot analysis (all lanes except Crude Extract). Crude Extract lane is equivalent to 1/20 of the amount of extract represented in the bead-associated lanes.
	Figure 8. Nck is critical for actin assembly initiated by Tir peptide 12aa(+PO4). Xenopus extracts were left untreated or were subjected to two rounds of immunodepletion with beads coated with either an anti-Nck antibody or with a rabbit IgG control antibody. Nck depletion was confirmed by immunoblot (not depicted). Recombinant GST-Nck was added to the Nck-depleted extract in the bottom panels. Extracts were supplemented with rhodamine actin, and beads coated with peptide 12aa(+PO4) were added.

Abe, Enteropathogenic Escherichia coli translocated intimin receptor, Tir, requires a specific chaperone for stable secretion. 1999, Pubmed

Abe, Enteropathogenic Escherichia coli translocated intimin receptor, Tir, requires a specific chaperone for stable secretion. 1999, Pubmed
Bladt, The murine Nck SH2/SH3 adaptors are important for the development of mesoderm-derived embryonic structures and for regulating the cellular actin network. 2003, Pubmed
Buday, The Nck family of adapter proteins: regulators of actin cytoskeleton. 2002, Pubmed
Campellone, A tyrosine-phosphorylated 12-amino-acid sequence of enteropathogenic Escherichia coli Tir binds the host adaptor protein Nck and is required for Nck localization to actin pedestals. 2002, Pubmed
Cantarelli, Talin, a host cell protein, interacts directly with the translocated intimin receptor, Tir, of enteropathogenic Escherichia coli, and is essential for pedestal formation. 2001, Pubmed
Celli, Enteropathogenic Escherichia coli (EPEC) attachment to epithelial cells: exploiting the host cell cytoskeleton from the outside. 2000, Pubmed
Crawford, The N-terminus of enteropathogenic Escherichia coli (EPEC) Tir mediates transport across bacterial and eukaryotic cell membranes. 2002, Pubmed
Deibel, EspE, a novel secreted protein of attaching and effacing bacteria, is directly translocated into infected host cells, where it appears as a tyrosine-phosphorylated 90 kDa protein. 1998, Pubmed
Donnenberg, Role of the eaeA gene in experimental enteropathogenic Escherichia coli infection. 1993, Pubmed
Ebel, Initial binding of Shiga toxin-producing Escherichia coli to host cells and subsequent induction of actin rearrangements depend on filamentous EspA-containing surface appendages. 1998, Pubmed
Elliott, The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69. 1998, Pubmed
Elliott, EspG, a novel type III system-secreted protein from enteropathogenic Escherichia coli with similarities to VirA of Shigella flexneri. 2001, Pubmed
Elliott, Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli. 1999, Pubmed
Foubister, The eaeB gene of enteropathogenic Escherichia coli is necessary for signal transduction in epithelial cells. 1994, Pubmed
Frankel, Intimin and the host cell--is it bound to end in Tir(s)? 2001, Pubmed
Frankel, Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements. 1998, Pubmed
Freeman, Interaction of the enteropathogenic Escherichia coli protein, translocated intimin receptor (Tir), with focal adhesion proteins. 2000, Pubmed
Frischknecht, Actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling. 1999, Pubmed
Goosney, Enteropathogenic E. coli translocated intimin receptor, Tir, interacts directly with alpha-actinin. 2000, Pubmed
Gruenheid, Enteropathogenic E. coli Tir binds Nck to initiate actin pedestal formation in host cells. 2001, Pubmed
Huang, Host focal adhesion protein domains that bind to the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC). 2002, Pubmed
Ide, Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli. 2001, Pubmed
Jerse, A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. 1990, Pubmed
Kenny, Enteropathogenic Escherichia coli (EPEC) Tir receptor molecule does not undergo full modification when introduced into host cells by EPEC-independent mechanisms. 2001, Pubmed
Kenny, Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells. 1997, Pubmed
Kenny, Targeting of an enteropathogenic Escherichia coli (EPEC) effector protein to host mitochondria. 2000, Pubmed
Kenny, Phosphorylation of tyrosine 474 of the enteropathogenic Escherichia coli (EPEC) Tir receptor molecule is essential for actin nucleating activity and is preceded by additional host modifications. 1999, Pubmed
Knutton, A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells. 1998, Pubmed
Kodama, The EspB protein of enterohaemorrhagic Escherichia coli interacts directly with alpha-catenin. 2002, Pubmed
Kresse, The EspD protein of enterohemorrhagic Escherichia coli is required for the formation of bacterial surface appendages and is incorporated in the cytoplasmic membranes of target cells. 1999, Pubmed
Liu, Point mutants of EHEC intimin that diminish Tir recognition and actin pedestal formation highlight a putative Tir binding pocket. 2002, Pubmed
Liu, The Tir-binding region of enterohaemorrhagic Escherichia coli intimin is sufficient to trigger actin condensation after bacterial-induced host cell signalling. 1999, Pubmed
Lommel, Actin pedestal formation by enteropathogenic Escherichia coli and intracellular motility of Shigella flexneri are abolished in N-WASP-defective cells. 2001, Pubmed
Ma, Corequirement of specific phosphoinositides and small GTP-binding protein Cdc42 in inducing actin assembly in Xenopus egg extracts. 1998, Pubmed , Xenbase
McDaniel, A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K-12. 1997, Pubmed
McNamara, Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function. 2001, Pubmed
Moreau, A complex of N-WASP and WIP integrates signalling cascades that lead to actin polymerization. 2000, Pubmed
Nataro, Diarrheagenic Escherichia coli. 1998, Pubmed
Rohatgi, Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway. 2001, Pubmed
Rohatgi, The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. 1999, Pubmed , Xenbase
Rosenshine, A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation. 1996, Pubmed
Sekiya, Supermolecular structure of the enteropathogenic Escherichia coli type III secretion system and its direct interaction with the EspA-sheath-like structure. 2001, Pubmed
Snapper, N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility. 2001, Pubmed
Taylor, Expression of the EspB protein of enteropathogenic Escherichia coli within HeLa cells affects stress fibers and cellular morphology. 1999, Pubmed
Tu, EspH, a new cytoskeleton-modulating effector of enterohaemorrhagic and enteropathogenic Escherichia coli. 2003, Pubmed
Wachter, Insertion of EspD into epithelial target cell membranes by infecting enteropathogenic Escherichia coli. 1999, Pubmed
Welch, Cellular control of actin nucleation. 2002, Pubmed
Wolff, Protein translocation into host epithelial cells by infecting enteropathogenic Escherichia coli. 1998, Pubmed