Interaction of Enterohemorrhagic Escherichia coli (EHEC) with Mammalian Cells: Cell Adhesion, Type III Secretion, and Actin Pedestal Formation

Pamela J. Savage1, Kenneth G. Campellone1, John M. Leong1

1 University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 5A.1
DOI:  10.1002/9780471729259.mc05a01s05
Online Posting Date:  June, 2007
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Infection by the food‐borne pathogen enterohemorragic Escherichia coli (EHEC) can lead to diarrhea, hemorrhagic colitis, and, in the most serious cases, renal failure. A critical step in colonization is the unusual interaction between EHEC and the intestinal epithelium. EHEC is able to adhere to mammalian cells, and then, by injecting bacterial proteins, or “effectors,” into the host cell via a type III secretion system, induces the formation of attaching and effacing (AE) lesions characterized by the accumulation of filamentous actin directly beneath bound bacteria. This unit describes methods to evaluate the ability of EHEC to adhere to cultured mammalian cells, to secrete type III effectors, and to promote the formation of actin “pedestals.” These methods can not only be used to evaluate the contribution of specific EHEC gene products to adherence, type III secretion, and mammalian cell signaling, but also facilitate the analysis of the eukaryotic pathways controlling fundamental cellular processes such as actin assembly.

Keywords: Escherichia coli; bacterial adherence; type III secretion; EHEC; actin assembly; attaching and effacing (AE) lesions; actin pedestals; Tir; EspFu

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Table of Contents

  • Basic Protocol 1: Adhesion of EHEC to Mammalian Cells
  • Basic Protocol 2: Fluorescent Staining of EHEC‐Induced Actin Pedestals
  • Support Protocol 1: Quantitation of Actin Pedestal Phenotype by Fluorescence Microscopy
  • Basic Protocol 3: Identification of Proteins Secreted by the EHEC Type III Secretion System
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Adhesion of EHEC to Mammalian Cells

  • HeLa cells (ATCC #CCL‐2)
  • HeLa cell growth medium (see recipe)
  • LB broth ( appendix 4A) containing appropriate antibiotic (Table 97.80.4717)
  • Enterohemorrhagic Escherichia coli (EHEC) strain of interest
  • DMEM/HEPES (see recipe) containing appropriate antibiotic
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • HeLa cell infection medium (see recipe)
  • 2.5% paraformaldehyde (see recipe)
  • PBS ( appendix 2A) containing 0.1% (v/v) Triton X‐100 (store at room temperature)
  • PBS ( appendix 2A) containing 1% (w/v) BSA (add 10 ml 10% w/v BSA to 90 ml PBS)
  • Anti‐0157 rabbit polyclonal antibody (Difco): resuspend lyophilized powder in 3 ml PBS ( appendix 2A), then dilute 1:500 (recommended) in PBS containing 1% BSA
  • 200 U/ml Alexa568‐phalloidin stock in methanol (Invitrogen)
  • 1 mg/ml 4′,6‐diamidino‐2‐phenyindole dilactate (DAPI) stock
  • 2 mg/ml goat anti‐rabbit‐Alexa488 (Invitrogen)
  • Mounting medium: 1:1 (v/v) PBS:glycerol or ProLong Gold Antifade Reagent (Invitrogen)
  • Clear nail polish
  • Sterile glass coverslips, 12 mm, circular
  • 24‐well tissue culture plates
  • Sterile forceps
  • 14‐ml snap‐cap culture tubes (e.g., Falcon)
  • Platform rocker
  • Centrifuge with plate carrier (e.g., Allegra 6 Benchtop Centrifuge; Beckman Coulter)
  • Glass microscope slides
  • Opaque slide box
  • Additional reagents and equipment for epifluorescence microscopy (UNIT 2A.1)

Basic Protocol 2: Fluorescent Staining of EHEC‐Induced Actin Pedestals

  • HeLa cell infection medium (see recipe), prewarmed
  • Desired primary antibody against host or bacterial protein of interest, diluted appropriately in PBS/1% BSA
  • 2 mg/ml Alexa488‐conjugated secondary antibody against species in which primary antibody was raised
  • Additional reagents and equipment for preparing EHEC‐infected HeLa cell monolayers ( protocol 1, steps 1 to 11) and quantitation of actin pedestal phenotype by fluorescence microscopy ( protocol 3)

Support Protocol 1: Quantitation of Actin Pedestal Phenotype by Fluorescence Microscopy

  • EHEC grown on HeLa cell monolayers and stained for actin pedestals ( protocol 2)
  • Additional reagents and equipment for epifluorescence microscopy (UNIT 2A.1)

Basic Protocol 3: Identification of Proteins Secreted by the EHEC Type III Secretion System

  • Enterohemorrhagic Escherichia coli (EHEC) strain(s) of interest and type III secretion–deficient mutant as control
  • Luria‐Bertani (LB) broth ( appendix 4A) containing appropriate antibiotics (Table 97.80.4717)
  • DMEM/HEPES (see recipe) or M9/glucose/bicarbonate medium (optional; see recipe)
  • 20% glycerol, sterile (see recipe)
  • 1× and 2× SDS sample buffer ( appendix 2A)
  • 100 mM phenylmethylsulfonyl fluoride (PMSF) stock solution
  • and 1 mg/ml pepstatin, leupeptin, aprotinin stock solutions (see recipe for protease inhibitor stock solutions)
  • 14‐ml snap‐cap culture tubes (Falcon)
  • Centrifuge (e.g., Allegra 6 Benchtop Centrifuge; Beckman Coulter)
  • 0.22‐µm sterile filters
  • Centrifugal filter concentrators with MWCO appropriate to the protein of interest (Amicon)
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, ), staining of proteins in gels (Sasse and Gallagher, ), and immunoblotting (Gallagher et al., )
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Literature Cited

Literature Cited
   Abe, A., Heczko, U., Hegele, R.G., and Brett Finlay, B. 1998. Two enteropathogenic Escherichia coli type III secreted proteins, EspA and EspB, are virulence factors. J. Exp. Med. 188:1907‐1916.
   Badea, L., Doughty, S., Nicholls, L., Sloan, J., Robins‐Browne, R.M., and Hartland, E.L. 2003. Contribution of Efa1/LifA to the adherence of enteropathogenic Escherichia coli to epithelial cells. Microb. Pathog. 34:205‐215.
   Campellone, K.G., Robbins, D., and Leong, J.M. 2004. EspFU is a translocated EHEC effector that interacts with Tir and N‐WASP and promotes Nck‐independent actin assembly. Dev. Cell. 7:217‐228.
   Campellone, K.G., Roe, A.J., Lobner‐Olesen, A., Murphy, K.C., Magoun, L., Brady, M.J., Donohue‐Rolfe, A., Tzipori, S., Gally, D.L., Leong, J.M., and Marinus, M.G. 2007. Increased adherence and actin pedestal formation by dam‐deficient enterohaemorrhagic Escherichia coli O157:H7. Mol. Microbiol. 63:1468‐1481.
   Charpentier, X., and Oswald, E. 2004. Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM‐1 beta‐lactamase as a new fluorescence‐based reporter. J. Bacteriol. 186:5486‐5495.
   Deng, W., Vallance, B.A., Li, Y., Puente, J.L., and Finlay, B.B. 2003. Citrobacter rodentium translocated intimin receptor (Tir) is an essential virulence factor needed for actin condensation, intestinal colonization and colonic hyperplasia in mice. Mol. Microbiol. 48:95‐115.
   Deng, W., Li, Y., Hardwidge, P.R., Frey, E.A., Pfuetzner, R.A., Lee, S., Gruenheid, S., Strynakda, N.C., Puente, J.L., and Finlay, B.B. 2005. Regulation of type III secretion hierarchy of translocators and effectors in attaching and effacing bacterial pathogens. Infect. Immun. 73:2135‐2146.
   DeVinney, R., Stein, M., Reinscheid, D., Abe, A., Ruschkowski, S., and Finlay, B.B. 1999. Enterohemorrhagic Escherichia coli O157:H7 produces Tir, which is translocated to the host cell membrane but is not tyrosine phosphorylated. Infect. Immun. 67:2389‐2398.
   Donnenberg, M.S., Tzipori, S., McKee, M.L., O'Brien, A.D., Alroy, J., and Kaper, J.B. 1993. The role of the eae gene of enterohemorrhagic Escherichia coli in intimate attachment in vitro and in a porcine model. J. Clin. Invest. 92:1418‐1424.
   Ebel, F., Podzadel, T., Rohde, M., Kresse, A.U., Kramer, S., Deibel, C., Guzman, C.A., and Chakraborty, T. 1998. Initial binding of Shiga toxin–producing Escherichia coli to host cells and subsequent induction of actin rearrangements depend on filamentous EspA‐containing surface appendages. Mol. Microbiol. 30:147‐161.
   Gallagher, S. R. 2006. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 75:10.2A.1‐10.2A.37.
   Gallagher, S. Winston, S.E., Fuller, S.A., and Hurrell, J.G.R. 2004. Immunoblotting and immunodetection. Curr. Protoc. Mol. Biol. 66:10.8.1‐10.8.24.
   Garmendia, J., Phillips, A.D., Carlier, M.F., Chong, Y., Schuller, S., Marches, O., Dahan, S., Oswald, E., Shaw, R.K., Knutton, S., and Frankel, G. 2004. TccP is an enterohaemorrhagic Escherichia coli O157:H7 type III effector protein that couples Tir to the actin‐cytoskeleton. Cell. Microbiol. 6:1167‐1183.
   Garmendia, J., Frankel, G., and Crepin, V.F. 2005. Enteropathogenic and enterohemorrhagic Escherichia coli infections: Translocation, translocation, translocation. Infect. Immun. 73:2573‐2585.
   Gruenheid, S., DeVinney, R., Bladt, F., Goosney, D., Gelkop, S., Gish, G.D., Pawson, T., and Finlay, B.B. 2001. Enteropathogenic E. coli Tir binds Nck to initiate actin pedestal formation in host cells. Nat. Cell. Biol. 3:856‐859.
   Jarvis, K.G., and Kaper, J.B. 1996. Secretion of extracellular proteins by enterohemorrhagic Escherichia coli via a putative type III secretion system. Infect. Immun. 64:4826‐4829.
   Jarvis, K.G., Giron, J.A., Jerse, A.E., McDaniel, T.K., Donnenberg, M.S., and Kaper, J.B. 1995. Enteropathogenic Escherichia coli contains a putative type III secretion system necessary for the export of proteins involved in attaching and effacing lesion formation. Proc. Natl. Acad. Sci. U.S.A. 92:7996‐8000.
   Kenny, B., Abe, A., Stein, M., and Finlay, B.B. 1997. Enteropathogenic Escherichia coli protein secretion is induced in response to conditions similar to those in the gastrointestinal tract. Infect. Immun. 65:2606‐2612.
   Knutton, S., Baldwin, T., Williams, P.H., and McNeish, A.S. 1989. Actin accumulation at sites of bacterial adhesion to tissue culture cells: Basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli. Infect. Immun. 57:1290‐1298.
   Levine, M.M., Nataro, J.P., Karch, H., Baldini, M.M., Kaper, J.B., Black, R.E., Clements, M.L., and O'Brien, A.D. 1985. The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor. J. Infect. Dis. 152:550‐559.
   Liu, H., Magoun, L., Luperchio, S., Schauer, D.B., and Leong, J.M. 1999. The Tir‐binding region of enterohaemorrhagic Escherichia coli intimin is sufficient to trigger actin condensation after bacterial‐induced host cell signalling. Mol. Microbiol. 34:67‐81.
   O'Connell, C.B., Creasey, E.A., Knutton, S., Elliott, S., Crowther, L.J., Luo, W., Albert, M.J., Kaper, J.B., Frankel, G., and Donnenberg, M.S. 2004. SepL, a protein required for enteropathogenic Escherichia coli type III translocation, interacts with secretion component SepD. Mol. Microbiol. 52:1613‐1625.
   Perna, N.T., Mayhew, G.F., Posfai, G., Elliott, S., Donnenberg, M.S., Kaper, J.B., and Blattner, F.R. 1998. Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7. Infect. Immun. 66:3810‐3817.
   Sasse, J. and Gallagher, S.R. 2003. Staining proteins in gels. Curr. Protoc. Mol. Biol. 63:10.6.1‐10.6.35.
   Sherman, P.M., and Soni, R. 1988. Adherence of Vero cytotoxin‐producing Escherichia coli of serotype O157:H7 to human epithelial cells in tissue culture: Role of outer membranes as bacterial adhesins. J. Med. Microbiol. 26:11‐17.
   Tatsuno, I., Horie, M., Abe, H., Miki, T., Makino, K., Shinagawa, H., Taguchi, H., Kamiya, S., Hayashi, T., and Sasakawa, C. 2001. toxB gene on pO157 of enterohemorrhagic Escherichia coli O157:H7 is required for full epithelial cell adherence phenotype. Infect. Immun. 69:6660‐6669.
   Torres, A.G., Giron, J.A., Perna, N.T., Burland, V., Blattner, F.R., Avelino‐Flores, F., and Kaper, J.B. 2002. Identification and characterization of lpfABCC'DE, a fimbrial operon of enterohemorrhagic Escherichia coli O157:H7. Infect. Immun. 70:5416‐5427.
   Wolff, C., Nisan, I., Hanski, E., Frankel, G., and Rosenshine, I. 1998. Protein translocation into host epithelial cells by infecting enteropathogenic Escherichia coli. Mol. Microbiol. 28:143‐155.
   Zhang, X., McDaniel, A.D., Wolf, L.E., Keusch, G.T., Waldor, M.K., and Acheson, D.W. 2000. Quinolone antibiotics induce Shiga toxin‐encoding bacteriophages, toxin production, and death in mice. J. Infect. Dis. 181:664‐670.
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