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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Abstract

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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Materials

Basic Protocol 1: Adhesion of EHEC to Mammalian Cells

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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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Figures

Videos

Literature Cited

Literature Cited
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