Pedestal Formation by Pathogenic Escherichia coli: A Model System for Studying Signal Transduction to the Actin Cytoskeleton
Dynamic rearrangement of the actin cytoskeleton in response to signal transduction plays a fundamental role in the regulation of cellular functions. Understanding actin dynamics therefore represents one of the challenges of modern cell biology. Several pathogens have evolved diverse strategies to trigger rearrangement of the host actin cytoskeleton to facilitate and enhance their infection processes. By manipulating the actin assembly machinery or signalling routes leading to its activation, pathogens can either block or induce phagocytosis, drive intracellular motility, and exploit their host cells in other ways. Analyses of host pathogen interactions have not only broadened our knowledge of how these pathogens cause disease, but have also emerged as model systems in the study of cellular actin dynamics (for examples, see Frischknecht and Way, 2001). The focus of this article is on the formation of cytoskeletal rearrangements called actin pedestals induced by the diarrhoeagenic extracellular bacterial pathogens enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) as systems to study signalling and actin assembly at the plasma membrane.
During infection of the intestinal mucosa, EPEC and EHEC induce specific, so-called attaching and effacing (A/E) lesions on intestinal epithelial cells (for review, see Nataro and Kaper, 1998). A/E lesions are characterized by a localized loss of microvilli and intimate adherence of bacteria to the cell surface followed by recruitment of the cellular actin assembly machinery to sites of bacterial attachment, resulting in the formation of actin-rich pseudopod-like structures termed pedestals to which the bacteria intimately adhere.
Importantly, the histopathological changes associated with the A/E phenotype in vivo can be mimicked in cell culture [(Knutton et al., 1987), see Fig. 1], which allows to define the molecular mechanisms employed by EPEC and EHEC to induce cytoskeletal rearrangements. The ability to form actin pedestals on cultured cells furthermore correlates with the ability of EPEC and EHEC to colonize the intestine and cause disease in human and other animal hosts (e.g., Donnenberg et al., 1993).
The genes necessary for A/E lesion formation in EPEC map to an about 35-kb chromosomal pathogenicity island, designated the locus of enterocyte effacement (LEE), which is highly conserved in EHEC. Although EPEC and EHEC produce highly similar lesions, EPEC in the small intestine and EHEC in the large intestine, the molecular mechanisms of pedestal formation employed by EPEC versus EHEC differ (for review, see Campellone and Leong, 2003).
Both pathogens translocate their own receptor, the translocated intimin receptor (Tir, EspE), which binds to the bacterial surface protein intimin, via a type III secretion system into the underlying host cell. EPEC Tir becomes tyrosine phosphorylated upon insertion into the host cell membrane and binding to intimin. This phosphorylation is critical for actin pedestal formation by EPEC (Kenny, 1999), as it allows recruitment of the cellular signalling adaptor protein Nck to bacterial attachment sites. Nck, in turn, triggers by a so far unknown mechanism recruitment and activation of NWASP. Both Nck and N-WASP are essential host proteins for pedestal formation induced by EPEC, as cells lacking either Nck or N-WASP are resistant to actin pedestal formation by EPEC (Gruenheid et al., 2001; Lommel et al., 2001) (for N-WASP, see Fig. 2). N-WASP is a member of the WASP/Scar family of cellular nucleation-promoting factors and has emerged as a central node protein that regulates actin polymerisation by activating the Arp2/3 complex, a main factor for nucleation of actin filaments in response to multiple upstream signals.
In contrast, EHEC Tir is not tyrosine phosphorylated and pedestals are formed independently of Nck. Despite that, EHEC-induced pedestal formation still depends on N-WASP function (Lommel et al., 2004) to promote Arp2/3 complex-mediated actin polymerisation. Thus, EPEC and EHEC have evolved different strategies to trigger cellular signalling routes leading to actin assembly, which converge in recruitment and activation of N-WASP to promote Arp2/3-mediated actin polymerisation.
The aim of this article is to outline a methodology for the analysis of actin pedestal formation by EPEC and EHEC using cultured cells as model systems to study regulatory mechanisms controlling actin assembly at the plasma membrane. This article describes protocols for the coculture of bacteria with cell lines and for basic immunofluorescence microscopy techniques that are used to examine bacterial-host cell interactions in terms of bacterial attachment and effects on the actin cytoskeleton. These may be combined with ectopical expression of host cell proteins tagged with green fluorescent protein (GFP) for analysis of subcellular localisation and dynamic reorganisation of a protein during the infection process using digital fluorescence microscopy (for examples, see Fig. 2). A further advance to unravel the molecular mechanism of pedestal formation is the use of cell lines derived from knockout mice. Cell lines derived from such mice can be reconstituted by expressing wild-type or mutated proteins tagged with GFP, which facilitates the analysis of the contribution of specific protein domains to actin pedestal formation (for examples, see Fig. 2).
II. MATERIALS AND INSTRUMENTATION
A Cell Lines, Bacterial Pathogen Strains, and Culture Media Reagents
Epithelial cell lines used commonly for EPEC and EHEC infection experiments are HeLa and HEp-2 cells, as well as the intestinal epithelial cell lines T84 or Caco-2 (available from American Type Culture Collection). EPEC and EHEC will also adhere and induce the formation of actin pedestals on mouse embryonic fibroblast cell lines (MEFs), which are used routinely in our laboratory. Embryonic fibroblast cell lines offer the advantage that such cell lines can be established quite easily from conditional or conventional knockout mice, thus allowing the analysis of specific host proteins in actin pedestal formation induced by EPEC and EHEC.
2. Bacterial Pathogen Strains
Prototype enteropathogenic and enterohaemorrhagic E. coli strains used in analyses of the molecular mechanism of actin pedestal formation are enteropathogenic E. coli strain E2348/69 (O127:H6) (Levine et al., 1978), enterohaemorrhagic E. coli strain 86/24 (O157:H7) [isolated from an outbreak in Walla Walla, WA., U.S.A. (Griffin et al., 1988)], and enterohaemorrhagic E. coli strain EDL933 (O157:H7) (American Type Culture Collection #700927; for genome sequence information, see Perna et al., 2001).
3. Cell and Bacterial Culture Reagents
Dulbecco's modified eagle medium (DMEM), low glucose (Invitrogen Corp., GIBCO #31885-023), fetal bovine serum (Sigma-Aldrich #F 7524), L-glutamine (Invitrogen Corp., GIBCO #25030-024), penicillin/ streptomycin (Invitrogen Corp., GIBCO #15070-063), Luria-Bertani (LB) broth agar (e.g., BD #244520), LB broth (e.g., BD #244620), and HEPES (Sigma-Aldrich #H 3375), fibronectin (pure) (Roche #1051407).
B. Constructs and Reagents for Expression of GFP-Tagged Host Proteins
C. Additionally Needed Reagents and Plasticware
General equipment and plasticware for molecular biology and cell culture techniques.
2. Cell Culture and Immunofluorescence Microscopy
Twenty-four-well cell culture plates (e.g., Corning Inc. #3524), 12-mm round glass coverslips (e.g., Assistent #1001, thickness 0.17mm), absolute ethanol (Sigma-Aldrich #E 7023), HCl (37%) (Sigma-Aldrich #H 7020), lint-free absorptive paper [e.g., GB002, Schleicher& Schuell #10427736 (58 × 68cm) and #10485285 (22.2 × 22.2cm)], large square plastic dish (e.g., 24.5 × 24.5-cm polystyrene dish with lid, Sigma-Aldrich #Z37,165-3), Parafilm M (e.g., Fisher Scientific #917 00 02), forceps with curved fine tips (e.g., coverslip forceps Dumont #11251-33), NaCl (Sigma-Aldrich #S 7653), KCl (Sigma-Aldrich #P 1338), Na2HPO4 (Sigma- Aldrich #S 7907), KH2PO4 (Sigma-Aldrich #P 0662), paraformaldehyde (PFA), (Sigma-Aldrich #P 6148), NaOH (Merck/VWR International #109913), Triton X- 100 (Sigma-Aldrich #T 8532), normal goat serum (Invitrogen Inc.: GIBCO #16210-064), bovine serum albumin (BSA, Sigma-Aldrich #A 2153), goat antimouse Alexa Fluor 350- or Alexa Fluor 488-conjugated secondary antibodies (Molecular Probes #A-11045 and #A-11001), DAPI (e.g., Molecular Probes, #D-1306), fluorophore- coupled phalloidin [e.g., Alexa Fluor 594 phalloidin (red fluorescence, Molecular Probes #A- 12381)], glycerol (87%, analytical grade) (e.g., Merck/VWR International #104094), Mowiol 4-88 (Calbiochem #475904), Tris base (e.g., Trizma base, Sigma-Aldrich #T 1503), n-propyl gallate (SigmaAldrich #P 3130), and SuperFrost microscope glass slides (Fisher Scientific #9161161).
D. Instrumentation and Laboratory Equipment
Tabletop centrifuge (e.g., centrifuge 5414D, Eppendorf #5425 000.219), centrifuge equipped with rotor suitable for microtiter plates, and 15- and 50-ml polypropylene tubes (e.g., centrifuge 5810, Eppendorf #5810 000.017 with rotor A-4-81 and A-4-81-MTP).
2. Clean Benches and Cell Incubators
Clean bench and cell culture incubator suitable for work with EPEC and EHEC pathogens in accordance with respective national safety regulations.
Inverted microscope (e.g., Axiovert 135TV, Carl Zeiss Jena GmbH) equipped for epifluorescence microscopy with 40×/1.3NA and 100×/1.3 NA Plan-NEOFLUAR oil immersion objectives, 1.6 and 2.5 optovar magnification, electronic shutters (e.g., Uniblitz Electronic 35-mm shutter including driver Model VMMD-1, BFI Optilas) to allow for computercontrolled opening of the light paths, excitation and emission filters (Omega Optical Inc. or Chroma Technology Corp.) to enable three-colour epifluorescence, and mercury short arc lamp (Osram, HBO103W/2) for fluorescence light path.
4. Data Acquisition
Preferably a back-illuminated, cooled chargecoupled device (CCD) camera (e.g., Princeton Research Instruments TKB 1000x800, SN J019820; Controller SN J0198609) driven, for instance, by IPLab (Scanalytics Inc.) or Metamorph software (Universal Imaging Corporation).
A. Basic Protocol: Infection of Cell Monolayers with EPEC or EHEC
EPEC and EHEC pose a significant threat to human health, especially EHEC with its low infectious dose of ~10-100cfu. When working with EPEC and EHEC, always wear protective clothing and work under an appropriate clean bench in accordance with national safety regulations. Decontaminate all materials that have been in contact with the pathogen.
B. Alternative Protocol: EPEC and EHEC Infections of Transiently Transfected Cell Monolayers
For experiments involving ectopic expression of host cell molecules, e.g., tagged with GFP, designed to analyse their role in actin pedestal formation induced by EPEC or EHEC (see general comments, Section IV), follow the basic protocol with the exceptions that cells are already seeded on day 1, and day 2 will be required for transfection of host cell monolayers with plasmid DNA in addition to starting bacterial overnight cultures.
Good transfection efficiency is a prerequisite for analyses involving bacterial infections, as the number of cells that are both transfected and infected may be too low. You may want to consider testing different lots of fetal bovine serum, as we have detected great variability in transfection rates using different sera. Alternatively, you may have to resort to fluorescenceassisted cell sorting to enrich for the population expressing the GFP-tagged protein of interest. In this case, transfect cells in regular cell culture plates and seed onto coverslips after sorting.
C. Immunofluorescence Microscopy
See general comments about immunofluorescence microscopy analysis of EPEC- and EHEC-infected cells.
IV. GENERAL COMMENTS ON IMMUNOFLUORESCENT ANALYSIS OF EPEC-AND EHEC-INFECTED CELLS
It is important to consider that polyclonal antibodies are likely to exhibit cross-reactivity against E. coli, which will lead to unspecific labeling of EPEC or EHEC bacteria and may confound the analysis of the role of a specific protein in actin pedestal formation by immunofluorescence microscopy. Thus, it may be difficult to discern between recruitment of a protein to the very tips of pedestals or bacterial attachment sites and unspecific labeling of bacteria, especially when using low magnification. Always test secondary reagents for unspecific labeling of bacteria. Consider using affinity-purified reagents. For polyclonal primary reagents, try addition of 0.1% Triton-X100 to PBS during washing steps. Cross-reaction of polyclonal reagents is no problem when immunolabelling is only performed to detect bacteria, as is the case in the examples shown in Fig. 2, in which EPEC bacteria were detected with monoclonal antibodies against bacterial EspE (Deibel et al., 1998) and Alexa Fluor 350 fluorescent-labelled goat antimouse secondary antibodies, whereas F-actin was stained with fluorescent Alexa Fluor 594 phalloidin, and the host cell protein Nck was visualized with the help of an EGFP tag. There are reports in the literature about preabsorption of antibodies against fixed bacteria prior to immunolabelling, which when tried in our laboratory was of little success. Another possible solution to overcome the problem may be the use of monoclonal secondary reagents (e.g., rat antimouse), which are available from various suppliers (e.g., Zymed Laboratories), although so far not as conjugates with AlexaFluor dyes.
An alternative approach to detect the subcellular localisation of a given protein during EPEC- or EHECinduced pedestal formation is the use of the enhanced green fluorescent protein (EGFP) as a tag (see Fig. 2). It is important to keep in mind that tagging may significantly alter folding, activity, and proper subcellular localisation of the protein of interest. Thus, we recommend testing N- and C-terminal fusions and confirming that the localization pattern of an EGFP fusion protein equals the localization pattern of the untagged protein as detected by immunofluorescence microscopy. In addition, with the increasing availability and use of knockout cell lines, testing an EGFP-tagged protein for its ability to complement a null mutation may become a feasible possibility to ensure proper function of the EGFP-tagged protein.
We thank Florian Gunzer for the transmission electron micrograph of EHEC-infected piglet colon for Fig. 1, Klemens Rottner and Theresia Stradal for support and helpful discussions, and Brigitte Denker for excellent technical assistance. J.W. was supported by the DFG (SFB 621) and the Fonds der Chemischen Industrie.
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