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.
|FIGURE 1 Actin pedestal formation induced by EHEC and EPEC in vivo and on the surface of cultured
cells. (A) Transmission electron micrograph showing the characteristic attaching and effacing (A/E) lesion
formation of the enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strain 86/24 observed in piglet colon.
Note the intimate attachment, localized loss of microvilli, and formation of a raised, pedestal-like structure
beneath the bacterium that characterizes this lesion (courtesy of Florian Gunzer, Institute of Medical Microbiology,
Hannover Medical School, Germany). (B) Scanning electron micrograph of enteropathogenic E. coli (EPEC) O127:H6 strain E2348/69 (pseudocoloured in red) sitting on top of pedestals induced on the surface
of cultured routine embryonic fibroblast cells upon infection that resemble A/E lesions formed by EPEC in
vivo. (C) EHEC O157:H7 strain 86/24-induced actin pedestal formation as visualized by fluorescence actin
staining using AlexaFluor 594 phalloidin to specifically label F-actin (shown in red). EHEC bacteria shown
in green were detected with monoclonal antibodies against EspE (Deibel et al., 1998) and AlexaFluor 488-
conjugated goat antimouse secondary antibodies. Bars: 1 µm.
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.
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.
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
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
A Cell Lines, Bacterial Pathogen Strains, and
Culture Media Reagents
|FIGURE 2 EGFP-tagging and knockout cell lines as tools for
analysis of the molecular mechanism of actin pedestal formation
induced by EPEC. Cultured routine embryonic fibroblasts expressing
(+/+, A) or lacking N-WASP (-/-, B and C) were infected with
EPEC and examined by immunofluorescence microscopy. F-actin is
shown in red as detected by fluorescence actin staining with Alexa-
Fluor 594 phalloidin, bacteria are shown in blue as detected with
anti-EspE monoclonal antibodies in combination with AlexaFluor
350-conjugated goat antimouse secondary antibodies, and the host
proteins Nck2 (A and B) and N-WASP (C) expressed ectopically with
an EGFP tag are shown in green. Whereas EPEC induced the formation
of prominent pedestals in N-WASP-expressing cells (+/+,
A), they were unable to trigger actin accumulation on N-WASPdefective
cells (-/-, B). The bacterial ability to direct actin reorganisation
in N-WASP-defective fibroblasts was restored by providing
EGFP-tagged N-WASP by transient transfection (C). Together, this
clearly demonstrates that N-WASP is a host cell protein essential for
pedestal formation induced by EPEC. Recruitment of the host cell
signalling adaptor protein Nck2 is triggered by EPEC independently
of actin accumulation, as was revealed by ectopic expression of
EGFP-tagged Nck2 in either N-WASP-expressing (+/+, A) or NWASP-
defective cells (-/-, B). Bar: 2µm, (valid for A-C).
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
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
(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.
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
- Transfection reagent, e.g., FuGENE 6 (Roche #1 814
- A set of vectors for construction of EGFP fusions is
available from Clontech (BD Biosciences).
- EGFP-N-WASP: as described (Lommel et al., 2001)
- EGFP-Nck2: as described (Scaplehorn et al., 2002)
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), Na2
Aldrich #S 7907), KH2
(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
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
A. Basic Protocol: Infection of Cell
Monolayers with EPEC or EHEC
- Cell culture growth media: Cell culture growth
medium suitable for the propagation of the cell line
chosen. We use DMEM, low glucose supplemented
with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine,
and 50 U/ml each of penicillin and streptomycin
for propagation of our embryonic fibroblast
- Cell culture growth media for bacterial infection
experiments: For bacterial infection experiments, omit
penicillin and streptomycin from growth media starting
a day prior to infection.
- Standard bacterial cultures: Prepare LB broth and
LB agar plates according to standard protocols (e.g.,
Maniatis et al., 1982).
- Preactivating culture of EPEC: DMEM, low
glucose for culturing EPEC prior to infection of cell
- Preactivating culture of EHEC: DMEM, low
glucose, supplemented with 100mM HEPES, pH
7.4, for culturing EHEC prior to infection of cell
- Phosphate-buffered saline (PBS): 140mM NaCl, 2.7
mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4
- Fixative: Prepare a 4% solution of PFA in PBS, pH
7.4. Paraformaldehyde is very toxic, work in fumehood
when preparing stock, do not inhale, and wear
gloves. To prepare 100ml, add 4g PFA to 90ml PBS. In
order for the PFA to dissolve, heat the solution to
60-65°C with continuous stirring. If necessary, adjust
the pH to 7.4 by adding NaOH (be patient!). Do not
heat solution above 70°C, as PFA will degrade. Let cool
to room temperature, check the pH, and adjust with
PBS to full volume. Filter through paper filter to
remove insoluble aggregates and store in aliquots (e.g.,
15 and 50ml) at -20°C.
- Cell permeabilization: Fixative supplemented with
0.1% Triton X-100 just prior to use. Make up a 10%
stock solution of Triton X-100 in PBS and store at 4°C.
- Antibody diluent: 1% BSA in PBS. Prepare and
store in suitable aliquots (1-2ml) at -20°C.
- Blocking solution: 10% normal goat serum in
PBS. Prepare from frozen aliquots of serum just prior
- Antibody Mixtures: Dilute antibodies just prior to
use to appropriate working concentration in 1% BSA
in PBS. The ideal concentration will result in a strong
signal with no or little background staining and has to
be established experimentally for each new antibody.
To start, follow instructions given by the supplier.
When using a concentrated primary antibody, a 1:100
dilution resulting in about 10µg/ml should be a good
starting point for immunofluorescence microscopy.
Secondary antibodies conjugated to fluorophores are
available from numerous suppliers. In our hands, secondary
reagents coupled to Alexa Fluor dyes from
Molecular Probes have worked very well. Note,
however, that most polyclonal antisera will exhibit
unspecific cross-reaction with EPEC and EHEC bacteria.
A way to avoid problems associated with unspecific labelling of bacteria is to use monoclonal reagents
(see general comment in Section IV). We store our antibodies
in the dark at 4°C in a refrigerator. For longterm
storage, antibodies may be stored at -20 or -80°C. Follow the recommendations given by the supplier.
- Mounting medium: Weigh out 6g glycerol and
add 2.4 g Mowiol 4-88. Stir thoroughly. Add 6 ml aqua
dest and mix for several hours at room temperature.
Add 0.2ml 0.2M Tris-Cl, pH 8.5, and heat to 60°C for
10 min. Remove insoluble material by centrifugation at
6000g for 30min. Store in aliquots at -20°C. In order
to reduce photobleaching during fluorescence microscopy,
add 2.5-5mg/ml n-propyl gallate prior to use
(Giloh and Sedat, 1982).
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
- Pretreat 12-mm round glass coverslips as
follows: wash coverslips in a mixture of 60% absolute
ethanol and 40% concentrated HCl for 30 min and rinse
extensively with aqua dest (heating in a microwave
oven is helpful). To dry coverslips, spread on lint-free
absorptive paper. Sterilize for tissue culture either by
autoclaving on the dry cycle at 220°C or by exposing
to ultraviolet light for 45 min in a culture dish.
- On day 1, streak EPEC and EHEC from frozen
glycerol stocks onto fresh LB agar plates and incubate
overnight at 37°C. Keep plates in the refrigerator until
liquid overnight cultures are started.
- On day 2, seed cells onto 12-mm pretreated coverslips
in a 24-well tissue culture plate in cell culture
growth media without antibiotics. Incubate overnight
in a tissue culture incubator supplemented with
CO2 to allow the cells to adhere to the coverslips. For
MEFs, we find microscopic analysis of actin pedestal
formation easiest if cells have reached about 80% confluency
at the time of analysis; the number of cells
seeded should be adjusted accordingly. To reduce
detachment of MEFs infected with EHEC, use
fibronectin coated coverslips.
- Start an overnight liquid culture of EPEC or
EHEC in 5ml LB medium with bacteria from the
streaked plate. In our experience, small inoculation
loads result in a higher infection efficiency. Grow
overnight with aeration at 37°C on a rotary shaker at
- On day 3, collect bacteria from the 0.5-ml
overnight culture by centrifugation for 3min at 4500
rpm in a tabletop centrifuge. Wash bacterial pellet
twice in DMEM and inoculate 25 ml DMEM (EPEC) or
25 ml DMEM supplemented with 100mM HEPES, pH
7.4 (EHEC). Grow at 37°C at 180rpm for 3 h.
Environmental cues such as temperature, pH, and
osmolarity, as well as growth phase, have been
described to influence the transcriptional regulation
of expression of virulence factors, e.g., of the type
III secretion system. Media conditions that appear
to stimulate virulence are those considered to mimic
the gastrointestinal tract (e.g., Beltrametti et al., 1999;
Kenny et al., 1997). In our experience, preactivation of
EPEC and EHEC by growth for 3 h in DMEM for EPEC
or DMEM supplemented with 100 mM HEPES, pH 7.4,
for EHEC is sufficient for reproducible efficient bacterial
infection of cultured cell monolayers. In our hands,
the morphological appearance of preactivated bacterial
cultures correlates with their infectious capability:
After 3h, EPEC should be found aggregated to small
clumps of bacteria but should not grow in long
rows. Aggregation likely reflects the expression of type
IV bundle-forming pili, rope-like appendages that
represent an important additional virulence factor
found to enhance initial adherence and virulence of
EPEC (Bieber et al., 1998). EHEC, which lack bundleforming
pili, should be present as single bacteria in
preactivation cultures, but again should not grow in
- In the meantime, exchange cell culture medium
of cell monolayers with 1 ml per well of fresh cell
culture medium without antibiotics.
- Add 10µl of the bacterial 3-h preactivation culture
into each well. Initiate infection by brief centrifugation
for 5 min at 650 g in a centrifuge prewarmed to
at least room temperature, allowing bacteria and cells
to make contact with each other.
- Place in an incubator supplemented with 7.5%
CO2 at 37°C for 1h. Incubation at lower temperatures
results in a reduced infection efficiency.
- Remove media containing nonattached bacteria
by aspiration and gently replace with 1 ml of fresh prewarmed
culture medium without antibiotics per well.
Be careful not to let the cells dry out when exchanging
- Repeat medium changes every hour until a total
infection time of 4.5 to 5 h has been reached.
In our hands, longer infection times generally result
in pedestals of increased length, facilitating analysis of pedestal composition. In addition, after short incubation
times, EPEC bacteria are found mostly adhered
in aggregates, which may confound analysis. Later
during infection, EPEC bacteria will distribute from
aggregates, making it easier to distinguish single
pedestals. However, too long incubation results in
increased cell death and detachment of cells, especially
when working with EHEC.
- After the incubation period, remove the
medium by aspiration and gently wash the coverslips
twice with PBS prewarmed to 37°C to remove unattached
- For fixation, immerse each coverslip with 500µl
of fixative prewarmed to 37°C and incubate at room
temperature for 20min.
- After fixation, gently wash twice with PBS and
store in PBS at 4°C until proceeding to cell permeabilization
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
- On day 1, seed cells in regular cell culture growth
media on coverslips as described in the basic protocol
with the modification that the number of cells seeded
should be reduced to allow for the additional day
needed for transfection. Again, about 80% confluency
of MEFs should be reached at the time of bacterial
- On day 2, prior to transfection, exchange the cell
culture growth medium for 0.5ml growth medium
without antibiotics. Transfect cells growing on coverslips
in 24-well plate 12-24h overnight according to
standard protocols, e.g., using FuGENE 6 transfection
reagent. We use 0.2µg of DNA per well of a 24-well
plate with a ratio of FuGENE 6 to DNA of 3:1. In our
hands, plasmid DNA prepared with MaxiPrep kits,
e.g., from Qiagen or Invitrogen Corp. is sufficiently
pure for cell transfection.
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
IV. GENERAL COMMENTS ON
OF EPEC-AND EHEC-INFECTED
- Permeablize cells in 0.1% Triton X-100 in PBS for
- Gently wash twice with PBS.
- Prepare a humid chamber: Lay out a large square
plastic dish (e.g., 24.5 × 24.5-cm polystyrene dish with
lid) with a moistened piece of absorptive filter paper
and coat with a layer of Parafilm M.
Perform all blocking and immunolabelling steps as
follows: Using forceps, place coverslips with the cell
side facing downward onto a drop of about 20µl of
blocking or antibody mixture spotted onto parafilm in
the humid chamber. This helps minimize the amount
of antibody needed without letting the cells dry out.
In order to minimize the loss of cells when moving the
coverslips after each incubation step, pipette a small
amount of PBS right next to the edge of each coverslip
after each incubation step. This will cause the coverslips
to float on top of the PBS and will allow easy
removal of the coverslips using forceps. Wash coverslips
by carefully submerging them successively into
three small beakers filled with PBS. In between individual
washing steps, drain residual PBS by carefully
streaking the edge of the coverslips across thin
absorptive filter paper without allowing the cells to
- Block with 10% normal goat serum in PBS for 20
min in humid chamber.
- Stain with primary antibody mixture for 1 h at
room temperature in humid chamber.
- Wash three times with PBS. For polyclonal
primary antibodies, include 0.1% Triton-X100 during
first two washing steps.
- Stain with secondary antibody mixture for 1h at
room temperature in humid chamber in the dark. To
specifically label F-actin, use fluorescent dye-coupled phalloidin, e.g., Alexa Fluor 594 phalloidin (red fluorescence),
added at a concentration of 1-2.5U/ml. To
detect Leatuis by staining bacterial DNA, blue fluorescent
DAPI may be added.
- Wash three times with PBS. For polyclonal
primary antibodies, include 0.1% Triton-X100 during
first two washing steps.
- To mount coverslips onto microscope glass
slides, place drops of about 5µl mounting medium
on ethanol-wiped slides. Avoid air bubbles. Using
forceps, gently place coverslips on mounting medium
with the cell side facing downward. Once in contact
with the mounting medium, avoid moving the coverslips
as this will cause cells to be ripped off. Carefully
remove access mounting media or residual PBS by
placing a piece of absorptive filter paper onto the glass
slides without moving the coverslips.
- Dry mount coverslips for 3-4h at room temperature
in the dark or overnight prior to microscopic
observation when using oil immersion lenses.
- Store samples in the dark at 4°C. It is advisable
to complete photography of samples within 1-2
weeks, as background fluorescence increases over
- Using epifluorescence and appropriate emission
filters, infected cells are detected readily by fluorescence
actin staining (FAS) of actin pedestals. Search
for interesting cells (e.g., both transfected and infected)
using low magnification (e.g., 40x). To resolve subcellular
structures such as pedestals, switch to higher
magnification (100x) with or without optovar magnification.
Always use minimal exposure time required
to get a good signal-to-noise ratio.
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
Beltrametti, F., Kresse, A. U., and Guzman, C. A. (1999). Transcriptional
regulation of the esp genes of enterohemorrhagic Escherichia coli
. J. Bacteriol
Bieber, D., Ramer, S. W., Wu, C.-Y., Murray, W. J., Tobe, T.,
Fernandez, R., and Schoolnik, G. K. (1998). Type IV pili, transient
bacterial aggregates, and virulence of enteropathogenic Escherichia coli
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