Download Enteropathogenic Escherichia coli (EPEC) adhesion to intestinal

Microbiology (2004), 150, 527–538
DOI 10.1099/mic.0.26740-0
Enteropathogenic Escherichia coli (EPEC)
adhesion to intestinal epithelial cells: role
of bundle-forming pili (BFP), EspA filaments
and intimin
Jennifer Cleary,1 Li-Ching Lai,2 Robert K. Shaw,1
Anna Straatman-Iwanowska,1 Michael S. Donnenberg,2
Gad Frankel3 and Stuart Knutton1
1
Institute of Child Health, University of Birmingham, Birmingham, UK
Correspondence
Stuart Knutton
2
[email protected]
3
Division of Infectious Diseases, University of Maryland, Baltimore, MD, USA
Centre for Molecular Microbiology and Infection, Department of Biosciences, Imperial College,
London, UK
Received 28 August 2003
Revised
18 November 2003
Accepted 25 November 2003
Enteropathogenic Escherichia coli (EPEC), an important paediatric diarrhoeal pathogen,
employs multiple adhesins to colonize the small bowel and produces characteristic ‘attaching
and effacing’ (A/E) lesions on small intestinal enterocytes. EPEC adhesins that have been
associated with A/E adhesion and intestinal colonization include bundle-forming pili (BFP),
EspA filaments and intimin. BFP are involved in bacteria–bacteria interaction and microcolony
formation but their role in cell adhesion remains unclear; EspA filaments are components of the
EPEC type III secretion system but since they interact directly with host cells they may also
function as adhesins; intimin is the well characterized intimate EPEC adhesin which binds the
translocated intimin receptor, Tir. However, other uncharacterized host cell receptors have been
implicated in intimin-mediated adhesion. In this study, the role of BFP, EspA filaments and
intimin in EPEC adhesion to intestinal brush border cells was assessed by observing adhesion
of wild-type EPEC strain E2348/69 and a set of isogenic single, double and triple mutants in
bfpA, espA and eae (intimin gene) to differentiated human intestinal Caco-2 cells. E2348/69
(bfpA+ espA+ eae+) adhered rapidly (<10 min) to the brush border of Caco-2 cells and
subsequently produced microcolonies and typical A/E lesions. Non-intimate brush border adhesion
of double mutant strain UMD880 (bfpA+ espA” eae”) also occurred rapidly, whereas adhesion
of strain UMD886 (bfpA” espA+ eae”) occurred later in the infection (>1 h) and with much
lower efficiency; confocal microscopy indicated BFP and EspA-mediated adhesion, respectively.
Strain UMD883 (bfpA” espA” eae+), which is unable to translocate Tir, was non-adherent
although this strain was able to form intimate attachment and A/E lesions when co-cultured
with strain CVD206 (bfpA+ espA+ eae”) which supplied Tir to the membrane. Single mutant
strains CVD206 (bfpA+ espA+ eae”) and UMD872 (bfpA+ espA” eae+) showed adherence
characteristics of strain UMD880 (bfpA+ espA” eae”), whilst triple mutant strain UMD888
(bfpA” espA” eae”) was totally non-adherent. These results support an adhesive role for BFP
and EspA in initial brush border cell attachment, and in typical EPEC which express both BFP
and EspA filaments suggest a predominant role for BFP; EspA filaments, however, could serve as
initial attachment factors in atypical EPEC which lacks BFP. The study found no evidence for an
independent host cell intimin receptor or for other adhesive factors able to support bacterial
adherence.
Abbreviations: A/E, attaching and effacing; BFP, bundle-forming pilus/pili; EPEC, enteropathogenic Escherichia coli ; LEE, locus of enterocyte
effacement; RBC, red blood cell; SEM, scanning electron microscopy; TTSS, type III secretion system; WGA, wheat germ agglutinin.
0002-6740 G 2004 SGM
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J. Cleary and others
INTRODUCTION
Enteropathogenic Escherichia coli (EPEC), an important
cause of infantile diarrhoea in the developing world (Nataro
& Kaper, 1998), colonizes the small bowel and produces
‘attaching and effacing’ (A/E) lesions on the brush border
surface of small intestinal enterocytes characterized by
effacement of brush border microvilli, intimate attachment
of bacteria and formation of an actin-rich pedestal beneath
intimately attached bacteria (Knutton et al., 1989; Moon
et al., 1983). Current evidence, derived mainly from in vitro
studies using cultured epithelial cell lines, supports a
model of A/E lesion formation which involves initial nonintimate bacterial adhesion, type III secretion and effector
protein translocation to the host cell followed by brush
border effacement, intimate bacterial attachment and
pedestal formation (Frankel et al., 1998). Several adhesins
have been implicated in A/E EPEC adhesion to epithelial
cells including the type IV bundle-forming pilus (BFP)
(Giron et al., 1991), type III secretion system (TTSS) EspA
filaments (Knutton et al., 1998) and the outer-membrane
adhesin, intimin (Donnenberg & Kaper, 1991).
BFP, encoded on a large ~80 kb EPEC adherence factor
plasmid (EAF) (Nataro et al., 1987b) present in typical EPEC
strains (Kaper, 1996), have been shown to be important for
EPEC pathogenicity (Bieber et al., 1998), to be responsible
for bacteria–bacteria interaction and microcolony formation (Giron et al., 1991) and also for dispersal of bacteria
from microcolonies (Bieber et al., 1998; Knutton et al.,
1999). Several studies have also implicated BFP in initial
binding of EPEC to host epithelial cells (Donnenberg et al.,
1992; Giron et al., 1991; Tobe & Sasakawa, 2001, 2002)
although other studies which used human intestinal organ
culture suggested that BFP were not involved in initial EPEC
adherence but only at a later stage to promote microcolony
formation (Hicks et al., 1998).
Genes encoding A/E lesion formation are localized to the
locus of enterocyte effacement pathogenicity island (LEE
PAI) (McDaniel et al., 1995). The LEE encodes a TTSS,
several secreted translocator and effector proteins and
proteins involved in intimate bacterial attachment (Frankel
et al., 1998). One secreted translocator protein, EspA, forms
a long filamentous extension to the TTSS needle complex
(Daniell et al., 2001; Sekiya et al., 2001), whereas two other
translocator proteins, EspB and EspD, are thought to provide a translocation pore in the host cell membrane, thereby
allowing translocation of LEE effector proteins into the
host cell cytosol (Frankel et al., 1998). Since EspA filaments
form a direct interaction with the host cell during early
stages of A/E lesion formation (Knutton et al., 1998), they
could function as an initial EPEC attachment factor,
particularly in the case of atypical EPEC which lacks EAF
plasmids (Kaper, 1996).
Intimin is the well characterized EPEC adhesin that promotes intimate attachment to host cells. However, to form
an intimate attachment and A/E lesion formation, the
528
LEE-encoded effector protein Tir (translocated intimin
receptor) has to be translocated and inserted into the host
cell membrane by the TTSS (Kenny et al., 1997); the
intimin–Tir interaction triggers the assembly of actin into
pedestals beneath intimately attached bacteria (Campellone
& Leong, 2003). Whilst Tir is a well characterized intimin
receptor, additional uncharacterized host cell receptors
have been implicated in intimin-mediated EPEC adhesion
(Frankel et al., 2001) and recently a specific host cell protein,
nucleolin, was shown to bind a specific intimin subtype,
intimin c, expressed by some EPEC and enterohaemorrhagic
E. coli strains (Sinclair & O’Brien, 2002).
The difficulty in assessing an adhesive role of BFP, EspA
filaments and intimin when they are being expressed
simultaneously in wild-type EPEC prompted us to generate
a set of single, double and triple mutants in bfpA, espA and
eae and to examine the role of these putative adhesins
individually and in pairs. Human intestinal brush border
cells provide a better model of EPEC infection than do
undifferentiated epithelial cells of non-intestinal origin.
Hence, in this study, we examined EPEC adhesion to
Caco-2 brush border cells. The study supports a role for
BFP and EspA filaments in initial brush border attachment of EPEC and intimin–Tir interaction in intimate
EPEC adhesion and A/E lesion formation. The study
found no evidence for other adhesins able to promote
EPEC adhesion.
METHODS
Bacterial strains. The strains and plasmids used in the study are
shown in Table 1. All mutants are derivatives of EPEC serotype
O127 : H6 strain E2348/69 (Levine et al., 1978). The following
mutant strains have been described previously: eae deletion mutant
strain CVD206, which does not produce intimin (Donnenberg &
Kaper, 1991); espA deletion mutant strain UMD872, which does
not produce EspA (Kenny et al., 1996); and bfpA mutant strain
UMD901, which has a substitution of serine for cysteine at amino
acid 129 that renders the bundlin pilin protein unstable and rapidly
degraded and therefore does not produce BFP (Zhang &
Donnenberg, 1996). The suicide vectors originally used to generate
these mutants were used to make the following by allelic exchange:
UMD880 is an espA eae double mutant; UMD883 is a bfpA espA
double mutant; UMD886 is a bfpA eae double mutant; and
UMD888 is a bfpA espA eae triple mutant. Plasmids pRPA100,
pMSD2 and pCVD438, used to transform bfp, espA and eae, respectively, have been described previously (Anantha et al., 2000;
Donnenberg & Kaper, 1991; Kenny et al., 1996). Bacteria were routinely grown overnight in Luria broth (LB) and then subcultured
(1 : 100) in tissue culture media for cell adhesion studies. Bacterial
motility assays were performed in plastic vials in LB and Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemented with 0?3 % agar
and read after 18 h incubation at 37 uC.
Infection of HEp-2 cells. Adhesion to HEp-2 cells was carried out
according to the method of Cravioto et al. (1979). Subconfluent cell
cultures on glass coverslips were washed and incubated with bacteria
[10 ml overnight LB culture per millilitre of tissue culture medium
(DMEM/5 % fetal calf serum)] for 3 h at 37 uC. After thorough
washing to remove non-adhering bacteria, cells were either fixed
in methanol and stained with Giemsa to assess the pattern of cell
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Microbiology 150
EPEC adhesins
Table 1. Strains and plasmids used in the study
Illustrated are strains, their bfpA espA eae genotypes, their phenotypes in HEp-2 and Caco-2 cell assays and their motility. HEp-2 cell
adhesion, 2 to +++++; LA, localized adherence; FAS, fluorescence actin staining test (+ indicates A/E lesion formation). Caco-2 cell
adhesion, mean±SD [relative to wild-type E2348/69 (100 %)].
Strains
Genotype
bfpA
E2348/69
UMD880
UMD886
UMD883
UMD888
UMD901
UMD872
CVD206
Plasmids
pRPA100
pMSD2
pCVD438
espA
+
+
2
2
2
2
+
+
+
2
+
2
2
+
2
+
Phenotype
eae
+
2
2
+
2
+
+
2
HEp-2 cells
Reference or source
+
+
+/2
+
+
+
+
+
Levine et al. (1978)
This study
This study
This study
This study
Zhang & Donnenberg (1996)
Kenny et al. (1996)
Donnenberg & Kaper (1991)
Caco-2 cells
Adhesion
LA
FAS
Adhesion
LA
FAS
++++
++++
+
2
2
+
++++
++++
+
+
2
2
2
2
+
+
+
2
2
2
2
+
2
2
100±4
110±4
8±1
0±0
0±0
7±1?5
72±4
59±3?5
+
+
2
2
2
2
+
+
+
2
2
2
2
+
2
2
Cloned bfpA
Cloned espA
Cloned eae
Anantha et al. (2000)
Kenny et al. (1996)
Donnenberg & Kaper (1991)
adhesion or fixed in 4 % formalin and A/E lesion formation assessed
using the fluorescent actin staining test (Knutton et al., 1989). Semiquantitative assessment of adhesion was made by counting 100 cells
in representative microscope fields and determining the percentage
of cells with adherent bacteria. Adhesion was scored from 2 (nonadherent) to +++++ (80–100 % cells with adherent bacteria).
Infection of Caco-2 cells. Caco-2 intestinal cells were grown in
DMEM/20 % fetal calf serum on 24 mm diameter Transwell permeable
culture supports for 12 days to ensure differentiation of the brush
border. EPEC infections were performed in HEPES-buffered Modified
Eagle’s Medium (MEM)/2 % fetal calf serum. Cell monolayers were
infected with an overnight LB culture of EPEC [10 ml broth culture
(ml culture medium)21] for up to 6 h at 37 uC; for long infections,
the medium was changed after 3 h. In co-culture experiments, 5 ml
broth culture (ml medium)21 of each strain was used. At the end of
the infection period, cells were washed three times in PBS for 1 min
on an orbital shaker and fixed in either 4 % formalin (for immunofluorescence studies) or 3 % buffered glutaraldehyde [for scanning
electron microscopy (SEM)]. In some experiments, a gentle washing
procedure was used. In this case cells were washed three times for
20 s with gentle rocking by hand. Quantitative assessment of adhesion was made from two separate infections by counting numbers of
bacteria in three representative microscope fields taken with a 636
objective lens. Adhesion (mean number of bacteria per field±SD) is
expressed relative to that of E2348/69 (100 %).
Infection of human red blood cell (RBC) monolayers. RBC
monolayers were produced as described previously (Knutton et al.,
2002). EPEC infections were performed in HEPES-buffered DMEM
and cell monolayers infected with an overnight LB culture of EPEC
[10 ml broth culture (ml culture medium)21] for 3 h at 37 uC. After
three washes in PBS, monolayers were fixed in 4 % formalin.
Infection of human intestinal mucosa. Normal paediatric duo-
denal mucosal biopsies taken with informed consent were transported to the laboratory in ice-cold transport medium as described
previously (Knutton et al., 1987) and used immediately. Bacteria
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Motility
grown for 3 h in DMEM express LEE genes and are ‘primed’ ready
to produce A/E lesions (Collington et al., 1998). Primed bacteria
were used to infect mucosal biopsies. Biopsies were placed in Bijoux
bottles with 1?5 ml primed bacterial culture and 1?5 ml fresh culture
medium (DMEM : NCTC135/10 % FCS) and the bottles placed on a
rotary mixer inside an incubator for 1?5 h at 37 uC. Biopsies were
washed three times in warm PBS on the rotary mixer and fixed in
3 % glutaraldehyde.
Antibodies and fluorescent stains. Polyclonal rabbit or mouse
antibodies to BFP (Knutton et al., 1999), EspA (Knutton et al.,
1998), intimin (Batchelor et al., 1999), Tir (Hartland et al., 1999)
and H6 flagellin (Yona-Nadler et al., 2003) were used for immunofluorescence staining in conjunction with Alexa488 (green) and
Alexa594 (red) goat anti-rabbit IgG or goat anti-mouse IgG
secondary antibody conjugates (Molecular Probes). In addition,
bacteria were stained with propidium iodide (red) or DAPI (blue)
(Molecular Probes), cell actin with fluorescein (green) or rhodamine
(red) conjugated phalloidin (Sigma) and RBCs and the brush border
membrane with rhodamine-conjugated wheat germ agglutinin
(WGA) (Sigma).
Immunofluorescence microscopy. To stain Tir and cytoskeletal
actin, Caco-2 cells were permeabilized with PBS containing 0?1 %
Triton X-100 for 5 min and washed three times in PBS prior to
immunostaining. All antibody dilutions and immune reactions
were carried out in PBS containing 0?2 % bovine serum albumin
(PBS/BSA). Formalin-fixed, permeabilized and washed cells were
incubated with antiserum (1 : 100) in PBS/BSA for 45 min at room
temperature. After three 5 min washes in PBS, samples were stained
with either Alexa488- or Alexa594-conjugated goat anti-rabbit IgG
or goat anti-mouse IgG (Molecular Probes) diluted 1 : 100 in PBS/
BSA for 45 min. Where appropriate, samples were simultaneously
stained with propidium iodide to stain bacteria and/or phalloidin to
stain cytoskeletal actin- or rhodamine-conjugated WGA to stain the
brush border membrane. RBC samples were stained with rhodamineconjugated WGA to stain the RBC membrane and with DAPI to
stain bacteria. Preparations were washed a further three times in
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J. Cleary and others
PBS and mounted in glycerol/PBS. HEp-2 and Caco-2 cell preparations were examined by confocal microscopy using a Leica TCS SP2
Spectral Confocal Microscope, equipped with an Argon (488 nm)
and two Helium/Neon (543 nm, 633 nm) lasers and 406 or 636
PL APO objectives. Confocal illustrations show either image sections
or image projections through the brush border region of the cell.
In triple-stained specimens using Alexa488, Alexa594 and propidium
iodide, the propidium iodide emission was artificially coloured blue
to distinguish it from Alexa594 (red). RBC samples were examined
by conventional epifluorescence microscopy using a Leica DMR
microscope equipped with a 636 PL APO objective and a Leica
DC200 digital camera.
Fig. 1. Horizontal xy (a) and vertical xz (b) confocal images of
uninfected Caco-2 cell monolayers stained for cellular actin
(FITC, green) and a scanning electron micrograph (c). Caco-2
cells grown on permeable filters have a well developed apical
brush border surface. Bars, 5 mm.
SEM. Glutaraldehyde-fixed Caco-2 cell monolayers and intestinal
mucosal biopsy tissue were post-fixed in 1 % osmium tetroxide,
dehydrated through graded alcohol solutions and critical-point-dried.
Mounted specimens were sputter-coated with platinum (Polaron)
and examined in a Philips XL30 scanning electron microscope
(Knutton, 1995).
Fig. 2. Confocal (a–g) and SEM (h) images of Caco-2 cell monolayers infected with E2348/69 (bfpA+ espA+ eae+). Within
10 min bacteria are seen adhering to the brush border (phalloidin–FITC, green) as single bacteria (propidium iodide, red) (a);
adherent bacteria express BFP (Alexa594, red), EspA filaments (Alexa488, green) and intimin (Alexa488, green, inset) (b).
BFP fibrils (Alexa488, green) appear to promote attachment of bacteria (propidium iodide, coloured blue) to the brush border
(phalloidin–TRITC, red) and also bacteria–bacteria aggregation (c). After 3 h, horizontal (d) and vertical (e) confocal images
following BFP (Alexa488, green), bacteria (propidium iodide, blue) and actin (phalloidin–TRITC, red) staining revealed
adherent three-dimensional microcolonies of bacteria connected by BFP. After 6 h, horizontal (f) and vertical (g) confocal
images following staining for bacteria (propidium iodide, red) and actin (phalloidin–FITC, green) showed A/E lesion formation
(actin accretion beneath bacteria) (f, g, arrows); bacterial microcolonies were no longer present. A/E lesion formation (h,
arrow) and an absence of microcolonies was also seen by SEM (h). Bars, (a–g) 5 mm; (h) 1 mm.
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EPEC adhesins
RESULTS
Characterization of mutants in HEp-2 cell
assays
Prior to examining EPEC interaction with Caco-2 brush
border cells, the collection of mutants in bfpA, espA and eae
was tested in HEp-2 cell assays for extent of adhesion,
pattern of adhesion (Nataro et al., 1987a) and fluorescent actin staining (Knutton et al., 1989). Expression
of BFP, EspA filaments and intimin were assessed by
immunofluorescence. In each case the observed phenotype
was consistent with the genotype (Table 1).
Wild-type EPEC adhere to Caco-2 cells and
form A/E lesions
Caco-2 cells grown on permeable filters for 12 days produce
a polarized sheet of columnar epithelial cells with a well
developed apical brush border surface that can be visualized
by confocal microscopy after staining the microvillous
membrane with WGA or the microvillous actin cytoskeleton
with phalloidin (Fig. 1a, b) or visualized by SEM (Fig. 1c).
To examine initial EPEC attachment to Caco-2 cells, we
examined cell monolayers at early time points (10 min)
when single bacteria were first seen bound to the brush
border surface (Fig. 2a). At this stage, fluorescence staining
of E2348/69 revealed expression of BFP fibrils and surface
expression of intimin; EspA filaments were expressed by
some but not all bacteria at this time point and they were
frequently shorter than mature EspA filaments seen at
later time points (Knutton et al., 1998). The longer and
more numerous BFP fibrils appeared to mediate bacterial
attachment to the brush border surface (Fig. 2b). Actin
accretion, indicative of A/E lesion formation (Knutton
et al., 1989), was not observed at this stage. Adhesion of
individual bacteria to Caco-2 cells was followed by bacterial
aggregation giving rise first to small aggregates (Fig. 2c)
and later after 3 h to larger microcolonies (Fig. 2d) typical
of localized adherence seen with HEp-2 cells (Table 1).
All bacteria within microcolonies expressed BFP and BFP
appeared to mediate bacteria–bacteria aggregation (Fig. 2d, e);
actin accretion beneath bacteria in contact with the apical
cell surface indicated that they had formed A/E lesions
(Fig. 2f, g). By 6 h only bacteria in contact with the cell
surface that had produced A/E lesions were observed,
indicating that the three-dimensional bacterial microcolonies had dispersed (Fig. 2g, h), a phenomenon we
demonstrated previously in E2348/69-infected HEp-2 cells
(Knutton et al., 1999). Since BFP, EspA filaments and
intimin were expressed when EPEC first adhered to Caco-2
cells, we examined double deletion mutant strains UMD880,
UMD886 and UMD883 (Table 1) to assess the role in
adhesion of BFP, EspA filaments and intimin independently.
BFP mediates rapid adherence of EPEC to
Caco-2 cells
Strain UMD880 (bfpA+ espA2 eae2) expressed BFP bundles
typical of the wild-type strain but there were no EspA
filaments and no surface intimin (Table 1). Strain UMD880
adhered rapidly (10 min) to Caco-2 cells, initially as single
bacteria (Fig. 3a) and BFP fibrils appeared to promote
brush border attachment of bacteria (Fig. 3b, c). BFP-like
fibrils connecting bacteria to brush border microvilli were
also seen by SEM (Fig. 3d). Subsequent bacterial aggregation resulted after 3 h in typical localized microcolonies
with bacteria interlinked by BFP (Fig. 3e, f ). Strain UMD880
did not induce actin accretion or A/E lesion formation but
the level of adhesion was comparable to that of the wildtype (Table 1). Interestingly, after 6 h UMD880 microcolonies were still present and had not dispersed (data not
shown), which suggests that the kinetics of dispersal are
different from those of the wild-type or that signals which
lead to dispersal are not produced by strain UMD880.
Fig. 3. Confocal (a–c, e, f) and SEM (d) images of UMD880 (bfpA+ espA” eae”) infected Caco-2 cell monolayers. Within
10 min individual bacteria (propidium iodide, red) adhere to the brush border (stained for actin, phalloidin–FITC, green) (a).
BFP fibrils (Alexa488, green) seen in horizontal (b, arrow) and vertical (c, arrow) confocal images appear to promote adhesion
of UMD880 to the brush border (phalloidin–TRITC, red). BFP-like fibrils connecting bacteria to brush border microvilli were
also seen by SEM (d, arrows). After 3 h, horizontal (e) and vertical (f) confocal images following BFP (Alexa488, green),
bacteria (propidium iodide, coloured blue) and actin (phalloidin–TRITC, red) staining revealed adherent three-dimensional
microcolonies of bacteria interlinked by BFP. Bars, (a–c, e, f ) 5 mm; (d) 1 mm.
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Fig. 4. Confocal images of UMD886 (bfpA” espA+ eae”) infected Caco-2 cell monolayers. UMD886 (propidium iodide, red)
adhered poorly to the Caco-2 cell brush border (WGA–FITC, green) even after 3 h (a). However, an ~10-fold increase in the
level of UMD886 adhesion was seen following a gentle wash procedure (b). In each case EspA filaments (Alexa488, green)
seen in horizontal (c, arrow) and vertical (d, arrow) confocal images appeared to promote adhesion of bacteria (propidium
iodide, coloured blue) to the cell brush border (phalloidin–TRITC, red). Bars, 5 mm.
Transformation of EspA (plasmid pMSD2) or intimin
(plasmid pCVD438) into strain UMD880 did not alter the
adherence characteristics of this strain.
EspA filaments mediate EPEC adherence to
Caco-2 cells
Strain UMD886 (bfpA2 espA+ eae2) produced EspA filaments but lacked both BFP and surface intimin (Table 1).
This strain adhered to Caco-2 cells but inefficiently compared to wild-type E2348/69 and UMD880. No adhesion
was detected after 10 min and only a low level of adhesion
was detected after 3 h (Fig. 4a, Table 1); bacteria adhered
individually and there was no aggregation into microcolonies. We noted previously greatly improved EspA
filament-mediated bacterial adhesion to HEp-2 cells using
a gentle washing procedure to remove non-adherent
bacteria following infection (Knutton et al., 1998). In a
similar manner, using the gentle washing procedure,
adhesion of UMD886 to Caco-2 cells was greatly improved
(Fig. 4b) and was ~10-fold greater than when using the
more vigorous washing procedure. In each case EspA
filaments appeared to mediate brush border adhesion of
strain UMD886 (Fig. 4c, d). Transformation of UMD886
with pRPA100 restored BFP expression, localized adhesion,
microcolony formation and good adhesion; transformation
with pCVD438 did not affect the overall level of adherence
but conferred A/E lesion formation (data not shown).
Intimin promotes Caco-2 cell adherence of
EPEC when Tir is present in the cell membrane
Strain UMD883 (bfpA2 espA2 eae+) did not produce BFP
or EspA filaments but did express surface intimin (Table 1).
This strain did not adhere to Caco-2 cells even after 6 h,
irrespective of the washing procedure used (Table 1).
Transformation of UMD883 with pRPA100 restored BFP
expression and microcolony formation whilst transformation with pMSD2 restored adherence comparable to strain
UMD886 and A/E lesion formation (data not shown).
532
Intimin is known to bind Tir following its translocation
and insertion into the host cell membrane; strain UMD883
lacks EspA filaments and thus a functional TTSS and so
is unable to translocate Tir. To assess the adhesive properties of UMD883 to cells possessing translocated Tir, we
performed co-culture infections with strain CVD206 or
UMD886, which lack the intimin gene but which possess a
functional TTSS and can translocate Tir. In co-culture with
CVD206 (Fig. 5) or UMD886, strain UMD883, identified
by surface intimin staining, was able to adhere intimately
to Caco-2 cells and produced typical A/E lesions. However,
the extent of UMD883 adhesion and A/E lesion formation was much more pronounced when co-cultured with
CVD206 compared to UMD886, presumably due to the
Fig. 5. Confocal (a–c) and SEM (d) images of Caco-2 cells
co-infected with UMD883 (bfpA” espA” eae+) and CVD206
(bfpA+ espA+ eae”) for 3 h and stained only for UMD883
bacteria (intimin staining). In horizontal (a) and vertical (b) confocal images of cells stained for actin (phalloidin–FITC, green),
UMD883 bacteria (Alexa594, red) were now adherent and produced A/E lesions (actin accretion beneath bacteria) (b, arrow).
Focused Tir (Alexa488, green) was also seen beneath intimately
attached UMD883 bacteria (Alexa594, red) (c). A/E lesions
produced by UMD883 (d, arrow) were also seen by SEM.
Bars, 1 mm.
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EPEC adhesins
Fig. 6. Confocal images showing Caco-2 cell monolayers infected with single mutant strains CVD206 (bfpA+ espA+ eae”)
(a), UMD872 (bfpA+ espA” eae+) (b) and UMD901 (bfpA” espA+ eae+) (c). Caco-2 cells were stained for actin
(phalloidin–FITC, green) and bacteria stained with propidium iodide (red). CVD206 adhered rapidly (10 min) to Caco-2 cells
(a) and adherent bacteria expressed BFP (Alexa594, red) and EspA filaments (Alexa488, green) (a, inset). UMD872 similarly
induced rapid Caco-2 cell adhesion (b) and adherent UMD872 produced BFP fibrils (Alexa594, red) but no EspA filaments
(b, inset). By 6 h, some UMD901 bacteria (propidium iodide, red) had formed A/E lesions (c, arrow). Other adherent bacteria
had not induced actin accretion (c, arrowhead) and in such bacteria EspA filaments (Alexa488, green) seen in vertical
confocal images appeared to mediate brush border (phalloidin–TRITC, red) adhesion of bacteria (c, inset). Bar, 5 mm; inset 1 mm.
much more efficient adhesion of CVD206 (Table 1).
Focused Tir translocated by CVD206 (Fig. 5c) or UMD886
was also detected beneath intimately attached UMD883
bacteria.
Adhesion of single mutants in bfpA, espA
and eae
To assess which might be the predominant EPEC adhesin,
we compared adhesion of the double mutants just described with adhesion of single mutants in bfpA, espA and
eae. Strain CVD206 (bfpA+ espA+ eae2) produced BFP and
EspA filaments but lacked surface intimin; strain UMD872
(bfpA+ espA2 eae+) produced BFP and expressed surface
intimin but lacked EspA filaments; strain UMD901 (bfpA2
espA+ eae+) produced EspA filaments and expressed surface intimin but lacked BFP (Table 1). Strains CVD206
and UMD872 exhibited Caco-2 cell adherence characteristic
of UMD880, i.e. rapid (10 min) bacterial adhesion
(Fig. 6a, b) followed by microcolony formation but there
was no A/E lesion formation. Adherence of strain UMD901
was characteristic of strain UMD886, i.e. no adhesion after
10 min and poor adhesion after 3 h. However, unlike strain
UMD886, adherent UMD901 bacteria do possess a functional TTSS and were able to translocate Tir and produce
A/E lesions on Caco-2 cells after 6 h (Fig. 6c). As was the
case for UMD886, a mild washing procedure to remove
non-adherent bacteria significantly increased the adherence
of strain UMD901.
Adhesion of a triple mutant in bfpA, espA
and eae
Strain UMD888 (bfpA2 espA2 eae2) lacked BFP, EspA
filaments and intimin and was non-adherent to Caco-2
cells after 6 h irrespective of which washing procedure
was used (Fig. 7d, Table 1). Transformation with plasmid
pRPA100 restored adherence and microcolony formation
typical of UMD880; transformation with pMSD2 conferred
a weak, non-localized cell adherence to cells typical of
UMD886; UMD888 transformed with pCVD438 remained
non-adherent (data not shown).
Fig. 7. Confocal images showing Caco-2 cell monolayers infected with EPEC strain E2348/69 (bfpA+ espA+ eae+) for
10 min (a), 3 h (b) and 6 h (c) and with strain UMD888 (bfpA” espA” eae”) for 6 h (d). Bacteria were stained with propidium
iodide (red) and flagella with an anti-flagellin antibody (Alexa488, green). All bacteria attached after 10 min possessed
flagella (a) but with increasing time fewer and fewer flagellate bacteria were present (b, c). After 6 h, strain UMD888 was
non-adherent (d) but flagellate bacteria were present in culture supernatants (d, inset). Bars, 5 mm.
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J. Cleary and others
Fig. 8. Fluorescence images showing EPEC adhesion to RBC monolayers. Bacteria were stained with DAPI (blue) and the
RBC membrane with WGA–TRITC (red). Strains UMD886 (bfpA” espA+ eae”) (d), UMD901 (bfpA” espA+ eae+) (f) and
CVD206 (bfpA+ espA+ eae”) (h) adhered to RBC monolayers in similar numbers to wild-type E2348/69 (bfpA+ espA+
eae+) (a), whereas strains UMD880 (bfpA+ espA” eae”) (b), UMD883 (bfpA” espA” eae+) (c), UMD888 (bfpA” espA”
eae”) (e) and UMD872 (bfpA+ espA” eae+) (g) did not adhere. Bar, 5 mm.
Flagella and EPEC adhesion to Caco-2 cells
Flagella were recently implicated in EPEC adhesion to
epithelial cells (Giron et al., 2002). Since strain E2348/69
produced flagella which might be contributing to adhesion,
we examined motility and flagella expression of all the
strains; all the strains were motile when grown in LB and in
the DMEM used for cell adhesion assays (Table 1) and in
each strain flagella expression was detected by immunofluorescence using an anti-flagellin antibody.
Flagella expression was also examined during cell adhesion.
Virtually all E2348/69 bacteria attaching initially to Caco-2
cells expressed flagella (Fig. 7a) but as bacteria formed
microcolonies and produced A/E lesions (3 h) the percentage of flagellated bacteria was greatly reduced (Fig. 7b);
after 6 h few flagella were observed (Fig. 7c). A similar
decrease in the number of flagellate bacteria following
initial attachment was seen with the other adherent
strains (UMD880, UMD872, UMD901, CVD206). Strains
UMD883 and UMD888 (Fig. 7d) were non-adherent to
Caco-2 cells but the presence of flagellate bacteria was
confirmed by immunofluorescence of bacteria present in
the culture medium (Fig. 7d, inset).
EPEC adhesion to RBC monolayers
In recent studies we demonstrated EPEC adhesion to RBC
monolayers, initially by EspA filaments and subsequently
with strains able to translocate Tir, by intimin–Tir intimate
534
interaction (Shaw et al., 2001, 2002). In this study we also
screened the collection of mutants in bfpA, espA and eae
for their ability to adhere to RBC monolayers. As reported
previously, wild-type E2348/69 adhered to RBC monolayers
(Fig. 8a) and induced a high level (~80 %) of haemolysis
(Shaw et al., 2001). Similar levels of RBC adhesion (and
haemolysis) were produced by strains UMD886 (Fig. 8d),
UMD901 (Fig. 8f ) and CVD206 (Fig. 8h), whereas strains
UMD880 (Fig. 8b), UMD883 (Fig. 8c), UMD888 (Fig. 8e)
and UMD872 (Fig. 8g) did not adhere to RBC monolayers
and did not induce haemolysis. Thus, adhesion to RBCs
was independent of BFP expression but correlated with
expression of EspA filaments; under the assay conditions
used, adhesion of the four positive strains was shown to be
mediated by EspA filaments (data not shown).
EPEC adhesion to human intestinal mucosa
Studies using human intestinal organ culture suggested that
BFP was not involved in initial EPEC adherence but only
at a later stage to promote microcolony formation (Hicks
et al., 1998). We showed previously that wild-type EPEC
strain E2348/69 will colonize the surface of cultured human
intestinal mucosa and produce typical A/E lesions after a
6–8 h incubation (Knutton et al., 1987). However, in these
studies we did not attempt to examine early stages of
adhesion. Here we examined early stages of E2348/69
adhesion to human intestinal mucosa (Fig. 9b) in comparison with CVD206 (bfpA+ espA+ eae2), which cannot
produce A/E lesions but which does produce BFP and
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EPEC adhesins
EspA filaments and may therefore be able to undergo
initial mucosal attachment if BFP and/or EspA filaments
are adhesins (Fig. 9c). After a shorter 1?5 h incubation
with E2348/69, SEM revealed adhesion of bacterial microcolonies to mucosal cells in which brush border microvilli
had become perturbed and elongated. Some bacteria
appeared deeply embedded in the brush border (Fig. 9b),
typical of bacteria that have formed intimate attachment
and A/E lesions. CVD206 adhered in similar small microcolonies and induced elongation of brush border microvilli
but did not appear to be intimately attached to the mucosal
surface (Fig. 9c). Uninfected control tissue had a uniformly
smooth appearance and lacked any adherent bacteria
(Fig. 9a).
DISCUSSION
Intestinal colonization and A/E lesion formation by EPEC
is a complex multi-stage process and several different well
characterized and putative adhesive factors involved in this
process have been described including BFP, EspA filaments
and intimin. Observation of the interaction with Caco-2
cells of wild-type EPEC strain E2348/69 and a set of defined
bfpA, espA and eae mutants and transformants supports
a role for BFP and EspA filaments in initial brush border
cell attachment of EPEC whereas intimin was required for
intimate EPEC adhesion and A/E lesion formation providing that Tir had been translocated to the host cell.
Several studies have implicated BFP in initial binding of
EPEC to host cells. The initial studies of Giron et al. (1991)
demonstrated a significant reduction in localized adherence of EPEC in the presence of BFP antibodies; residual
adherence can now be explained by EspA filament and
intimin–Tir adherence. Tobe & Sasakawa (2001) implicated
BFP in host cell attachment by demonstrating preferential
binding of BFP, producing EPEC to the Caco-2 cell surface
rather than to existing EPEC microcolonies formed during
an earlier infection. These authors also concluded that
differential EPEC adherence to cells of different species
origin was due to BFP (Tobe & Sasakawa, 2002). This
present study, which examined EPEC strains at an early
stage when they first adhere to cells, also supports such a
cell adhesive role for BFP. Early rapid adhesion of individual bacteria correlated with the expression of BFP and,
by immunofluorescence, BFP fibrils appeared to connect
bacteria to the brush border surface. Subsequently, individual bacteria aggregated to produce small and then larger
microcolonies with BFP appearing to link bacteria in the
aggregates. Thus, these morphological data also support a
direct role for BFP in initial cell attachment in addition
to their recognized role in microcolony formation and
dispersion. An adhesive role for BFP implies a specific host
cell BFP receptor. Numerous candidate BFP receptors have
been proposed, including a variety of oligosaccharides and,
most recently, the phospholipid phosphatidylethanolamine
(PE) (Nougayrede et al., 2003). This study also confirmed
our previous conclusion that EPEC adhesion to RBCs is
independent of BFP (Shaw et al., 2001) and indicates a BFP
receptor that is lacking on the RBC surface. A PE receptor
would be consistent with these RBC data because in the
erythrocyte membrane PE is predominantly localized on the
cytosolic side of the membrane.
A study by Hicks et al. (1998) employing in vitro intestinal
organ culture to examine EPEC adhesion to human small
intestinal mucosa concluded that BFP was not involved in
initial EPEC adherence but only at a later stage to promote
microcolony formation. This conclusion was based on
the fact that strain JPN15, which lacks the large plasmid
encoding BFP but expresses EspA and intimin, adhered
to cultured paediatric small intestinal mucosa in twodimensional microcolonies and produced A/E lesions
whereas strain CVD206 (bfpA+ espA+ eae2) did not
adhere. We also demonstrated previously A/E adhesion of
a plasmid-cured derivative of E2348/69 to human intestinal
Fig. 9. SEM images of uninfected small intestinal mucosa (a) and following a 1?5 h infection with primed E2348/69 (bfpA+
espA+ eae+) (b) and CVD206 (bfpA+ espA+ eae”) (c) bacteria. Uninfected mucosa had a smooth mucosal surface and no
adherent bacteria were present (a). E2348/69 adhered in microcolonies to mucosal cells in which the brush border had been
perturbed and microvilli elongated. Some bacteria appeared deeply embedded in the brush border, typical of those that form
intimate attachment and A/E lesions (b). CVD206 similarly adhered in microcolonies and induced elongation of brush border
microvilli but did not appear intimately attached to the mucosal surface (c). Bars, (a) 10 mm; (b, c) 5 mm.
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J. Cleary and others
mucosa and noted that the extent of adhesion was highly
attenuated compared to that of the wild-type (Knutton
et al., 1987). This could now be explained by a lack of
BFP-mediated initial attachment with initial attachment
of the plasmid-cured strain being promoted by the much
less efficient EspA filaments. In this study we examined early
stages of EPEC adhesion to human intestinal mucosa and,
in contrast to Hicks et al. (1998), we did observe adhesion
of strain CVD206. CVD206, unlike E2348/69, lacks intimin
and so cannot form an intimin–Tir intimate attachment
and A/E lesions but it does express BFP and EspA filaments which could be promoting non-intimate adhesion.
Although the CVD206 adhesins involved were not identified, the observation that CVD206 did adhere does cast
doubt on the conclusions of Hicks et al. that BFP does not
promote initial mucosal attachment of EPEC. Failure to
detect mucosal adhesion of CVD206 by Hicks et al. may
have been due to differences in the adhesion assay protocols
used and/or to poor BFP expression by the CVD206 strain
used. It was particularly noticeable in the study by Hicks
et al. that in a 3 h HEp-2 cell adhesion assay they only
demonstrated adhesion of individual CVD206 bacteria to
the HEp-2 cell surface, whereas others had observed adhesion of large bacterial microcolonies by 3 h (Donnenberg
& Kaper, 1991; Knutton et al., 1999).
EspA filaments are a component of the EPEC TTSS whose
primary function is the delivery of virulence proteins into
host cells (Daniell et al., 2001). However, since EspA
filaments interact with host cells during the early stages of
A/E lesion formation, it has been proposed that they may
also function as adhesins. We showed previously that EspA
filaments promoted attachment of strains lacking BFP to
epithelial cells and to RBCs (Knutton et al., 1998; Shaw
et al., 2001) and similar results supporting a role of EspA
filaments as initial attachment factors were demonstrated
with Shiga toxin-producing E. coli (Ebel et al., 1998) which
also lacks BFP. This present study also supports an adhesive
role for EspA filaments in EPEC adhesion to brush border
cells since adhesion correlated with EspA filament expression and, by immunofluorescence, EspA filaments appeared
to connect bacteria to the brush border surface. However,
adhesion was much less efficient than with strains expressing BFP. The extent of EspA-filament-mediated adhesion
was dependent on the washing procedure used, suggesting
that EspA-filament-mediated adhesion is weak compared
to BFP and intimin–Tir-mediated adhesion where the
washing procedure had no effect on levels of adhesion.
Compared to BFP, the less efficient EspA-filament-mediated
adhesion probably reflects the small number of EspA
filaments produced (~12 EspA filaments per bacterium)
(Daniell et al., 2001) and the nature of their interaction
with host cells which is currently unknown. EPEC have
been divided into typical EPEC which possess EAF plasmids
and BFP and atypical EPEC which lack EAF plasmids and
BFP (Kaper, 1996). The use of EspA filaments as initial
attachment factors could explain why atypical EPEC is
still able to colonize the gut and produce A/E lesions but,
536
due to a lack of BFP, is a much less efficient colonizer than
typical EPEC (Levine et al., 1985).
Intimin expressed on the bacterial surface binds Tir
following its translocation and insertion into the host cell
membrane; intimin–Tir interaction produces the characteristic intimate EPEC attachment and triggers subsequent
A/E lesion formation (Campellone & Leong, 2003; Frankel
et al., 1998). Strain UMD883 expressing only intimin did
not adhere to Caco-2 cells although it was able to bind and
form an intimate attachment and A/E lesions following
translocation of Tir into the Caco-2 cell membrane. The
adhesive role of intimin binding to translocated Tir is
well established (Kenny et al., 1997). However, there is also
a considerable body of evidence for a host cell intimin
receptor, not least of which is the binding of the cell-binding
domain of intimin to epithelial cells in the absence of Tir
(Frankel et al., 2001) and the recent demonstration, in the
case of E. coli O157 : H7, of a host cell surface protein,
nucleolin, which specifically binds intimin c expressed by
this serotype (Sinclair & O’Brien, 2002). Although available evidence supports the presence of host cell intimin
receptors, the inability of strain UMD883 to adhere to Caco2 and HEp-2 cells in the absence of Tir clearly indicates the
lack of a receptor able to support bacterial adhesion.
The present study examined three particular EPEC adhesive
factors. However, other putative EPEC adhesive factors
have been proposed, including other fimbrial antigens
(Giron et al., 1993), Efa1/LifA (Badea et al., 2003) and
flagella (Giron et al., 2002). Other than type 1 fimbriae,
which have been demonstrated not to be involved in
epithelial cell adhesion, strain E2348/69 has not been
reported to produce other recognized fimbriae (Elliott
& Kaper, 1997). Efa1/LifA, a protein unique to EPEC
(Klapproth et al., 2000) and other A/E pathogens, appears
to have cell-binding activity and might contribute to
adhesion in some manner. However, the lack of adhesion
of UMD888 (bfpA2 espA2 eae2) implies the absence of
other adhesive factors sufficient to promote adhesion of
EPEC to intestinal brush border cells. A direct role for
flagella in EPEC adhesion was suggested recently (Giron
et al., 2002). We thought previously that adherent EPEC
did not express flagella because we did not see flagella by
SEM. However, it is now clear from the immunofluorescence staining performed as part of this study that adherent
EPEC do possess flagella which could therefore play a
role in adhesion. Nevertheless, this study found no direct
evidence for an adhesive role for flagella since strains
UMD883 (bfpA2 espA2 eae+) and UMD888 (bfpA2 espA2
eae2) produced flagella and yet were totally non-adherent.
However, it is clear that EPEC flagella can interact with
host cells and stimulate host immune responses (Zhou
et al., 2003) and thus a contribution to adhesion cannot be
ruled out although this study indicates that flagella, in the
absence of other adhesins, cannot by themselves support
bacterial adhesion.
In summary, the results of this study, using defined EPEC
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Microbiology 150
EPEC adhesins
E2348/69 mutants and Caco-2 brush border cells, are
consistent with data obtained previously using undifferentiated epithelial cells of non-intestinal origin and indicate
that BFP, EspA filaments and, in a Tir-dependent manner,
intimin can each support bacterial adhesion to intestinal
epithelial cells; no evidence was found for other adhesins
which could support EPEC adhesion. BFP appeared to
be the predominant initial attachment factor but, in the
absence of BFP, EspA filaments were able to promote a less
efficient initial bacterial attachment. Intimin functioned as
an adhesin in the presence of translocated Tir but we found
no evidence for an independent host cell intimin receptor
capable of promoting EPEC adhesion.
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ACKNOWLEDGEMENTS
Giron, J. A., Torres, A. G., Freer, E. & Kaper, J. B. (2002). The flagella
We thank Ilan Rosenshine for the H6 flagella antiserum and the
Wellcome Trust for financial support. M. S. D. and L.-C. L. were
supported by Public Health Service Awards AI37606 and AI32074 from
the National Institutes of Health.
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