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Journal of Medical Microbiology (2003), 52, 861–867
DOI 10.1099/jmm.0.05229-0
Cytoskeletal rearrangements in gastric epithelial
cells in response to Helicobacter pylori infection
Bin Su, Peter J. M. Ceponis and Philip M. Sherman
Correspondence
Philip M. Sherman
Research Institute, Hospital for Sick Children, Departments of Paediatrics and Laboratory Medicine
and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5G 1X8
sherman@sickkids.ca
Received 20 February 2003
Accepted 13 June 2003
Helicobacter pylori causes host epithelial cell cytoskeletal rearrangements mediated by the
translocation and tyrosine phosphorylation of an outer-membrane protein, CagA, and by the
vacuolating cytotoxin, VacA. However, the mechanisms by which H. pylori mediates cytoskeletal
rearrangements in infected host cells need to be more clearly defined. The aim of this study was to
determine the effects of H. pylori isolates from children on the architecture of host gastric epithelial
cells. Gastric epithelial (AGS) cells were infected with type I (cagEþ , cagAþ , VacAþ ) H. pylori, a type
II H. pylori strain (cagE , cagA , VacA ) or a cagE isogenic mutant. Double-labelled immune
fluorescence was used to detect adherent H. pylori and the distribution of F-actin, Æ-actinin and
Arp3. Both type I and type II H. pylori strains induced stress fibres in gastric epithelial cells that were
not observed in uninfected cells. Type I H. pylori also induced cell elongation (hummingbird
phenotype) after 4 h of infection, whereas the type II H. pylori strain did not. Less elongation
occurred when AGS cells were exposed to a cagE isogenic mutant, compared with the parental
strain. Confocal microscopy showed Arp3 accumulation in AGS cells infected with wild-type
H. pylori, but not in response to infection with the cagE mutant. These findings indicate that type I
H. pylori induce a stress fibre-like phenotype in infected gastric epithelia by a mechanism that is
different from the induction of host-cell elongation. In addition to CagA and VacA, cagE also impacts
on the morphology of infected gastric epithelial cells.
INTRODUCTION
Helicobacter pylori is a Gram-negative bacterium that infects
the human stomach and causes gastritis, peptic ulceration
and gastric cancers (Blaser & Berg, 2001). Type I H. pylori
strains contain a cag pathogenicity island (PAI) that encodes
proteins that constitute a type-IV secretion system and the
putative virulence factors cagA and cagE (Censini et al.,
2001). Studies using isogenic mutants have demonstrated
that certain genes encoded on the cag PAI, including cagE but
not cagA, are responsible for nuclear factor-kB (NF-kB)
activation, resulting in the transcription of pro-inflammatory genes such as interleukin-8, interleukin-1, interferon-ª
and tumour necrosis factor-Æ (Maeda et al., 2001). H. pylori
vacuolating cytotoxin (VacA) is also involved in inducing
cytoskeletal rearrangements in infected epithelia (Pai et al.,
2000; Ashorn et al., 2000).
H. pylori delivers CagA into the cytoplasm of host cells,
through the type-IV secretion system, where it is phosphorylated on tyrosine residues (Backert et al., 2001; Odenbreit
et al., 2000). CagA induces a growth factor-like phenotype,
referred to as ‘hummingbird’ (Segal et al., 1999), in gastric
epithelial cells. This morphological change is similar to that
induced by hepatocyte growth factor (HGF), which occurs in
an Src kinase-dependent fashion (Selbach et al., 2002). Once
CagA is translocated into the host-cell cytosol and tyrosinephosphorylated, the SHP-2–Rho pathway is activated to
cause changes in host-cell morphology (Higashi et al., 2002;
Lacalle et al., 2002). Backert et al. (2001) showed that,
although the translocation and phosphorylation of CagA is
important for the induction of the hummingbird phenotype
in gastric (AGS) cells, it is not sufficient. This finding
suggests, therefore, that there are additional bacterial
factors involved in eukaryotic cellular responses to H. pylori
infection.
In this study, we report that infection with paediatric
H. pylori clinical isolates induces the accumulation of Arp3
in epithelial cells in a cagE-dependent manner. In addition to
CagA and VacA, cagE is necessary for the induction of the
hummingbird cell-elongation phenotype in response to
H. pylori infection.
METHODS
Reagents and antibodies. Polyclonal H. pylori rabbit antiserum was
purchased from DAKO. Monoclonal anti-Æ-actinin (IgM) antibody,
Abbreviation: PAI, pathogenicity island.
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861
B. Su, P. J. M. Ceponis and P. M. Sherman
fluorescent (FITC)-conjugated phalloidin and FITC-conjugated antimouse IgM were purchased from Sigma. Polyclonal anti-human Arp3
and goat anti-rabbit antiserum conjugated to rhodamine (TRTIC) were
purchased from Santa Cruz Biotechnology. Secondary antibodies
against rabbit IgG or against goat IgG conjugated with Cy5 or Cy3,
respectively, were purchased from Jackson Laboratory.
Bacterial growth and infection conditions. H. pylori strains em-
ployed in this study included the type I strain LC11 (cagAþ , cagEþ ,
VacAþ ) and the type II strain LC20 (cagA , cagE , VacA ), both
originally isolated from children in Toronto, respectively with duodenal
ulceration and gastritis alone (Dytoc et al., 1993). H. pylori strain 8823
(cagAþ , cagEþ , VacAþ ) and its isogenic cagE mutant were a kind gift
from Richard Peek, Jr (Vanderbilt University, Nashville, TN, USA).
Bacteria were cultured for 72 h on Columbia agar plates containing 5 %
sheep blood (PML Microbiologicals) under microaerophilic conditions
(5 % O2 , 85 % N2 and 10 % CO2 ) and then inoculated into Brucella
broth supplemented with 10 % fetal bovine serum (FBS) at 37 8C under
microaerophilic conditions with shaking overnight. For the cagE
mutant, the culture medium contained 20 ìg kanamycin ml1 (Gibco).
For harvesting, bacteria were washed once with sterile PBS (pH 7·4) and
then resuspended in antibiotic-free F-12 medium (Gibco) with 0·1 %
FBS.
Tissue culture. Human gastric carcinoma-derived AGS epithelial cells
(CRL-1739; ATCC, Manassas, VA, USA) were grown in Ham’s F-12
medium (Gibco) with 10 % FBS at 37 8C in 5 % CO2 . T84 cells (ATCC
CCL-248), a polarized intestinal epithelial cell line derived from a colon
cancer, were grown in a 1 : 1 mixture of Dulbecco’s minimum essential
medium (Gibco) and Ham’s F12 medium. HEp-2 cells (epithelial cells
originally derived from a carcinoma of the larynx; ATCC CCL-23) were
cultured in MEM medium. Each of the cell lines was cultured in medium
containing 2 mM L-glutamine, 10 % FBS, penicillin (100 U ml1 ) and
streptomycin (100 ìg ml1 ) (all from Gibco).
Immune fluorescence. Tissue-culture cells were seeded onto cover-
slips and placed into 24-well plates (Nunc) in medium containing 0·1 %
FBS without antibiotics for 20 h at 37 8C prior to bacterial infection.
Cells then were washed once with sterile PBS and H. pylori was added at
an m.o.i. of 100 : 1, for varying times at 37 8C. At the end of the infection
period, tissue-culture cells were washed six times with PBS to remove
non-adherent bacteria. Cells were then fixed in 2 % paraformaldehyde
for 15 min and permeabilized with 0·2 % Triton X-100 for 10 min.
F-actin was stained by FITC-labelled phalloidin at 20 ng ml1 and Æactinin was detected with anti-Æ-actinin mAb IgM (1 : 100) for 30 min
followed by incubation with FITC-labelled anti-mouse IgM (1 : 100) for
30 min. Arp2/3 was detected with polyclonal anti-human Arp3 followed
by anti-goat antibody conjugated with Cy3. HEp-2 cells were stained for
the signal transducer and activator of transcription (Stat)-6, which was
employed as a marker for cytoplasmic protein in epithelial cells
(Schindler, 2002), using a specific polyclonal antibody (Santa Cruz) at
a dilution of 1 : 100.
Adherent bacteria were visualized by staining bacteria with polyclonal
anti-H. pylori antibody (1 : 100) followed by goat anti-rabbit antibody
conjugated with TRITC (1 : 100). Alternatively, H. pylori was stained
green by using anti-rabbit antibody conjugated with Cy5 (Santa Cruz
Biotechnology).
Images were detected either under fluorescent microscopy (Leitz Dialux
22; Leica) or under laser scanning confocal microscopy performed using
a Zeiss LSM 510. Data presented represent findings arising from three
separate experiments.
RESULTS
Induction of a stress fibre phenotype in AGS cells by
both type I and type II H. pylori strains
Stress fibres were observed when AGS cells were infected with
a type I H. pylori, strain LC11 (30 min, m.o.i. 100 : 1) (Fig. 1).
By double immunostaining, H. pylori-infected AGS cells
showed stress fibre formation, compared with uninfected
control cells. This change in morphology was observed as
early as 30 min following infection and lasted for up to 4 h
post-infection. The altered morphology was observed for
more than 80 % of AGS cells following incubation with
H. pylori. These changes occurred before the time when
bacterial infection causes reduced viability of the gastric
epithelia by inducing programmed cell death (Jones, 1999).
Fig. 1. Type I H. pylori induces stress fibres in
AGS cells. Gastric epithelial cells were incubated
with H. pylori strain LC11 for 30 min and then
stained for F-actin with FITC-labelled phalloidin
(green) and bacteria were stained with anti-H.
pylori immune serum followed by goat anti-rabbit
serum conjugated with rhodamine (red). Images
were recorded by fluorescent microscopy (Leitz
Dialux 22). (a) AGS cells alone. (b) Attached
bacteria and stress fibres. Results are representative of three separate experiments. Approximate original magnifications, 31000.
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Journal of Medical Microbiology 52
Morphology of H. pylori-infected epithelium
Confocal microscopy of horizontal sections cut sequentially
from the apical to basal aspect of AGS cells (Z-sections)
showed that H. pylori adhered to and entered host cells and
induced the accumulation of actin filaments in host epithelia
(Fig. 2).
The stress fibre phenotype was observed in more than 90 % of
AGS cells infected with either a type I H. pylori, strain LC11
(Fig. 1), or a type II H. pylori, strain LC20 (Fig. 2). These data
indicate that H. pylori-induced epithelial cell stress fibre
formation occurred independently of the VacA, cagA and
cagE status of the infecting organism.
Type I H. pylori-induced elongation of AGS cells is
dependent on cagE
Analysis of cell morphology was also performed by the
detection of Æ-actinin using immunofluorescence and corresponding phase-contrast images. Epithelial cells infected
with a type I H. pylori strain displayed extended, slender and
elongated cell processes (Fig. 3c, d), also referred to as the
hummingbird phenotype (Segal et al., 1999), which were not
observed in uninfected cells (Fig. 3a, b). Epithelial cell
elongation occurred after infection with H. pylori strain
LC11 (m.o.i. 100 : 1) for 4 h, but not after 30 min. In
addition, cell elongation was not observed when AGS cells
were infected under the same experimental conditions with
the type II H. pylori strain LC20 (cagA , cagE , VacA ) (data
not shown).
In order to delineate further the virulence factors involved in
changing cell morphology, an isogenic cagE mutant was
incubated with AGS cells for 4 h. As shown in Fig. 3, the type I
wild-type H. pylori strain 8823 caused elongation of AGS cells
(Fig. 3e, f), compared with uninfected controls. However,
less elongation occurred when AGS cells were infected with
the isogenic cagE mutant (Fig. 3g, h).
In order to determine whether H. pylori induced cytoskeletal
rearrangements in other epithelial cell lines, immunofluorescent analysis was undertaken of T84 cells and HEp-2 cells
infected with H. pylori strain LC11 (4 h, m.o.i. 100 : 1). As
Fig. 2. Type II H. pylori induces stress fibres in AGS cells. Confocal microscopy of a horizontal section from top to bottom of the cell (Zsections) showing double staining of actin filaments (green) and H. pylori (red). Sections are presented in sequence from the apical
side (a) to the basolateral aspect (l) of an AGS cell. Images were recorded by using a Zeiss LSM 510 confocal microscope.
Approximate original magnifications, 3640.
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B. Su, P. J. M. Ceponis and P. M. Sherman
then stained for Arp3 to determine whether the F-actinrelated protein is also affected. As shown by confocal
microscopy (Fig. 5), H. pylori-infected AGS cells showed
Arp3 protein accumulation that co-localized with adherent
bacteria. Cells infected with a cagE mutant strain did not colocalize with Arp3, indicating that H. pylori-induced accumulation of Arp3 protein occurred in a cagE-dependent
manner.
DISCUSSION
This study shows that H. pylori strains induce morphological
changes in multiple epithelial cell lines. There was evidence of
both stress fibre formation (after 30 min of infection) and
elongation of infected cells (after 4 h post-challenge).
H. pylori-induced stress fibre formation was independent
of the cagA, cagE or VacA status of the infecting strain. In
contrast, elongation was observed only when cells were
infected with a type I H. pylori strain. Furthermore, this
elongation was characterized by Arp3 accumulation beneath
adherent bacteria in a cagE-dependent manner.
Fig. 3. Type I H. pylori-induced elongation in infected AGS cells is
dependent on cagE. (a)–(d) AGS cells were incubated with culture
medium only (a, b) or with H. pylori strain LC11 (cagAþ , cagEþ , VacAþ )
(c, d) for 4 h. Strain LC11 induced elongation of cells and the
formation of processes extending from the elongated cells (c, d),
whereas strain LC20 did not (data not shown). (e)–(h) AGS cells were
incubated with wild-type strain 8823 (e, f) or with the cagE mutant
(8823cagE : : kan) (g, h) for 4 h, demonstrating the induction of cell
elongation with the wild-type and the absence of changes in morphology with the mutant. Panels (a), (c), (e) and (g) represent phasecontrast microscopy images. Panels (b), (d), (f) and (h) show cells
stained for Æ-actinin. Images were recorded by fluorescent microscopy (Leitz Dialux 22). Representative fields are shown from the
results of four separate experiments. Approximate original magnifications, 3400.
shown in Fig. 4, Æ-actinin staining of H. pylori-infected T84
cells (Fig. 4a, b) and staining of the cytoplasmic protein Stat6
in infected HEp-2 cells (Fig. 4c, d) showed elongation of
about 80 % of cells in all three cell lines. These data indicate
that multiple epithelial cell lines elongate in response to
infection with a type I H. pylori isolate.
H. pylori induces accumulation of Arp3 in AGS cells
in a cagE-dependent manner
AGS cells were infected with H. pylori (4 h, m.o.i. 100 : 1) and
864
Stress fibre formation has been reported previously in
epithelial cells infected with H. pylori (Segal et al., 1999).
Herein, we observed similar effects with both type I and type
II H. pylori strains, thereby confirming the work of Palovuori
et al. (2000). Cell morphology changes were observed not
only using AGS cells, but also in other epithelial cell types,
including HEp-2 cells and T84 cells that have previously been
used as models of H. pylori infection (Papini et al., 1998;
Fahey et al., 2002). These findings indicate that the morphological changes induced by H. pylori are not restricted to
gastric-derived tissues.
Isogenic pairs of H. pylori strains were compared to demonstrate that elongation in infected epithelial cells was dependent on cagE. Previous studies have shown the role of CagA in
mediating morphological changes (Segal et al., 1999), where
the protein is translocated into host cells through a type-IV
secretion system to become phosphorylated on tyrosine
residues (Censini et al., 2001). Our findings, using type I
and type II clinical isolates from infected children, support
the observation that CagA is involved in inducing host-cell
cytoskeletal rearrangements. CagE, its gene encoded in the
cag PAI, mediates the activation of NF-kB and interleukin-8
secretion (Maeda et al., 2001). The present study shows that
cagE can also be involved in manipulation of the host-cell
cytoskeleton. It remains to be determined whether cagE acts
as a transporter for CagA and thereby contributes to the
observed changes in host-cell morphology (Guillemin et al.,
2002). Regardless, these findings indicate that multiple genes
on the cag PAI of H. pylori are involved in mediating
cytoskeletal rearrangements in infected epithelial cells.
The molecular control of H. pylori-induced cytoskeletal
rearrangements involves activation of Rho-GTPase Rac1
and Cdc42 (Churin et al., 2001; Hotchin et al., 2000). The
Arp2/3 complex is a seven-subunit protein complex containing two actin-related proteins, Arp2 and Arp3, that initiates
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Journal of Medical Microbiology 52
Morphology of H. pylori-infected epithelium
Fig. 4. T84 cells and HEp-2 cells elongate in
response to H. pylori infection. T84 (a, b) and
HEp-2 (c, d) cell monolayers were incubated with
medium alone (a, c) or with H. pylori strain LC11
(b, d) for 4 h at 37 8C. Both cell lines showed
elongated morphology in response to H. pylori
infection, comparable to that observed using
AGS cells. Cells in (a) and (b) were stained for
Æ-actinin and those in (c) and (d) were stained
with antibody to detect the cytoplasmic protein
Stat6. Findings presented represent the results
of three separate experiments. Approximate
original magnifications, 3600.
Fig. 5. Accumulation of Arp3 beneath adherent
H. pylori in infected AGS cells. AGS cells were
infected with H. pylori strain LC11 (4 h, m.o.i.
100 : 1) and stained for H. pylori (a, d, g; fluorescein-conjugated secondary antibody) and
Arp3 (b, e, h; red). Merged images are shown in
(c), (f) and (i). AGS cells were uninfected (a–c) or
infected with wild-type strain 8823 (d–f) or the
mutant 8823cagE : : kan (g–i). Arp3 accumulated beneath adherent bacteria (e, f). cagE is
involved in cell elongation because the cagE
mutant did not induce cell changes (g–i), compared with the morphological changes induced
by the wild-type parental strain (d–f). Images
were recorded on a Zeiss LSM 510 confocal
microscope. Approximate original magnifications, 3640.
the formation of actin-filament networks in response to
intracellular signals (Dayel et al., 2001). Bacterial pathogens
have developed a variety of mechanisms to utilize the
cytoskeleton of host cells to their benefit (Steele-Mortimer
et al., 2000). For example, induction of ruffles at the plasma
membrane allows the invasion of non-phagocytic cells by
Salmonella typhimurium (Jones et al., 1993). Shigella flexneri
induces epithelial cell signalling through activation of small
GTPases of the Rho family and c-Src to cause major
cytoskeletal rearrangements leading to bacterial entry. The
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surface protein IcsA of Shigella flexneri binds and activates
neuronal Wiskoff–Aldrich syndrome protein (N-WASP) to
induce F-actin rearrangements in an Arp2/3-dependent
mechanism (Sansonetti, 2001). Also, the surface protein
ActA of Listeria monocytogenes directly activates the Arp2/3
complex (Cossart, 2000; Boujemaa-Paterski et al., 2001).
Our findings indicate that H. pylori induces the accumulation of Arp3 underneath adherent bacteria and suggest,
therefore, that Arp3 is important in altering host-cell
morphology.
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865
B. Su, P. J. M. Ceponis and P. M. Sherman
The Yersinia enterocolitica protein invasin binds Æ5 1 integrin to cause actin polymerization through Cdc42 activation
and recruitment of a WASP and Arp2/3 complex
(Wiedemann et al., 2001) to achieve internalization into
cells (Alrutz et al., 2001). The Æ5 1 integrin mediates
H. pylori adherence and invasion into cultured cells, which
also requires tyrosine kinase activity and actin polymerization (Su et al., 1999). Thus, H. pylori binding to Æ5 1 integrin
could lead to cytoskeletal rearrangements through Cdc42
activation and recruitment of the Arp2/3 complex.
In summary, this study shows that, in addition to cagA and
VacA, cagE is involved in the cytoskeletal rearrangements of
host cells that occur in response to infection with H. pylori
strains originally isolated from children. In addition, there is
Arp2/3 accumulation beneath adherent bacteria that occurs
in a cagE-dependent fashion.
ACKNOWLEDGEMENTS
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This study was supported by a Canadian Institutes of Health Research
(CIHR)-University Industry (Canadian Association of Gastroenterology/AstraZeneca Canada) Research Initiative Award and by an operating grant from the CIHR. P. J. M. C. is the recipient of a CIHR doctoral
studentship award and is a CIHR Strategic Training Fellow in Cell
Signaling in Mucosal Inflammation and Pain (STP-53877). P. M. S. is
the recipient of a Canadian Research Chair in Gastrointestinal Disease.
Helicobacter pylori induces gastric epithelial cell apoptosis in association
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