Systematic Analysis of Phosphotyrosine Antibodies Recognizing Single Phosphorylated EPIYA-Motifs in CagA of Western-Type Helicobacter pylori Strains
et al. (2014) Systematic Analysis of Phosphotyrosine Antibodies Recognizing Single Phosphorylated
EPIYA-Motifs in CagA of Western-Type Helicobacter pylori Strains. PLoS ONE 9(5): e96488. doi:10.1371/journal.pone.0096488
Systematic Analysis of Phosphotyrosine Antibodies Recognizing Single Phosphorylated EPIYA-Motifs in CagA of Western-Type Helicobacter pylori Strains
Judith Lind 0
Steffen Backert 0
Klaus Pfleiderer 0
Douglas E. Berg 0
Yoshio Yamaoka 0
Heinrich Sticht 0
Nicole Tegtmeyer 0
Ivo G. Boneca, Institut Pasteur Paris, France
0 1 Friedrich Alexander University Erlangen-Nuremberg, Department of Biology, Division of Microbiology, Erlangen, Germany, 2 Division of Infectious Disease, Department of Medicine, University of California San Diego, La Jolla, California, United States of America, 3 Oita University Faculty of Medicine, Department Environmental and Preventive Medicine, Yufu, Japan, 4 Friedrich Alexander University Erlangen-Nuremberg, Bioinformatics, Institute for Biochemistry , Erlangen , Germany
The clinical outcome of Helicobacter pylori infections is determined by multiple host-pathogen interactions that may develop to chronic gastritis, and sometimes peptic ulcers or gastric cancer. Highly virulent strains encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation at EPIYA-sequence motifs, called A, B and C in Western-type strains, by members of the oncogenic Src and Abl host kinases. Phosphorylated EPIYA-motifs mediate interactions of CagA with host signaling factors - in particular various SH2-domain containing human proteins - thereby hijacking multiple downstream signaling cascades. Observations of tyrosine-phosphorylated CagA are mainly based on the use of commercial phosphotyrosine antibodies, which originally were selected to detect phosphotyrosines in mammalian proteins. Systematic studies of phosphorylated EPIYA-motif detection by the different antibodies would be very useful, but are not yet available. To address this issue, we synthesized phospho- and non-phosphopeptides representing each predominant Western CagA EPIYA-motif, and determined the recognition patterns of seven different phosphotyrosine antibodies in Western blots, and also performed infection studies with diverse representative Western H. pylori strains. Our results show that a total of 9-11 amino acids containing the phosphorylated EPIYA-motifs are necessary and sufficient for specific detection by these antibodies, but revealed great variability in sequence recognition. Three of the antibodies recognized phosphorylated EPIYA-motifs A, B and C similarly well; whereas preferential binding to phosphorylated motif A and motifs A and C was found with two and one antibodies, respectively, and the seventh anti-phosphotyrosine antibody did not recognize any phosphorylated EPIYA-motif. Controls showed that none of the antibodies recognized the corresponding non-phospho CagA peptides, and that all of them recognized phosphotyrosines in mammalian proteins. These data are valuable in judicious application of commercial antiphosphotyrosine antibodies and in characterization of CagA phosphorylation during infection and disease development.
Competing Interests: The authors have declared that no competing interests exist.
Posttranslational modification of proteins by kinases is
important in many cell signaling processes. Although phosphorylation of
some serine, threonine and histidine residues occurs both in
prokaryotes and eukaryotes, tyrosine phosphorylation is believed
generally to be mostly restricted to higher organisms, where it
plays important roles in signal transduction and developmental
regulation . In fact, typical tyrosine kinase genes have been
reported in only very few sequenced bacterial genomes [2,3].
However, there are numerous reports of effector proteins from
pathogenic bacteria that undergo tyrosine phosphorylation after
translocation into eukaryotic host cells, which is a remarkable
example of convergent evolution, not just descent from a common
ancestor [2,46]. In various cases, effector protein
phosphotyrosines together with some flanking residues act as recognition
motifs for eukaryotic signaling factors. They recruit in particular
cellular binding partners that contain SH2 (Src homology 2)
domains, but not PTB (phosphotyrosine binding) domains, and
thereby target and subvert eukaryotic signal transduction
pathways in ways that benefit the pathogen [2,4]. This virulence
strategy is well-established for six different bacterial pathogens:
enteropathogenic Escherichia coli (EPEC), Helicobacter pylori,
Chlamydia trachomatis, Bartonella henselae, Anaplasma phagocytophilum and
Ehrlichia chaffeensis .
The virulence factor CagA of the gastric pathogen H. pylori
provides a prime example of such tyrosine phosphorylatable
effector proteins [8,1418]. CagA is delivered to host cells via a
type IV secretion system (T4SS), a complex syringe-like pilus
device whose synthesis is encoded in the cag pathogenicity island
and induced on contact with target cells . A hallmark of
cultured AGS gastric epithelial cells infected by CagA-producing
H. pylori strains is the development of the so-called
hummingbird or elongation cell phenotype [8,23,24]. This in vitro
phenotype is likely to reflect several in vivo signaling processes that
control immune responses, wound healing, metastasis and invasive
growth of cancer cells [25,26]. CagA interacts with more than 20
host cell proteins, variously in phosphorylation-dependent and
phosphorylation-independent manners . Sequence analysis and
site-directed mutagenesis identified a series of EPIYA
(Glu-Pro-IleTyr-Ala) motifs near the CagA carboxy-terminus as
phosphorylation sites and showed that phospho-CagA is essential for AGS
cell elongation [23,24,2730]. Four specific EPIYA-motifs termed
A, B, C and D have been identified based primarily on relative
positions in CagA and adjoining amino acid sequences despite
some diversity in flanking sequences and even in EPIYA-motifs
themselves [5,6,31-33]. Whereas EPIYA-A and EPIYA-B motifs
are found in all CagA proteins, EPIYA-C is mainly found in
strains of African and Indo-European ancestry, whereas CagA
from most East-Asian strains contain the more potent EPIYA-D
motif in place of EPIYA-C. Although most CagA proteins contain
only three EPIYA-motifs, some strains have additional
EPIYAcopies [5,6], due to recombination between repetitions in flanking
DNA sequences [31,3444]. Two-dimensional gel electrophoresis
(2-DE) of phospho-CagA proteins from infections of AGS or
MKN-28 cells by Western H. pylori strains with 3-4 EPIYA-motifs
has shown that only one or two tyrosines (but not three) can be
phosphorylated per CagA molecule [45,46].
The host tyrosine kinases active on these CagA EPIYA-motifs
were identified as members of the Src [24,47] and Abl [48,49]
families. We found that c-Src only phosphorylated EPIYA-C or
EPIYA-D, while c-Abl phosphorylated EPIYA-A, EPIYA-B,
EPIYA-C, and EPIYA-D. Further analysis revealed that none of
the phosphorylated EPIYA-motifs alone was sufficient for inducing
AGS cell elongation . Site-directed mutagenesis has shown
that the preferred combination of phosphorylated EPIYA-motifs
in Western strains was EPIYA-A and EPIYA-C, either across two
CagA molecules or simultaneously on one . These studies thus
identified a tightly regulated hierarchic phosphorylation model for
CagA starting at EPIYA-C/D, followed by phosphorylation at
EPIYA-A or EPIYA-B. However, the observation that CagA can
undergo tyrosine phosphorylation in host cells is mainly based on
the use of commercial a-phosphotyrosine antibodies in Western
blots [8,1417]. These antibodies had been selected years ago to
specifically detect a broad range of phosphorylated tyrosine
residues in mammalian proteins. Three of these
phosphotyrosine-specific antibodies have been shown to exhibit a similar
phosphotyrosine-binding preference in mammalian proteins,
preferably with a proline residue at position +3 and a leucine at
position -1 . However, proline and leucine residues are not
present at this position in the corresponding phosphorylation sites
of CagA [2,5,6]. In addition, systematic studies on the specific
recognition patterns of phosphotyrosine motifs in the delivered
bacterial effectors such as CagA by a large number of different
antibodies are not yet available. Therefore, we addressed this
important question and synthesized phospho- and non-phospho
peptides of each CagA EPIYA-motif from Western strains to
investigate the recognition specificity by seven commercially
available phosphotyrosine antibodies. We also performed infection
studies with H. pylori to investigate the recognition patterns of
phosphorylated CagA upon delivery into host target cells.
Materials and Methods
Bacterial strains and culture conditions
All wild-type H. pylori strains were typical type-I isolates
expressing CagA. The generation of an isogenic DcagA mutant
has been described . H. pylori was grown in thin layers on horse
serum GC agar plates supplemented with vancomycin (10 mg/
mL), nystatin (1 mg/mL), and trimethoprim (5 mg/mL) as
described previously [51,52]. All antibiotics were obtained from
Sigma-Aldrich (St. Louis, MO, USA). Bacteria were grown at
37uC for 2 days in an anaerobic jar containing a Campygen gas
mix of 5% O2, 10% CO2, and 85% N2 (Oxoid, Wesel, Germany)
Synthesis of phospho- and non-phospho CagA peptides
The C-STEPIYAKVNK (EPIYA-A), C-STEPI(pY)AKVNK
(phospho-EPIYA-A), C-PEEPIYTQVAK (EPIYA-B),
C-PEEPI(pY)TQVAK (phospho-EPIYA-B), C-SPEPIYATIDD (EPIYA-C)
and C-SPEPI(pY)ATIDD (phospho-EPIYA-C) sequences were
synthesized by Jerini AG (Berlin, Germany) and the
C-TEPI(pY)AKVN, C-EPI(pY)AKV and C-PI(pY)AK peptides by
Biosyntan GmbH (Berlin, Germany). These 11-mer peptides were
chosen to compare the three EPIYA-motifs because
a-phosphotyrosine antibodies typically recognize short phosphopeptides
[35,50,55,56]. Commonly, 11-mer peptides are also used for
immunizations to generate phospho-specific antibodies, which
then recognize the corresponding phosphopeptides bound to
affinity columns and in ELISA (Biogenes, Berlin, Germany). All
above EPIYA peptides were purified by HPLC, and full-length
synthesis as well as purity of each peptide was confirmed by mass
spectrometry by Jerini AG and Biosyntan AG. The peptides were
resolved at a concentration of 1 mg/mL in DMSO and stored at
Cloning and purification of recombinant CagA
A cagA gene fragment of 891 bp (corresponding to amino acid
positions 890-1,186 in the C-terminus of CagA from H. pylori
strain 26695) including the EPIYA-motifs A, B and C was
synthesized by Geneart (Regensburg, Germany). This cagA
fragment was then subcloned into the pGEX-2T vector using
the restriction enzymes BamHI and EcoRI. The resultant construct
was transformed into the E. coli strain BL21. Protein expression
was carried out in 800 mL of pre-warmed LB medium, which
were inoculated with 8 mL of an overnight culture. When cells
reached an OD600 nm of 0.4, protein expression was induced with
1 mM IPTG. After 4 hours, the cells were harvested and the pellet
was stored at 220uC. Prior to cell disruption by sonication, the
pellet was suspended in 30 mL PBS (phosphate buffered saline)
supplemented with 1 mM DTT. To remove cell debris, the
sample was centrifuged at 25,000 rpm for 50 min. Subsequently,
the supernatant was loaded onto a 1 mL GSTrap HP column (GE
Healthcare, Munich, Germany) and bound target protein was
eluted with 50 mM Tris-HCl pH 8.0 containing 10 mM reduced
Glutathione and 1 mM DTT. Fractions were analyzed by
SDSPAGE and Western blotting. Selected CagA-positive fractions
were pooled and further purified using HiLoad 16/60 Superdex
75 (GE Healthcare) equilibrated with 20 mM Tris-HCl pH 8.0,
150 mM NaCl and 1 mM DTT. Peak fractions were analyzed by
SDS-PAGE and Western blot. Fractions containing the purified
CagA fragment showed no detectable impurities by other proteins.
In vitro phosphorylation of CagA with recombinant c-Abl
1010 cells of H. pylori strain 26695 expressing wild-type CagA (or
isogenic DcagA mutant as control) were harvested in 1 mL of
kinase buffer as described previously . A total of two units of
recombinant human c-Abl tyrosine kinase (NEB GmbH,
Frankfurt/M., Germany) and 1 mmol/L of ATP were mixed with 30 mL
H. pylori lysate or 10 mL recombinant CagA and incubated for 30
min at 30uC as described previously . The reactions were
terminated by a 5 min denaturing step at 95uC.
Twenty mg of each CagA peptide or 30 ml of the in vitro kinase
reaction products described above were mixed in 1 mL of blotting
buffer (192 mM Glycin; 20 mM Tris-HCl, pH 8,4; 0.1% SDS;
20% Methanol). These peptide samples were spotted onto
Immobilon-P membrane (Merck Millipore, Darmstadt, Germany)
using the BioDot SF apparatus (Bio-Rad, Munich, Germany). The
resulting Dotblots were dried and subjected to antibody detection
as described below for Western blots.
Eukaryotic cell culture and elongation phenotype
Human adherent gastric adenocarcinoma epithelial cells (AGS,
ATCC CRL-1730) were grown in 6-well plates containing RPMI
1640 medium (Invitrogen) supplemented with 25 mM HEPES
buffer and 10% heat-inactivated FBS (Biochrom, Berlin,
Germany) for 2 days to approximately 70% confluence [57,58]. Cells
were serum-deprived overnight and infected with H. pylori at a
MOI of 50 for 6 hours. After infection, the cells were harvested in
ice-cold PBS containing 1 mmol/L Na3VO4 (Sigma-Aldrich).
Elongated AGS cells in each experiment were quantitated in 10
different 0.25-mm2 fields using an Olympus IX50 phase contrast
microscope. All experiments were performed in triplicate and
subjected to statistical analysis as described below.
SDS-PAGE and immunoblot analysis
AGS cell pellets with attached bacteria were mixed with equal
amounts of 2 x SDS-PAGE buffer and boiled for 5 minutes.
Proteins were separated by SDS-PAGE on 6% polyacrylamide
gels and blotted onto PVDF membranes (Immobilon-P, Merck
Millipore). Membranes were blocked in TBST with 3% BSA or
5% skim milk for 1 hour at room temperature. Membranes were
incubated with the seven a-phosphotyrosine antibodies (Table 1)
or mouse monoclonal a-CagA antibody (Austral Biologicals, San
Ramon, CA, USA) according to the instructions of the
manufacturer. Phosphorylated and non-phosphorylated CagA proteins
were detected using horseradish peroxidaseconjugated
antimouse or anti-rabbit polyvalent sheep immunoglobulin secondary
antibodies in the ECL Plus chemoluminescence Western blot
system of GE Healthcare .
Quantification of Dotblot and Western blot signals
Spot or band intensities on blots probed with the different
aphosphotyrosine antibodies were quantitated with the
LumiImager F1 (Roche Diagnostics, Mannheim, Germany).
Densitometric measurement of signal intensities revealed the percentage of
phosphorylation per sample . The strongest spot on Dotblots
was set at 100% as indicated in the corresponding Figures.
All data were evaluated using Student t-test with SigmaStat
statistical software (version 2.0). All error bars shown in figures and
b oh :io
those quoted following the +/2 signs represent standard
Strong recognition of 9-mer and 11-mer CagA
phosphopeptides by a-phosphotyrosine antibodies
The majority of CagA proteins in Western type clinical isolates
contain three EPIYA-motifs, such as that of the first fully
sequenced H. pylori strain 26695 (Figure 1A). Commercial
phosphotyrosine antibodies typically recognize short mammalian
phosphopeptides; in some cases even five amino acids were
previously shown to be sufficient for recognition [35,50,55,56]. To
systematically analyze the recognition capabilities of
phosphorylated CagA EPIYA-motifs by a-phosphotyrosine antibodies we
first synthesized a series of peptides derived from the EPIYA-A
motif exhibiting the phosphotyrosine residue in the middle +/2
five, four, three or two flanking amino acids, including the
STEPIYAKVNK (11-mer), TEPIYAKVN (9-mer), EPIYAKV
(7mer) and PIYAK (5-mer) sequences as indicated (Figure 1B, top).
Twenty mg of each EPIYA-peptide was immobilized per spot on
PVDF membranes using the Dotblot method followed by probing
with the a-phosphotyrosine antibodies a-PY99 and a-PY20 (BD).
The results show that both antibodies recognized 11-mers and
9mers with similar strong intensity, while recognition of 7-mers and
5-mers was significantly reduced (Figures 1B). An 11-mer of the
corresponding non-phospho-EPIYA peptide did not produce
signals in a parallel control experiment (Figure 1B). In further
tests, the efficient Dotblot approach detected full-length CagA in
bacterial lysates or recombinant C-terminal CagA when
phosphorylated by c-Abl in in vitro kinase reactions (Figure 1C). This
experiment also confirmed that the a-phosphotyrosine antibodies
do not cross-react with non-phospho CagA proteins in control
reactions without c-Abl kinase (Figure 1C and data not shown), as
Recognition of phosphopeptides from EPIYA-A, -B and -C
by a-phosphotyrosine antibodies
After validating the Dotblot approach using phospho-CagA
peptides functions, we synthesized 11-mer phospho- and
nonphosphopeptides of EPIYA-B (PEEPIYTQVAK) and EPIYA-C
(SPEPIYATIDD) motifs as indicated (Figure 2A, top) and probed
them with a set of seven different commercially available
aphosphotyrosine antibodies (Table 1). The results confirmed that
these antibodies do not cross-react with the corresponding
nonphospho CagA peptides (Figure 2A), and showed, interestingly,
that all antibodies except a-PY350 predominantly recognized the
EPIYA-A phosphopeptide, which did not recognize any of the
CagA-derived phosphopeptides. Some remarkable differences,
however, were seen in the abilities of the antibodies to react with
the EPIYA-B and EPIYA-C phosphopeptides. In particular, three
antibodies (a-PY99, a-PY20-BD, a-PY20-SC) recognized all three
phosphorylated EPIYA-A, EPIYA-B and EPIYA-C peptides with
similar strong signals, while two others (a-PY100, a-PY69)
recognized the phosphorylated EPIYA-A peptide preferentially,
and phospho-EPIYA-B and phospho-EPIYA-C more weakly
(Figure 2A). A sixth antibody (a-PY102) recognized
phosphorylated EPIYA-A strongly and EPIYA-C very weakly, while the
seventh antibody, a-PY350, did not recognize any phosphorylated
EPIYA-peptide, as noted above (Figure 2A). This inability to
detect signals with the a-PY350 antibody was unaffected by
binding five-fold more peptide (0.1 mg) to membranes or use of
twice as much antibody (data not shown), even though we
confirmed that our batch of a-PY350 effectively recognizes
phosphorylated host proteins, as discussed below. The
quantification of Dotblot data from three independent experiments is
presented in Figure 2B. Taken together, the results revealed
enormous variability in the capability of seven a-phosphotyrosine
antibodies to recognize phosphorylated EPIYA-motifs A, B and/
Recognition of phosphorylated CagA protein during
infection of AGS cells
Next, we aimed to investigate recognition patterns of
phosphorylated CagA proteins during infection. For this purpose, we
selected eight representative H. pylori strains from different
continents, including Africa, Europe, Asia, North America and
South America (Table 2). Sequence alignment of CagA
carboxyterminal regions establishes that all three EPIYA-motifs A, B and
C are present, although with some heterogeneity in flanking amino
acid sequences (Figure 3). AGS cells were infected for 6 hours with
these H. pylori strains and the elongation phenotype of infected
cells was monitored over time, to indicate successful CagA delivery
and phosphorylation . About 60270% of AGS cells
exhibited the elongation phenotype after infection with each H.
pylori strain, suggesting that high amounts of phospho-CagA are
produced (Figure 4). Next, protein lysates were prepared from
infected AGS cells and analyzed with the different antibodies.
First, the samples were probed with a monoclonal a-CagA
antibody recognizing both non-phosphorylated and
phosphorylated forms of CagA, to ensure that similar amounts of CagA
protein were loaded in each lane (Figure 5, top). The various CagA
variants exhibit different band sizes between 1302150 kDa as
expected from the different strains (Table 2). The samples were
then probed with the seven a-phosphotyrosine antibodies. The
results confirmed that all antibodies recognized host cell proteins
(Figure 5, asterisks), whereas phospho-CagA was recognized by
only some of the antibodies (Figure 5, arrows). The quantification
data for phospho-CagA from three independent experiments are
presented in Table 3. Two antibodies (a-PY99 and a-PY20-BD)
produced strong phospho-CagA bands from all eight strains with
little background in the 1252150 kDa region (Figure 5). This
indicates that sufficient detectable phospho-CagA was generated
during infection and confirmed the elongation phenotype data for
each strain noted above. Interestingly, phospho-CagA from two
strains (26695 and Sat464) was strongly recognized by six of the
seven a-phosphotyrosine antibodies. Antibodies a-PY20-SC,
aPY100 and a-PY102, which recognized all three phospho-EPIYAs
in the above Dotblots gave mixed results. Although these three
antibodies produced strong bands with phospho-CagA from H.
pylori strains 26695 and Sat464, they produced only weak signals
with phospho-CagA from strains P310, Lit75, B8, 2002-370 and
Oki61 (Figure 5). In addition, it should be noted that the
phosphoCagA patterns among these three antibodies were not identical.
For example, phospho-CagA from strain Gam94/24 was strongly
recognized by a-PY20-SC, but only weakly by a-PY100 and not
by a-PY102. The seventh antibody, a-PY350, did not recognize
phosphorylated CagA from any H. pylori strain tested, in
agreement with results obtained using EPIYA phosphopeptides,
although it did recognize major 125, 150 and 170 kDa host
phosphoproteins (Figure 5, bottom). Antibody a-PY69 also
appears to be not very useful for studying CagA phosphorylation
in AGS cells because of the presence of a cross-reacting host
phosphoprotein at about 140 kDa, which is in the size range of
various phospho-CagA bands (Figure 5).
Phospho-EPIYA recognition patterns are influenced both
by length and amino acid sequence
The binding specificities of the a-PY20 and a-PY100 antibodies
have been experimentally characterized using microarrays of
phosphopeptide libraries derived from mammalian proteins .
This knowledge allowed to investigate the sequence features
responsible for differences in binding affinity observed for the
EPIYA-B phosphopeptide in more detail. This peptide is
recognized by a-PY100 with lower affinity, whereas the other
phosphopeptides (EPIYA-A and EPIYA-C) are recognized by
aPY20 and a-PY100 with similar affinity (Figure 1, Table 3). One
striking feature of phospho-EPIYA-B is the presence of a
negatively charged glutamate residue in the -4 position, which is
highly conserved in CagA among different H. pylori strains
(Figure 3, shaded with green). The peptide array data from Tinti
and co-workers  indicates that the presence of a glutamate at
this position negatively affects a-PY100 binding, but not a-PY20
binding. Thus, the differences in a-PY20 and a-PY100 binding of
phospho-EPIYA-B in our experiments are in line with data from
mammalian phosphoproteins and can most likely be attributed to
this sequence position. In addition, phosphorylated CagA from
different H. pylori strains during infection is generally detected
better by a-PY20 than by a-PY100 (Figure 5 and Table 3). These
differences in a-PY100 binding most likely result from
strainspecific sequence variations in the vicinity of the EPIYA-A motif,
which is generally less well-conserved than in the vicinity of the
EPIYA-motifs B and C (Figure 3). Phospho-CagA produced by the
H. pylori strains P310 and Gam94/24 is bound by a-PY100 with
relatively low affinity (Figure 5), which can be attributed at least in
part to a glutamate at the -4 position of the EPIYA-A motif
(Figure 3, shaded with green). Further inspection of the overall
data in Table 3 suggests that additional features also affect
aPY100 binding specificity, as evidenced by the low affinity of
phospho-CagA from H. pylori strains B8 and 2002-370 produced
during infection. These features are likely determined by residues
flanking the EPIYA core motif, as suggested by the significantly
weaker binding of the 5-mer and 7-mer peptides described above.
antibodies and exposed as described in the Material & Methods section.
(B) Quantification of spot intensities on Dotblots. Signal intensities were
measured densitometrically with the Lumi-Imager F1 and revealed the
percentage of phosphorylation signal per sample. The strongest spot
on every Dotblot was set at 100% for each of the different
aphosphotyrosine antibodies as indicated. Quantitation results are
shown for three independent experiments.
The CagA protein and its EPIYA-motifs are known for long
time as important virulence markers of H. pylori [5,6,18,23,24,27
34,67,68]. These EPIYA-repeat motifs were originally described in
1993 by the group of Antonello Covacci . Even before these
sequences were identified to be the sites of CagA tyrosine
phosphorylation , certain variations within the EPIYA-region
at the sequence level have been reported and associated with
gastric disease [70,71]. After the discovery of CagA tyrosine
phosphorylation about 15 years ago , intensive efforts were
undertaken to identify the involved host cell kinases . Mammals
encode about 90 protein tyrosine kinases , which
phosphorylate their mammalian substrates with specificity depending on
amino acid sequences next to the targeted tyrosine residue .
The EPIYA-motifs in CagA commonly have isoleucine at the 1
position and the small amino acid alanine at the +1 position, which
is similar to the phosphorylation consensus motif EEIYG/E of the
host kinase c-Src . Indeed, several lines of evidence have then
demonstrated that c-Src and also c-Abl family kinases mediate
CagA phosphorylation in vitro and in vivo [24,4649,74]. However,
progress in this research field has been hampered by a lack of
standardized commercial EPIYA-specific phospho-antibodies and
lack of knowledge about which phospho-EPIYAs are recognized
by the set of available a-phosphotyrosine antibodies (Table 1).
Systematic examination of which phosphotyrosine residues in the
three different EPIYA-motifs are recognized by these various
antibodies has not yet been reported. Thus, despite the years of
research, detailed, clear-cut conclusions about CagA
phosphorylation patterns in clinical strains have not been possible. Here, we
investigated for the first time the recognition specificity of seven
commercially available a-phosphotyrosine antibodies with regard
to CagA EPIYA-motifs A, B and C in Western H. pylori strains. We
found that these antibodies exhibit a remarkable variety in
recognition of the various phosphorylated EPIYA-motifs. These
results shed light on the usefulness of these antibodies in research
and provide valuable new insights for future studies on CagA
phosphorylation sites and downstream signaling.
It is well known that the a-phosphotyrosine antibodies were
originally developed for mammalian proteins and typically
recognize short amino acid stretches containing the
phosphorylated tyrosine residue, including synthetic phospho-peptides
[35,50,55,56]. We therefore proposed that phospho-peptides
derived from the CagA EPIYA-motifs would be useful for studying
the recognition capabilities by seven commercial antibodies. Using
this strategy we found that 9-mers and 11-mers of
EPIYAphosphopeptides are necessary and sufficient for strong antibody
recognition. In addition, three a-phosphotyrosine antibodies
(aPY99, a-PY20-BD and a-PY20-SC) recognized all three 11-mer
phospho-EPIYA peptides (A, B and C) with similar and very
strong affinity, confirming that this approach works for peptides
derived from bacterial effector proteins in addition to mammalian
peptides. Overall, this observation nicely correlated with the
pronounced recognition of phospho-CagA in cell lysates produced
after infection with eight different H. pylori strains (Table 3).
Another antibody (a-PY100) reacted preferentially with
phosphoH. pylori strain
Not evaluated directly
[Non-atrophic gastritis assumed]
Not evaluated directly
[Non-atrophic gastritis assumed]
EPIYA peptides A and C, and also produced acceptable
phosphoCagA patterns by Western blotting of extracts from infected cells.
In addition, antibody a-PY102 strongly recognized
phosphoEPIYA peptide A and very weakly phospho-EPIYA peptide C, but
reacted only with six of eight phospho-CagAs in infected cells.
Although a-PY69 also recognized phospho-EPIYA-A
preferentially (with weak signals for B and C), it also strongly reacted with
host cell proteins in the 125140 kDa range and is therefore not
useful for studying CagA phosphorylation during infection.
Importantly, the antibodies a-PY99, a-PY20-BD, a-PY20-SC,
aPY100 and a-PY102 did not react with AGS host cell proteins in
the 130150 kDa range, another important criterion that makes
CagA EPIYA type
D. Kersulyte and D. Berg
D. Kersulyte and D. Berg
D. Kersulyte and D. Berg
Figure 4. AGS cell elongation induced during infection with
different clinical H. pylori strains. AGS cells were infected for
6 hours with various CagA-expressing H. pylori strains as indicated. (A)
The number of elongated cells in each experiment was quantitated in
triplicate in 10 different 0.25-mm2 fields . (B) Representative
phase contrast micrographs of AGS cells infected with the different
strains as indicated.
them useful for H. pylori studies. These results allow us to
recommend the use of up to five a-phosphotyrosine antibodies for
studies of infection by H. pylori (a-PY99, a-PY20-BD and
a-PY20SC, and if needed, also a-PY100 and a-PY69) based on their
ability to recognise a broad spectrum of different phospho-CagAs,
and thereby to clarify what EPIYAs are phosphorylated.
Most knowledge about phosphotyrosine-based protein-protein
interactions is derived from use of a-phosphotyrosine antibodies to
investigate mammalian signaling factors . Recent studies have
applied the microarray technology to characterize the substrate
specificity of widely used a-phosphotyrosine antibodies (including
a-PY100 and a-PY20) in human phosphopeptides . The
conclusions that were drawn from this analysis are: (i) the
antibodies exhibit a similar phosphotyrosine binding specificity
whilst at the same time showing specific binding preference
depending on some flanking amino acids; and (ii) the similar
phosphotyrosine binding specificity is rather broad although
proline residues are preferred at position +3 and leucine at
position 1 . In addition, the investigations by Tinti and
coworkers indicated that the presence of a negatively charged residue
such as glutamate at the 4 position specifically affects interaction
with a-PY100, but not with a-PY20 . Interestingly, EPIYA-B
has a glutamate residue at the 4 position (and sometimes at
EPIYA-A), which is highly conserved in CagA among the different
H. pylori strains (Figure 3). However, inspection of our overall data
in Table 3 suggests that additional features also affect a-PY100
binding specificity, as evidenced by the low affinity to
phosphoCagA from strains B8 and 2002-370. Both H. pylori strains differ at
several sequence positions in the vicinity of EPIYA-motif A
rendering it difficult to clearly correlate antibody affinity with a
single sequence position. This also suggests the existence of other
structural features, like secondary structure of the motif and its
vicinity, which may additionally affect a-phosphotyrosine antibody
How could one apply this knowledge of binding preferences of
a-phosphotyrosine antibodies for certain EPIYA-motifs? The use
of a-phosphotyrosine antibodies by probing one-dimensional
SDS-PAGE blots of lysates from H. pylori infected cells provides
a useful first picture of CagA phosphorylation events .
However, this picture is not completely useful because increased
phospho-CagA signal intensities that can arise over time could
result either from increased amounts of translocated CagA
molecules undergoing phosphorylation at a specific site, from
increased phosphorylation of multiple sites per CagA molecule, or
both possibilities that cannot be distinguished on 1-DE gels.
Using 2-DE gel separation of different CagA protein spots, we
have recently shown that CagA can be simultaneously
phosphorylated either on one or two EPIYAs per molecule . This
suggests the appearance of multiple differentially phosphorylated
CagA protein species in host cells, each with different functions,
and thus could explain how CagA achieves signaling to many
different host binding partners . To investigate this hypothesis
further and to obtain better tools for clinical applications, it will be
desirable in the near future to generate phospho-specific a-CagA
antibodies for each EPIYA-motif. In this context, the a-PY69 and
a-PY102 antibodies may already be useful because they
predominantly detect phospho-EPIYA-A as shown here. A very few
studies have reported generation of phospho-specific a-CagA
antibodies designed for one specific EPIYA-motif, although to our
knowledge these are not commercially available. A first study
showed phosphorylation of EPIYA-motif B after infection and
after transfection of CagA from strain NCTC11637 ; and
another study reported on specific phosphorylation of EPIYA-C in
CagA from strains 26695 and P12, including respective
phenylalanine substitution controls . In addition, the generation of a
phosphospecific antibody against EPIYA-C motif in CagA from
strain NCTC11637 was described, but this antibody also
recognized the phosphorylated CagA forms of strains GC401
and G501, which lack the EPIYA-motif C, a result suggesting that
this antibody may be rather unspecific, able to recognize other
phosphorylation sites . Thus, the production of more reliable
EPIYA-site specific phospho-antibodies could yield highly valuable
Our studies focused on the EPIYA-motifs A, B and C of
Western H. pylori strains. However, it will be also important to
investigate the phosphorylation of the generally more potent CagA
proteins East-Asian strains with their EPIYA-A, B and D motifs,
even though the East-Asian 11-mer EPIYA-D sequence
(SPEPIYATIDF) is similar to the Western EPIYA-C sequence
(SPEPIYATIDD) . We recently showed that c-Src kinase
only phosphorylates CagA EPIYA-C and EPIYA-D motifs, not
EPIYA-A and EPIYA-B, using Western blots and the a-PY99
antibody . This result suggests that phospho-EPIYA-D maybe
recognized by many or all antibodies that recognize
phosphoEPIYA-C. However, this has not yet been tested experimentally,
and more studies are certainly warranted to understand the CagA
phosphorylation sites in greater detail. A detailed analysis of the
various East-Asian EPIYAs is currently underway in our
laboratories. One additional option would be to use the
phosphopeptide microarray technology to characterize all known
individual phospho-EPIYA-motifs and associated amino acid
polymorphisms, as was described for human proteins , and
to test for antibody recognition and host effector protein binding.
This would help to better understand the role of single
EPIYAmotifs for CagA function and possibly allow correlations and risk
predictions for the development of diverse gastric diseases in the
future. In addition to CagA, further studies should also focus on
the investigation of EPIYA-like phosphorylation sites and
downstream signaling used by other bacterial effector proteins from
pathogens such as EPEC, Chlamydia, Bartonella, Anaplasma and
Ehrlichia species, which collectively represents a fascinating new
research area [5,713].
We thank Dr. Martin Griebl (FAU Erlangen, Germany) for technical
advice in CagA purification, Dr. Javier Torres (Unidad de Investigacion en
Enfermedades Infecciosas UMAE Pediatria, IMSS, Mexico) for providing
H. pylori strain 2002-370 and Dr. Gabriele Rieder (University Salzburg,
Austria) for providing strain B8.
Conceived and designed the experiments: JL HS SB NT. Performed the
experiments: JL KP HS SB NT. Analyzed the data: NT HS SB DB YY.
Contributed reagents/materials/analysis tools: DB KP YY. Wrote the
paper: NT. Made figures: NT.
1. Hunter T ( 2009 ) Tyrosine phosphorylation: thirty years and counting . Curr Opin Cell Biol 21 : 140 - 146 .
2. Backert S , Selbach M ( 2005 ) Tyrosine-phosphorylated bacterial effector proteins: the enemies within . Trends Microbiol 13 : 476 - 484 .
3. Cousin C , Derouiche A , Shi L , Pagot Y , Poncet S , et al. ( 2013 ) Protein-serine/ threonine/tyrosine kinases in bacterial signaling and regulation . FEMS Microbiol Lett 346 : 11 - 19 .
4. Selbach M , Paul FE , Brandt S , Guye P , Daumke O , et al. ( 2009 ) Host cell interactome of tyrosine-phosphorylated bacterial proteins . Cell Host Microbe 5 : 397 - 403 .
5. Backert S , Tegtmeyer N , Selbach M ( 2010 ) The versatility of Helicobacter pylori CagA effector protein functions: The master key hypothesis . Helicobacter 15 : 163 - 176 .
6. Hayashi T , Morohashi H , Hatakeyama M ( 2013 ) Bacterial EPIYA effectorswhere do they come from? What are they? Where are they going? Cell Microbiol 15 : 377 - 385 .
7. Kenny B , DeVinney R , Stein M , Reinscheid DJ , Frey EA , et al. ( 1997 ) Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells . Cell 91 : 511 - 520 .
8. Segal ED , Cha J , Lo J , Falkow S , Tompkins LS ( 1999 ) Altered states: involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori . Proc Natl Acad Sci U S A 96 : 14559 - 14564 .
9. Clifton DR , Fields KA , Grieshaber SS , Dooley CA , Fischer ER , et al. ( 2004 ) A chlamydial type III translocated protein is tyrosine-phosphorylated at the site of entry and associated with recruitment of actin . Proc Natl Acad Sci U S A 101 : 10166 - 10171 .
10. Schulein R , Guye P , Rhomberg TA , Schmid MC , Schroder G , et al. ( 2005 ) A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells . Proc Natl Acad Sci U S A 102 : 856 - 561 .
11. Ijdo JW , Carlson AC , Kennedy EL ( 2007 ) Anaplasma phagocytophilum AnkA is tyrosine-phosphorylated at EPIYA motifs and recruits SHP-1 during early infection . Cell Microbiol 9 : 1284 - 1296 .
12. Lin M , den Dulk-Ras A , Hooykaas PJ , Rikihisa Y ( 2007 ) Anaplasma phagocytophilum AnkA secreted by type IV secretion system is tyrosine phosphorylated by Abl-1 to facilitate infection . Cell Microbiol 9 : 2644 - 57 .
13. Rikihisa Y , Lin M ( 2010 ) Anaplasma phagocytophilum and Ehrlichia chaffeensis type IV secretion and Ank proteins . Curr Opin Microbiol 13 : 59 - 66 .
14. Odenbreit S , Pu ls J, Sedlmaier B , Gerland E , Fischer W , et al. ( 2000 ) Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion . Science 287 : 1497 - 1500 .
15. Stein M , Rappuoli R , Covacci A ( 2000 ) Tyrosine phosphorylation of the Helicobacter pylori CagA antigen after cag-driven host cell translocation . Proc Natl Acad Sci U S A 97 : 1263 - 1268 .
16. Asahi M , Azuma T , Ito S , Ito Y , Suto H , et al. ( 2000 ). Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells . J Exp Med 191 : 593 - 602 .
17. Backert S , Ziska E , Brinkmann V , Zimny-Arndt U , Fauconnier A , et al. ( 2000 ) Translocation of the Helicobacter pylori CagA protein in gastric epithelial cells by a type IV secretion apparatus . Cell Microbiol 2 : 155 - 164 .
18. Covacci A , Rappuoli R ( 2000 ) Tyrosine-phosphorylated bacterial proteins: Trojan horses for the host cell . J Exp Med 191 : 587 - 592 .
19. Rohde M , Puels A , Buhrdorf R , Fischer W , Haas R ( 2003 ) A novel sheathed surface organelle of the Helicobacter pylori cag type IV secretion system . Mol Microbiol 49 : 218 - 234 .
20. Tanaka J , Suzuki T , Mimuro H , Sasakawa C ( 2003 ) Structural definition on the surface of Helicobacter pylori type IV secretion apparatus . Cell Microbiol 5 : 395 - 404 .
21. Backert S , Meyer TF ( 2006 ) Type IV secretion systems and their effectors in bacterial pathogenesis . Curr Opin Microbiol 9 : 207 - 217 .
22. Kwok T , Zabler D , Urman S , Rohde M , Hartig R , et al. ( 2007 ) Helicobacter exploits integrin for type IV secretion and kinase activation . Nature 449 : 862 - 866 .
23. Backert S , Moese S , Selbach M , Brinkmann V , Meyer TF ( 2001 ) Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells . Mol Microbiol 42 : 631 - 644 .
24. Stein M , Bagnoli F , Halenbeck R , Rappuoli R , Fantl WJ , et al. ( 2002 ) c-Src/Lyn kinases activate Helicobacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs . Mol Microbiol 43 : 971 - 980 .
25. Ridley AJ , Schwartz MA , Burridge K , Firtel RA , Ginsberg MH , et al. ( 2003 ) Cell migration: integrating signals from front to back . Science 302 : 1704 - 1709
26. Schneider S , Weydig C , Wessler S ( 2008 ) Targeting focal adhesions: Helicobacter pylori-host communication in cell migration . Cell Commun Signal 6 : 2 .
27. Higashi H , Tsutsumi R , Muto S , Sugiyama T , Azuma T , et al. ( 2002 ) SHP2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein . Science 295 : 683 - 686 .
28. Puels M , Fischer W , Haas R ( 2002 ) Activation of Helicobacter pylori CagA by tyrosine phosphorylation is essential for dephosphorylation of host cell proteins in gastric epithelial cells . Mol Microbiol 43 : 962 - 969 .
29. Mimuro H , Suzuki T , Tanaka J , Asahi M , Haas R , et al. ( 2002 ) Grb2 is a key mediator of Helicobacter pylori CagA protein activities . Mol Cell 10 : 745 - 755 .
30. Asahi M , Tanaka Y , Izumi T , Ito Y , Naiki H , et al. ( 2003 ) Helicobacter pylori CagA containing ITAM-like sequences localized to lipid rafts negatively regulates VacA-induced signaling in vivo . Helicobacter 8 : 1 - 14 .
31. Xia Y , Yamaoka Y , Zhu Q , Matha I , Gao X ( 2009 ) A comprehensive sequence and disease correlation analyses for the C-terminal region of CagA protein of Helicobacter pylori . PLoS One 4 : e7736 .
32. Backert S , Selbach M ( 2008 ) Role of type IV secretion in Helicobacter pylori pathogenesis . Cell Microbiol 10 : 1573 - 1581 .
33. Yamaoka Y ( 2010 ) Mechanisms of disease: Helicobacter pylori virulence factors . Nat Rev Gastroenterol Hepatol 7 : 629 - 641 .
34. Aras RA , Lee Y , Kim SK , Israel D , Peek RM Jr, et al. ( 2003 ) Natural variation in populations of persistently colonizing bacteria affect human host cell phenotype . J Infect Dis 188 : 486 - 496 .
35. Kim M , Shin DS , Kim J , Lee YS ( 2010 ) Substrate screening of protein kinases: detection methods and combinatorial peptide libraries . Biopolymers 94 : 753 - 762 .
36. Argent RH , Zhang Y , Atherton J ( 2005 ) Simple method for determination of the number of Helicobacter pylori CagA variable-region EPIYA tyrosine phosphorylation motifs by PCR . J Clin Microbiol 43 : 791 - 795 .
37. Naito M , Yamazaki T , Tsutsumi R , Higashi H , Onoe K , et al. ( 2006 ) Influence of EPIYA-repeat polymorphism on the phosphorylation-dependent biological activity of Helicobacter pylori CagA . Gastroenterology 130 : 1181 - 1190 .
38. Panayotopoulou EG , Sgouras DN , Papadakos K , Kalliaropoulos A , Papatheodoridis G , et al. ( 2007 ) Strategy to characterize the number and type of repeating EPIYA phosphorylation motifs in the carboxyl terminus of CagA protein in Helicobacter pylori clinical isolates . J Clin Microbiol 45 : 488 - 495 .
39. Basso D , Zambon CF , Letley DP , Stranges A , Marchet A , et al. ( 2008 ) Clinical relevance of Helicobacter pylori cagA and vacA gene polymorphisms . Gastroenterology 135 : 91 - 99 .
40. Schmidt HM , Goh KL , Fock KM , Hilmi I , Dhamodaran S , et al. ( 2009 ) Distinct cagA EPIYA motifs are associated with ethnic diversity in Malaysia and Singapore . Helicobacter 14 : 256 - 263 .
41. Miura M , Ohnishi N , Tanaka S , Yanagiya K , Hatakeyama M ( 2009 ) Differential oncogenic potential of geographically distinct Helicobacter pylori CagA isoforms in mice . Int J Cancer 125 : 2497 - 2504 .
42. Truong BX , Mai VT , Tanaka H , Ly le T , Thong TM , et al. ( 2009 ) Diverse characteristics of the CagA gene of Helicobacter pylori strains collected from patients from southern vietnam with gastric cancer and peptic ulcer . J Clin Microbiol 47 : 4021 - 4028 .
43. Jones KR , Joo YM , Jang S , Yoo YJ , Lee HS , et al. ( 2009 ) Polymorphism in the CagA EPIYA motif impacts development of gastric cancer . J Clin Microbiol 47 : 959 - 968 .
44. Furuta Y , Yahara K , Hatakeyama M , Kobayashi I ( 2011 ) Evolution of cagA oncogene of Helicobacter pylori through recombination . PLoS ONE 6 : e23499 .
45. Backert S , Muller EC , Jungblut PR , Meyer TF ( 2001 ) Tyrosine phosphorylation patterns and size modification of the Helicobacter pylori CagA protein after translocation into gastric epithelial cells . Proteomics 1 : 608 - 617 .
46. Mueller D , Tegtmeyer N , Brandt S , Yamaoka Y , De Poire E , et al. ( 2012 ) c-Src and c-Abl kinases control hierarchic phosphorylation and function of the CagA effector protein in Western and East Asian Helicobacter pylori strains . J Clin Invest 122 : 1553 - 1566 .
47. Selbach M , Moese S , Hauck CR , Meyer TF , Backert S ( 2002 ) Src is the kinase of the Helicobacter pylori CagA protein in vitro and in vivo . J Biol Chem 277 : 6775 - 6778 .
48. Poppe M , Feller SM , Romer G , Wessler S ( 2007 ) Phosphorylation of Helicobacter pylori CagA by c-Abl leads to cell motility . Oncogene 26 : 3462 - 3472 .
49. Tammer I , Brandt S , Hartig R , Konig W , Backert S ( 2007 ) Activation of Abl by Helicobacter pylori: a novel kinase for CagA and crucial mediator of host cell scattering . Gastroenterology 132 : 1309 - 1319 .
50. Tinti M , Nardozza AP , Ferrari E , Sacco F , Corallino S , et al. ( 2012 ) The 4G10, pY20 and p-TYR-100 antibody specificity: profiling by peptide microarrays . N Biotechnol 29 : 571 - 517 .
51. Kumar Pachathundikandi S , Brandt S , Madassery J , Backert S ( 2011 ) Induction of TLR-2 and TLR-5 expression by Helicobacter pylori switches cagPAI-dependent signalling leading to the secretion of IL-8 and TNF-a . PLoS One 6 : e19614 .
52. Wiedemann T , Hofbaur S , Tegtmeyer N , Huber S , Sewald N , et al. ( 2012 ) Helicobacter pylori CagL dependent induction of gastrin expression via a novel avb5-integrin-integrin linked kinase signalling complex . Gut 61 : 986 - 996 .
53. Hirsch C , Tegtmeyer N , Rohde M , Rowland M , Oyarzabal OA , et al. ( 2012 ) Live Helicobacter pylori in the root canal of endodontic-infected deciduous teeth . J Gastroenterol 47 : 936 - 940 .
54. Tegtmeyer N , Rivas Traverso F , Rohde M , Oyarzabal OA , Lehn N , et al. ( 2013 ) Electron microscopic, genetic and protein expression analyses of Helicobacter acinonychis strains from a Bengal tiger . PLoS One 8 : e71220 .
55. Blaydes JP , Vojtesek B , Bloomberg GB , Hupp TR ( 2000 ) The development and use of phospho-specific antibodies to study protein phosphorylation . Methods Mol Biol 99 : 177 - 189 .
56. Houseman BT , Huh JH , Kron SJ , Mrksich M ( 2002 ) Peptide chips for the quantitative evaluation of protein kinase activity . Nat Biotechnol 20 : 270 - 274 .
57. Conradi J , Tegtmeyer N , Wozna M , Wissbrock M , Michalek C , et al. ( 2012 ) An RGD helper sequence in CagL of Helicobacter pylori assists in interactions with integrins and injection of CagA . Front Cell Infect Microbiol 2 : 70 .
58. Hoy B , Geppert T , Boehm M , Reisen F , Plattner P , et al. ( 2012 ) Distinct roles of secreted HtrA proteases from gram-negative pathogens in cleaving the junctional protein and tumor suppressor E-cadherin . J Biol Chem 287 : 10115 - 10120 .
59. Tegtmeyer N , Hartig R , Delahay RM , Rohde M , Brandt S , et al. ( 2010 ). A small fibronectin-mimicking protein from bacteria induces cell spreading and focal adhesion formation . J Biol Chem 285 : 23515 - 23526 .
60. Boehm M , Krause-Gruszczynska M , Rohde M , Tegtmeyer N , Takahashi S , et al. ( 2011 ) Major host factors involved in epithelial cell invasion of Campylobacter jejuni: role of fibronectin, integrin beta1, FAK, Tiam-1, and DOCK180 in activating Rho GTPase Rac1 . Front Cell Infect Microbiol 1 : 17 .
61. Boehm M , Hoy B , Rohde M , Tegtmeyer N , Baek KT , et al. ( 2012 ) Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin . Gut Pathog 4 : 3 .
62. Traverso FR , Bohr UR , Oyarzabal OA , Rohde M , Clarici A , et al. ( 2010 ) Morphologic, genetic, and biochemical characterization of Helicobacter magdeburgensis, a novel species isolated from the intestine of laboratory mice . Helicobacter 15 : 403 - 415
63. Tegtmeyer N , Wittelsberger R , Hartig R , Wessler S , Martinez-Quiles N , et al. ( 2011 ) Serine phosphorylation of cortactin controls focal adhesion kinase activity and cell scattering induced by Helicobacter pylori . Cell Host Microbe 9 : 520 - 531 .
64. Backert S , Schwarz T , Miehlke S , Kirsch C , Sommer C , et al. ( 2004 ) Functional analysis of the cag pathogenicity island in Helicobacter pylori isolates from patients with gastritis, peptic ulcer, and gastric cancer . Infect Immun 72 : 1043 - 1056 .
65. Brandt S , Wessler S , Hartig R , Backert S ( 2009 ) Helicobacter pylori activates protein kinase C delta to control Raf in MAP kinase signalling: role in AGS epithelial cell scattering and elongation . Cell Motil Cytoskeleton 66 : 874 - 892 .
66. Tegtmeyer N , Zabler D , Schmidt D , Hartig R , Brandt S , et al. ( 2009 ) Importance of EGF receptor, HER2/Neu and Erk1/2 kinase signalling for host cell elongation and scattering induced by the Helicobacter pylori CagA protein: antagonistic effects of the vacuolating cytotoxin VacA . Cell Microbiol 11 : 488 - 505 .
67. Backert S , Clyne M , Tegtmeyer N ( 2011 ) Molecular mechanisms of gastric epithelial cell adhesion and injection of CagA by Helicobacter pylori . Cell Commun Signal 9 : 28 .
68. Smolka AJ , Backert S. ( 2012 ) How Helicobacter pylori infection controls gastric acid secretion . J Gastroenterol 47 : 609 - 618 .
69. Covacci A , Censini S , Bugnoli M , Petracca R , Burroni D , et al. ( 1993 ) Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer . Proc Natl Acad Sci U S A . 90 : 5791 - 5795 .
70. Yamaoka Y , Kodama T , Kashima K , Graham DY , Sepulveda AR. ( 1998 ) Variants of the 3' region of the cagA gene in Helicobacter pylori isolates from patients with different H. pylori-associated diseases . J Clin Microbiol . 36 : 2258 - 2263 .
71. Yamaoka Y , El-Zimaity HM , Gutierrez O , Figura N , Kim JG , et al. ( 1999 ) Relationship between the cagA 3' repeat region of Helicobacter pylori, gastric histology, and susceptibility to low pH . Gastroenterol . 117 : 342 - 349 .
72. Robinson DR , Wu YM , Lin SF ( 2000 ) The protein tyrosine kinase family of the human genome . Oncogene 19 : 5548 - 5557 .
73. Songyang Z , Carraway KL3rd , Eck MJ , Harrison SC , Feldman RA , et al. ( 1995 ) Catalytic specificity of protein-tyrosine kinases is critical for selective signaling . Nature 373 : 536 - 539 .
74. Posselt G , Backert S , Wessler S ( 2013 ) The functional interplay of Helicobacter pylori factors with gastric epithelial cells induces a multi-step process in pathogenesis . Cell Commun Signal 11 : 77 .
75. Tegtmeyer N , Backert S ( 2011 ) Role of Abl and Src family kinases in actincytoskeletal rearrangements induced by the Helicobacter pylori CagA protein . Eur J Cell Biol 90 : 880 - 890 .
76. Tomb JF , White O , Kerlavage AR , Clayton RA , Sutton GG , et al. ( 1997 ) The complete genome sequence of the gastric pathogen Helicobacter pylori . Nature 388 : 539 - 547 .
77. Moese S , Selbach M , Kwok T , Brinkmann V , Konig W , et al. ( 2004 ) Helicobacter pylori induces AGS cell motility and elongation via independent signaling pathways . Infect Immun 72 : 3646 - 3649 .
78. Moese S , Selbach M , Brinkmann V , Karlas A , Haimovich B , et al. ( 2007 ) The Helicobacter pylori CagA protein disrupts matrix adhesion of gastric epithelial cells by dephosphorylation of vinculin . Cell. Microbiol 9 : 1148 - 1161 .
79. Hoy B , Lower M , Weydig C , Carra G , Tegtmeyer N , et al. ( 2010 ) Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E-cadherin to disrupt intercellular adhesion . EMBO Rep . 11 : 798 - 804 .
80. Brandt S , Kwok T , Hartig R , Konig W , Backert S ( 2005 ) NF-kappaB activation and potentiation of proinflammatory responses by the Helicobacter pylori CagA protein . Proc Natl Acad Sci U S A 102 : 9300 - 9305 .
81. Farnbacher M , Jahn T , Willrod D , Daniel R , Haas R , et al. ( 2010 ) Sequencing, annotation, and comparative genome analysis of the gerbil-adapted Helicobacter pylori strain B8 . BMC Genomics 11 : 335 .
82. Matsunari O , Shiota S , Suzuki R , Watada M , Kinjo N , et al. ( 2012 ) Association between Helicobacter pylori virulence factors and gastroduodenal diseases in Okinawa, Japan . J Clin Microbiol 50 : 876 - 583 .