A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

Abstract

Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk.

Full text

RESEARCH ARTICLE
A Specific A/T Polymorphism in Western
Tyrosine Phosphorylation B-Motifs Regulates
Helicobacter pylori CagA Epithelial Cell
Interactions
Xue-Song Zhang
1
*, Nicole Tegtmeyer
2
, Leah Traube
1
, Shawn Jindal
1
, Guillermo Perez-
Perez
1
, Heinrich Sticht
3
, Steffen Backert
2
, Martin J. Blaser
1
1Departments of Medicine and Microbiology, New York University School of Medicine and VA Medical
Center, New York, New York, United States of America, 2Friedrich Alexander University Erlangen,
Department of Biology, Division of Microbiology, Erlangen, Germany, 3Friedrich Alexander University
Erlangen, Bioinformatics, Institute for Biochemistry, Erlangen, Germany
*xuesong.zhang@nyumc.org
Abstract
Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human
health. The CagA protein, a key host-interaction factor, is translocated by a type IV secre-
tion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs
(TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades.
The CagA TPMs have been described as type A, B, C or D, each with a specific conserved
amino acid sequence surrounding EPIYA. Database searching revealed strong non-ran-
dom distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates.
In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT
B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By
generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated
the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host
cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase
(PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA
with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than
the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound
to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain
hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-
culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly atten-
uated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic
strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate
CagA activity through interfering with host signaling pathways related to carcinogenesis,
thus influencing cancer risk.
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 1/25
OPEN ACCESS
Citation: Zhang X-S, Tegtmeyer N, Traube L, Jindal
S, Perez-Perez G, Sticht H, et al. (2015) A Specific A/
T Polymorphism in Western Tyrosine
Phosphorylation B-Motifs Regulates Helicobacter
pylori CagA Epithelial Cell Interactions. PLoS Pathog
11(2): e1004621. doi:10.1371/journal.ppat.1004621
Editor: Steven R. Blanke, University of Illinois,
UNITED STATES
Received: August 16, 2014
Accepted: December 16, 2014
Published: February 3, 2015
Copyright: © 2015 Zhang et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: The work of XSZ, LT, SJ, GPP, and MJB is
supported by the National Institutes of Health
(R01GM63270), by the Knapp Family Fund, the Ziff
Foundation, and the Diane Belfer Program for Human
Microbial Ecology. The work of NT, SB, and HS is
supported through DFG grants (projects B10 and A2
of CRC-796). The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.

Author Summary
As the dominant bacterium living in the human stomach, Helicobacter pylori has mixed
roles in host health. One significant pathogenic risk factor is the CagA protein, which in-
terferes with multiple host cell signaling pathways through its EPIYA tyrosine phosphory-
lation motifs (TPMs). Through database searching and silico analysis, we reveal a strong
non-random distribution of the EPIYA B motif polymorphisms (including EPIYT and
EPIYA) in Western H. pylori isolates, and provide evidence that the EPIYT are significant-
ly less associated with gastric cancer than the EPIYA. By constructing a series of H. pylori
cagA isogenic mutants and isogenic complementation plasmids, generating specific anti-
bodies, co-culturing with human AGS cells, performing biochemical and modeling analy-
sis, we demonstrate that CagA B-motif phosphorylation status is essential for its
interaction with host PI3-kinase during colonization and that CagA with an EPIYT B-
motif had significantly attenuated induction of interleukin-8 and the hummingbird phe-
notype, had higher affinity with PI3-kinase, and enhanced induction of AKT compared to
the EPIYA. These findings provide insight into how Western H. pylori CagA regulates can-
cer-related activity inside host cells through the A/T polymorphisms at the functionally
important B motif.
Introduction
Helicobacter pylori, a spiral-shaped, microaerophilic gram-negative bacterium, persistently
colonizes the human gastric mucosa [1,2]. H. pylori is carried by about 50% of the world’s pop-
ulation, and it exhibits extensive genetic diversity and distinct phylogeographic features [3,4].
Colonization increases risk of peptic ulcer disease and gastric carcinoma [5,6], and has been as-
sociated with diminished risk for esophageal inflammatory and neoplastic lesions [7,8], and
childhood-onset asthma [9,10]. In 1995, the cytotoxin-associated gene A (CagA) protein of H.
pylori was first associated with increased risk of gastric cancer [11], and since then, its patho-
genic effects have been intensely studied [1,12].
The 120–145 kDa CagA protein is encoded by the cagA gene, located within the *40 kb H.
pylori cag pathogenicity island (cagPAI) [13,14], along with a type IV secretion system that in-
jects it into host gastric epithelial cells [15]. The carboxy-terminal region of CagA has several
Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs which are strongly correlated to gastric disease outcomes
[16,17]. The carboxy-terminal region of CagAs exhibit geographical, structural, and functional
diversity, which is the result of the evolution of this protein through various modes of recombi-
nation mechanism [18].
In host cells, CagA molecules are associated with the inner surface of the plasma membrane
and are dimerized via the carboxy-terminal EPIYA motif-containing regions [19,20]. CagA
molecules undergo tyrosine phosphorylation (pCagA) at the EPIYA motifs by host Src-family
kinases (SFKs) and Abl kinase [21–23]. CagA interacts with multiple host signaling factors
through its EPIYA TPMs in a phosphorylation-dependent or -independent manner [24,25], af-
fecting cell proliferation, motility, polarity, apoptosis, inflammation and nuclear responses,
which may promote gastric carcinogenesis [26–28]. By mimicking host substrates through its
C-terminal sequence, CagA inhibits PAR1/MARK family kinase pathways [29], and by associa-
tion with the human tumor suppressor apoptosis-stimulating protein of p53-2 (ASPP2)
through its N-terminal sequence, CagA inhibits apoptosis of host cells co-colonized with H.
pylori [30]. Through phosphorylated EPIYA TPMs, pCagA binds to the Src homology 2 (SH2)
domains of host signaling factors [26,28]. In this way pCagA activates the tyrosine phosphatase
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 2/25
Competing Interests: The authors have declared
that no competing interests exist.

Shp2, which affects cell proliferation by inducing the ERK MAP kinase cascade [31–33], and
also leads to cell elongation (producing the hummingbird phenotype) by inhibition of focal
adhesion kinase (FAK) [34–36]. Phosphorylated TPMs also facilitate CagA interactions with
C-terminal Src kinase (CSK), which inhibits SFK activity and negatively regulates CagA-Shp2
interaction [37]. The phosphorylated CagA TPMs directly bind other tyrosine phosphatases
such as Shp1, phosphatidylinositide 3-kinase (PI3-kinase) and GTPase activating protein Ras
GAP1, as well as adaptor proteins Crk-I, Crk-II, Crk-L, Grb2, and Grb7 [12,26]. Transgenic
mice expressing wild-type CagA but not tyrosine-phosphorylation-resistant CagA developed
gastric and small intestinal epithelial hyperplasia and neoplasia and B cell lymphomas and
myeloid leukemias [38], supporting a critical role of CagA tyrosine phosphorylation in H.
pylori-induced oncogenesis.
In addition to phosphorylation-dependent effects, CagA also associates with the polarity-
regulating kinase partitioning-defective 1 (PAR1) protein through its C-terminal CagA-multi-
merization motif (CM), which overlaps with the EPIYA C- or D-TPM sequences [33,39]. The
interaction between CagA and Par1 disrupts gastric epithelial cell tight junctions and apical-
basal polarity [33], and enhances CagA TPM-phosphorylation-dependent interactions by sta-
bilizing complex structures such as CagA-Shp2 [20]. In total, both phosphorylation-dependent
and -independent [33–35] interactions affect host signaling pathways.
H. pylori has extensive genetic diversity [40–42]; isolates from different populations exhibit
distinct biogeographic features, reflecting ancient human migrations [43]; cagA also possesses
population-specific polymorphisms with major East Asian and Western groupings [44,45].
Four distinct CagA EPIYA TPMs (A, B, C or D), have conserved flanking sequences [46]. East
Asian CagA include A-, B-, and D-TPMs, while Western CagA has A-, B-, and C-TPMs [47].
The East Asian CagAs are more interactive with host cells than Western CagAs, largely due to
the higher affinity of the strongly phosphorylated D-TPM to Shp-2 than the Western C-TPMs
[47,48]. Western CagA includes one or multiple C-TPMs, while East Asian CagA only has one
D-TPM [12].
Regardless of C/D type, most CagA molecules include single A- and B-TPMs that undergo
later and not simultaneous tyrosine phosphorylation [25]. The phosphorylated A- or B-TPMs
have distinct host interaction partners from C- or D-TPMs and from each other [26], suggest-
ing unique signaling functions. Here we report and characterize the functional importance of a
specific A/T polymorphism present only within the Western CagA EPIYA B-TPMs.
Results
Polymorphisms in the CagA tyrosine phosphorylation EPIYA motif
Based on the CagA sequences published in Genbank, we investigated the variability within the
C-terminal EPIYAs. A total of 2,561 complete or partial H. pylori CagA protein sequences were
analyzed for polymorphisms within the EPIYAs. Our analysis indicated that the CagA B-TPM
exhibits the highest variability (Table 1). EPIYA represents only 72.6% of 2,617 B-TPM se-
quences, with 23 alternative sequences present, including EPIYT, ESIYT, ESIYA and GSIYD;
EPIYT is the most frequent alternative. In contrast, very few alternative sequences are identi-
fied in the A- and C-TPMs (Table 1), and none in the 1,196 type D-TPMs; only a single alter-
native sequence (EPIYV) is observed in the 1,620 C-TPMs (Table 1). All 25 independent CagA
sequences with the EPIYV C-TPM show the same rare B-TPM (GSIYD). Only one low fre-
quency (0.2%) alternative sequence (EPIYT) is observed in the A-TPMs (Table 1). In total,
these results indicate specific polymorphisms in the CagA tyrosine phosphorylation (EPIYA)
sequences, especially involving the B-TPMs. Among the 35 fully-sequenced H. pylori genomes
and 81 ongoing partially-sequenced H. pylori genomes possessing the cagPAI (based on the
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 3/25

NCBI Bioproject Database), EPIYA represents 52.6%, while EPIYT represents 34.5% of the
B-TPMs.
The EPIYA- B-TPM of Western type CagAs has alternative sequences
Next, analyzing the B-TPMs in East Asian CagA possessing D-TPMs or Western type CagA
possessing C-TPMs, we found that the distributions of the identified alternative sequences
were significantly different (Table 2). In the Western CagA EPIYA B-TPMs, there are two
major sequences (EPIYA; 55.5% and EPIYT; 32.9%). Other alternative TPMs comprise about
11.6% of the sequences. In contrast, among East Asian CagA B-TPMs, EPIYA is the major se-
quence (91.1%), EPIYT is at low (1.7%) frequency, and ESIYA is the alternative TPM with the
highest frequency (6.0%) (Table 2). In total, Western CagAs have more alternative B-TPM
EPIYA sequences than do East Asian CagAs.
Since Western CagAs may have more than one C-TPM, we asked whether the presence of
the alternative EPIYA B-TPMs is related to C-TPM number. There was no link between the al-
ternative B-TPM sequences and the number of C-TPMs, except for ESIYT, which co-appears
Table 1. Distribution of alternative sequences in the four types of H. pylori CagA TPMs
a
.
Number
TPM type Total EPIYA EPIYT ESIYA ESIYT Other
(n = 7954) (n = 7207) (%) (n = 478) (n = 96) (n = 60) (n = 113)
A 2521 2517 (99.8) 4 0 0 0
B
b
2617 1899 (72.6) 474 96 60 88
c
C 1620 1595 (98.5) 0 0 0 25
d
D 1196 1196 (100) 0 0 0 0
a
From review of Genbank entries to Aug. 8, 2013.
b
B-TPM bolded, since most variable.
c
B-domain other TPMs include: GSIYD (31), EPVYA (13), ELIYA (12), ESIYD (7), ETIYA (3), EPIYD (3), EHIYA (2), EPTYA (2), EPIYS (2), EPIYV (2),
ESVYA (2), DPIYA (1), DPIYD (1), EPVYT (1), QPIYP (1), EPIFT (1), EPIHA (1), EPINA (1), ESIYS (1), EFIYT (1).
d
The C-domain other TPMs all are EPIYV (25), which only is present when the B-domain sequence is GSIYD (p<0.0001).
doi:10.1371/journal.ppat.1004621.t001
Table 2. Distribution of alternative TPMs in 2617 CagA B-TPMs, by H. pylori geographic origin.
B-TPM Number (% of total)
a
Sequence type Total East Asian Western p-value
b
(n = 2617) (n = 1190) (n = 1337)
EPIYA 1899 1084 (91.1) 742 (55.5) <0.001
EPIYT 474 20 (1.7) 440 (32.9) <0.001
ESIYA 96 71 (6.0) 26 (1.9) <0.001
ESIYT 60 7 (0.6) 49 (3.7) <0.001
Others
C
88 8 (0.7) 80 (6.0) <0.001
a
For 93 Genbank records, strain origin was not recorded.
b
By Chi-square test.
c
See Table 1.
doi:10.1371/journal.ppat.1004621.t002
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 4/25

with 2 C-TPMs on the same Western CagAs at significantly high frequency (Table 3). These
findings indicate a common previously unrecognized TPM polymorphism with strongly
non-random distribution in the available census of strains.
Pathological association with the Western B-domain TPM alternatives
To assess the relationship of B-TPM sequence and clinical outcome, we analyzed a total of 364
Western CagAs, which were reported to be present in patients with defined gastrointestinal pa-
thology, according to the descriptions from Genbank and the indicated publications (Table 4).
Compared with gastritis alone, gastric cancer was significantly associated with the EPIYA B-
TPMs, whereas duodenal ulcers were significantly associated with the EPIYT B-TPM
(Table 4). That these polymorphisms in B-TPM are associated with different diseases suggest
that EPIYT and EPIYA may differentially regulate the CagA pathophysiologic roles in Western
H. pylori strains that interact with host cells; while the CM and CRPIA motifs are commonly
present in both forms of CagA.
Codon usage in the Western B-domain TPMs
To assess whether the EPIYA/T polymorphisms at the protein level were random, we com-
pared the codons in which the A/T polymorphisms were present (S1 Table in S1 Text and
Fig. 1). Only one major (91.1%) set of codons (TAT ACT) encodes the YT of EPIYT B-TPM.
In contrast, there are two major sets of codons (TAC GCT and TAT GCT) that encode the YA
Table 3. Relation of B-domain TPM sequence type to number of C-domain TPMs in 1322 Western CagA sequences.
B-TPM Sequence type Total
a
C-TPM number (%)
(n = 1322) 1 (n = 1035) 2 (n = 254) 3 (n = 33) p-value
EPIYA 730 582(79.7) 124(17.0) 24(3.3) NS
b
EPIYT 436 339(77.8) 90(20.6) 7 (1.6) NS
ESIYA 26 21 (80.8) 4(15.4) 1 (3.8) NS
ESIYT 50 14 (28.0) 35(70.0) 1 (2.0) <0.001
Others 80 79 (98.8) 1 (1.2) 0 (0.0) NS
a
For some Western strains (n = 16), there are >3 C-TPMs.
b
NS, P>0.05.
doi:10.1371/journal.ppat.1004621.t003
Table 4. Relation of Western CagA B-TPM sequence type to gastrointestinal ailments.
Clinical Status
a
Number (% of total)
Total EPIYA EPIYT p-value
b
(n = 364) (n = 194) (53.3%) (n = 170) (46.7)
Gastritis alone 171 88 (51.5) 83 (48.5) (Reference)
Duodenal Ulcer 102 43 (42.2) 59 (57.8) 0.02
Gastric Ulcer 24 13 (54.2) 11 (45.8) NS
Gastric Cancer 46 35 (76.1) 11 (23.9) 0.0019
Esophagitis 21 15 (71.4) 6 (28.6) NS
a
Based on the descriptions from the Genbank database or the associated publications.
b
By X
2
analysis; NS, P>0.05.
doi:10.1371/journal.ppat.1004621.t004
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 5/25

of the EPIYA B-TPMs with similar frequencies (59.4% and 40.4%, respectively). Compared
with YA and YT codons in the other loci in the H. pylori 26695 genome, this distribution is sig-
nificantly non-random (p<0.001). This non-random distribution suggests that the polymor-
phisms have been selected rather than being stochastic, potentially providing for different
CagA functional roles.
Construction of H. pylori isogenic cagA mutants with TPM
polymorphisms
To investigate how the major (EPIYT) alternative TPM affects CagA functions, we created a se-
ries of isogenic H. pylori mutants that express a Western CagA with variant TPMs. The
Figure 1. Polymorphisms of the Western CagA B-TPMs and their codons. Panel A: The sequences for
1027 Western CagA B-TPMs were aligned and a sequence logo was produced (upper), and amino acids
(lower,*: E-P/S-I-). Panel B: The distribution of codons for the Western CagA B-TPM YA and YT
polymorphisms. The proportions of major (TA) and minor (CA) codons of YT are significantly different
(p<0.0001). The proportions of major (CG) and minor (CA) codons of YA and also are significantly different
(p = 0.0075).
doi:10.1371/journal.ppat.1004621.g001
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
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Western-type cagA gene from H. pylori strain 147C, originally isolated from a human antrum
corpus [44], was used as the CagA parent gene for the constructions, created in a site-directed
manner using a recombinant PCR technique (S1A Fig. in S1 Text). This 147C cagA gene
possesses one A-, B- and C-TPM. The gene product CagA
147C
has been previously shown to in-
duce both AGS cell hummingbird phenotype and IL-8 production [44,49]. We replaced the
H. pylori 26695 cagA ORF with isogenic cagA genes in its native genetic locus via transforma-
tion. The set of 6 isogenic H. pylori 26695 mutants all possess cagA with EPIYA, EPIYT, or
EPIAT (as a control: presumed to be inactive) B-TPMs, and with EPIYA or EPIAA forms of
the A- or C-TPM (S1A Fig. in S1 Text). These mutants exhibit the same physiological charac-
ters, transformation frequency and growth rates in vitro as the parental 26695 strains. Western
blotting confirmed that each isogenic mutant expressed a CagA molecule (S1B Fig. in S1 Text),
and sequencing of each cagA ORF confirmed that the sequences surrounding the target site(s)
were identical.
CagA EPIYT B-TPM is phosphorylated during H. pylori co-culture
To investigate CagA B-TPM phosphorylation status and function during co-culture, we first
generated phospho-specific and non-phospho antibodies, α-pCagA-EPIYT-918 (phospho)
and α-CagA-EPIYT-918 (non-phospho) against the EPIYT B-TPM motif of CagA, corre-
sponding to the amino acid residues derived from strain 26695 (S2 Fig. in S1 Text). Our analy-
sis indicated that the α-pCagA-EPIYT-918 (phospho) or α-CagA-EPIYT-918 (non-phospho)
antibodies recognize the B-TPM including both EPIYT B-TPM and EPIYA B-TPM, but not
the A-TPM or C-TPM (control) peptides (S2 Fig. in S1 Text). To investigate whether this
CagA EPIYT B-TPM can be phosphorylated during co-culture, AGS cells were co-incubated
for 6 h with a set of clinical H. pylori strains, which vary in their CagA carboxy-terminal TPM
sites. Phosphorylation of CagA at B-TPM was examined using the now-confirmed phospho-
specific α-pCagA-EPIYT-918 antibody. Results indicated that the EPIYT-motif can be phos-
phorylated during co-culture, but to varying extents (Fig. 2).
The alternative B-TPM EPIYT has enhanced PI3-kinase/AKT pathway
induction
To investigate how this alternative B-TPM affects the role of CagA in host signaling pathways,
we co-cultured the isogenic H. pylori mutants with AGS cells for 24 h and analyzed cell ly-
sates for CagA-mediated protein binding and signal activation by immunoblotting and co-
immunoprecipitation. Co-culture with the EPIYT isogenic strain induced the phosphorylation
of serine/threonine kinase AKT (also called protein kinase B, PKB) at threonine residue 308
(T-308) 2.4 ± 0.10 fold, compared with 1.7 ± 0.14 fold for the EPIYA strain (Fig. 3), indicating
intensified induction of PI3-kinase/AKT activation. Neither co-culture with the isogenic H.
pylori strain possessing a B-domain with an abolished tyrosine phosphorylation site, nor co-
culture with the cagA knockout strain significantly increased AKT phosphorylation at T-308
(Fig. 3), suggesting that CagA-positive H. pylori can activate the PI3-kinase/AKT pathway with
activity dependent on B-domain TPM phosphorylation.
The EPIYT site at B-TPM of CagA is necessary for interaction with PI3-
kinase
To investigate the interaction between the CagA B-TPM and PI3-kinase, we first used α-CagA
antibodies to perform immunoprecipitation after co-culture of AGS cells with isogenic H.
pylori 26695 strains with cagA variations. Western blotting using α-PI3-kinase antibody
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 7/25

revealed that only wild-type CagA can bind to PI3-kinase but not EPIYA-ABC
Y>F
or EPIYT-
B
Y>F
mutants (S3 Fig. in S1 Text). These data indicate that EPIYT B-TPM, but not EPIYA A-
or EPIYA C-TPMs, is necessary for this interaction. The α-pY-99 control blot shows that the
wild-type CagA is phosphorylated, while phosphorylated CagA with the EPIYT-B
Y>F
mutation
cannot interact with PI3-kinase (S3 Fig. in S1 Text). In a similar experiment, AGS cells were
co-cultured with several isogenic CagA-expressing H. pylori strains including the EPIYT-
AC
Y>F
and EPIYT-B
Y>F
mutants, followed by α-CagA immunoprecipitation. For both CagA
variants (EPIYT-AC
Y>F
and EPIYT-B
Y>F
), the phosphorylation signal was as expected. West-
ern blotting using α-PI3-kinase antibody revealed that only CagA (wt) and EPIYT-AC
Y>F
(with intact B-TPM) can bind to PI3-kinase, but not the EPIYT-B
Y>F
mutant (Fig. 4). These
findings indicate that EPIYT B-motif can be phosphorylated, which is necessary for the PI3-ki-
nase-CagA B-TPM interaction. To further confirm our observation, CagA presence and phos-
phorylation at the EPIYT-site was examined using phospho-specific α-pCagA-EPIYT-918 and
α-CagA antibodies when all samples contained similar amounts of PI3-kinase (Fig. 5A and B).
Only the lane with H. pylori expressing wild-type CagA revealed a signal for CagA and
Figure 2. Sequence comparison of the three TPM sites in CagA proteins from different clinical H.
pylori strains and specific detection of phosphorylated EPIYT-motif during co-culture. Panel A: CagA
proteins of H. pylori vary in their carboxy-terminal TPM sites. These EPIYA-repeats serve as tyrosine
phosphorylation sites of CagA and can be targeted by c-Abl and c-Src kinases. Three EPIYA- or EPIYT-
segments at position A, B and C are shaded with yellow. One striking feature of B-TPM is the presence of a
threonine residue in the +1 position (shaded with blue) relative to the phosphorylated tyrosine residue, which
is highly conserved in most but not all H. pylori strains and may affect the capabilities of binding the p85
subunit of PI3-kinase, as discussed in the text. The CagA protein sequences were obtained from databases
and sequence alignment wasdone using the ClustalW2 program (http://www.ebi.ac.uk/Tools/msa/clustalw2/).
Panel B: To investigate whether the EPIYT-motif can be phosphorylated during co-culture, AGS cells were
co-incubated with the indicated CagA-expressing H. pylori strains for 6 h. Phosphorylation of CagA was
examined using the phospho-specific α-pCagA-EPIYT-918 antibody. Loading of equal amounts of protein in
each sample was confirmed by probing with monoclonal α-CagA and α-GAPDH antibodies.
doi:10.1371/journal.ppat.1004621.g002
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 8/25

Figure 3. Analysis of the PI3-kinase-AKT pathway after co-culture of human AGS cells with isogenic
H. pylori strains containing the engineered CagA molecules. Panel A: After 24 h co-culture with
H. pylori isogenic cagA mutants, AGS cells were washed 5 times with ice-cold PBS buffer to remove H. pylori
cells, and whole cell lysates were separated by SDS-PAGE, followed by immunodetection with antibodies
(α-CagA, α-p-AKT, or α-AKT). Panel B: Targeted bands were quantified with ImageJ software to calculate
the relative level of AKT phosphorylation.
doi:10.1371/journal.ppat.1004621.g003
Figure 4. The EPIYT site at B-TPM of CagA is phosphorylated and necessary for interaction with
PI3-kinase. Panel A: Site-directed mutagenesis of CagA TPM-motifs A, B and C was performed to generate
the indicated phospho-resistant variants. Tyrosine residues in adjacent TPM-motifs were replaced by
phenylalanines. The resulting single and double mutants are indicated and complemented into the H. pylori
ΔcagA mutant. Panel B: AGS cells were co-cultured with the various CagA-expressing H. pylori strains for
6 h as indicated. Cell extracts were harvested and subjected to immunoprecipitation (IP) using α-CagA
antibodies. CagA phosphorylation in the IPs was examined using α-pY-99 and α-CagA antibodies (arrows).
All strains expressed similar amounts of CagA, and H. pylori expressing CagA wild-type (wt), EPIYT-AC
Y>F
,
and EPIYT-B
Y>F
all showed phosphorylation signal. Western blotting using α-PI3-kinase antibody revealed
that only CagA wt and EPIYT-AC
Y>F
can bind to PI3-kinase, but not the EPIYT-B
Y>F
mutant, suggesting that
EPIYT-B is phosphorylated and necessary for the interaction.
doi:10.1371/journal.ppat.1004621.g004
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 9/25

phosphorylation at EPIYT B-TPM in the immunoprecipitation. These findings provide further
evidence that phosphorylated EPIYT B-TPM is necessary for the interaction with PI3-kinase.
The alternative B-TPM EPIYT has enhanced PI3-kinase affinity
Co-immunoprecipitation assays indicated that in AGS cells, CagAs possessing the EPIYA or
EPIYT B-TPM interacted with PI3-kinase (p85), while CagA possessing EPIAT did not
Figure 5. PI3-kinase can interact with B-TPM of CagA during co-culture. Panel A: Site-directed
mutagenesis of CagA TPM-motifs A, B and C was performed to generate the indicated phospho-resistant
variants. Tyrosine residues in adjacent TPM-motifs were replaced by phenylalanines. The resulting single
and triple mutants were named as indicated and complemented into the H. pylori ΔcagA mutant. Panel B:
AGS cells were co-cultured with the various CagA-expressing H. pylori strains for 6 h as indicated. Cell
extracts were harvested and subjected to reverse immunoprecipitation (IP) using α-PI3-kinase antibodies. All
samples contained similar amounts of PI3-kinase in the input control. CagA presence and phosphorylation at
the EPIYT-site in the IPs was examined using phospho-specific α-pCagA-EPIYT-918 and α-CagA antibodies
(arrows). Only the lane with H. pylori expressing CagA wt revealed a signal for CagA and phosphorylation at
EPIYT-918 in the IP, indicating that phosphorylated EPIYT-B is necessary for the interaction with PI3-kinase.
Panel C: After 24 h co-culture of AGS cells with the isogenic H. pylori strains containing the engineered CagA
molecules, whole cell lysates were subjected to immunoprecipitation with an anti-CagA antibody. The anti-
CagA immunoprecipitates (IP) were separated on SDS-PAGE, followed by western blot with anti-PI3-kinase
(p85), which indicated that the engineered CagA B-EPIYA and CagA B-EPIYT molecules have different
affinity to the PI3-kinase protein in AGS cells.
doi:10.1371/journal.ppat.1004621.g005
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 10 / 25

(Fig. 5C), suggesting that CagA activates PI3-kinase/AKT signaling pathways by interacting
with the kinase via a functional B-motif. In AGS cells, CagA molecules possessing the B-do-
main EPIYT had higher affinity for PI3-kinase than those with EPIYA (Fig. 5C). These results
suggest that the CagA interaction with PI3-kinase has activating, rather than inhibiting effects
on its major downstream effector, AKT. The CagA molecules without a C-TPM sequence (as
in CagA
147A
) did not induce AKT phosphorylation at T-308, indicating that the C-TPM se-
quence is necessary for expression of the B-domain function. The C-domain enhancement of
B-domain activation of the PI3-kinase/AKT pathway is not dependent on the C-domain tyro-
sine phosphorylation since the B-domain EPIYT (in HPXZ1066) and B-domain EPIYA (in
HPXZ1067) without C-domain activity activated the PI3-kinase/AKT pathway. This suggests
the importance of maintaining the CagA dimerization state via the CagA multimerization
(CM) sequence [39].
Molecular modeling of CagA B-TPM interaction with the PI3-kinase SH2
domain
Molecular modeling of the CagA B-TPM EPIYTQVA sequence in complex with the N-termi-
nal PI3-kinase SH2-domain reveals that the threonine residue at the pY+1 position forms a
side chain hydrogen bond with an asparagine residue (N-417) of PI3-kinase (Fig. 6A). The re-
spective hydrogen bond cannot be formed for the “EPIYAQVA”motif, because the alanine
present at the respective sequence position lacks the hydroxyl group side chain required for an
interaction (Fig. 6B). Therefore, a T>A substitution at the pY+1 position is expected to signifi-
cantly decrease binding affinity of the B-TPM motif to PI3-kinase. This model is also sup-
ported by experimental peptide binding studies, that show that threonine at the pY+1 position
forms a stronger interaction than alanine with the respective SH2-domain [50]. Notably, PI3-
kinase also contains a second SH2 domain, in which the asparagine required for ligand binding
is conserved (N-707), suggesting that both PI3-kinase SH2-domains possess similar binding
specificity for the pY+1 position.
The alternative B-domain TPM EPIYT attenuates induction of the AGS
cell hummingbird phenotype
CagA-positive H. pylori co-cultured with AGS cells induce an elongated cell morphology
known as the hummingbird phenotype which is associated with effects on host cell polarity,
migration, and adhesion [36,51]. Next we evaluated whether the major alternative B-domain
TPM EPIYT affected CagA-induced hummingbird cell formation by co-culturing AGS cells
with the isogenic H. pylori 26695 cagA mutants based on a comparable bacterial/host cell popu-
lation level. We found that the isogenic cagA+ H. pylori strains induced significantly more
hummingbird-type AGS cells than did an H. pylori ΔcagA mutant, but abolishing the A- and
C- (EPIYA) TPMs significantly decreased hummingbird phenotype, indicating their involve-
ment in hummingbird induction (Fig. 7A and B). In the presence of functional A and C TPMs,
CagA with the B-domain EPIYA induced significantly more hummingbird cells than the CagA
possessing the B-domain EPIYT or EPIAT (Fig. 7A and B). This observation suggests that the
B-domain EPIYT has functional differences as compared to EPIYA. In the presence of non-
functional (EPIAA) A- and C-TPMs, strains with CagA possessing EPIYT or EPIYA B-TPM
induced significantly more hummingbird cells than the strain possessing the EPIAT B-TPM
(p<0.05) (Fig. 7B). B-TPM functions may be affected by the A- and C-TPMs since the signifi-
cant differential effects of EPIYT and EPIYA B-TPM were lost when we abolished those tyro-
sine phosphorylation sites.
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 11 / 25

The alternative B-domain TPM EPIYT attenuates AGS IL-8 induction
Interleukin-8 (IL-8), a neutrophil-activating chemokine [52], may play an important role link-
ing chronic inflammation and carcinogenesis [53]. The IL-8 induction effect is also associated
with the number of C domains in Western CagA+ strains [49,54]. Here, we evaluated whether
the major alternative B-domain TPM sequence, EPIYT, affects CagA-induced IL-8 production
by co-culturing AGS cells with the isogenic H. pylori 26695 cagA mutants based on a compara-
ble bacterial/host cell population level. At 24 h, AGS cells co-cultured with H. pylori ΔcagA
mutants had the same IL-8 level as the AGS cells without H. pylori co-culture (control), but
Figure 6. Model of the CagA B-TPM motif variants bound to PI3-kinase. The interactions of the
EPIYTQVA motif (Panel A) are compared to that of the EPIYAQVA motif (Panel B). The threonine residue at
the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by
alanine. The motif is shown in stick presentation and colored according to atom type. The PI3-kinase SH2-
domain is shown in yellow space-filled presentation and N-417 is colored by atom type.
doi:10.1371/journal.ppat.1004621.g006
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 12 / 25

Figure 7. Analysis of the hummingbird phenotype and IL-8 induction after co-culture of human AGS
cells with isogenic H. pylori strains containing the engineered CagA molecules. Panel A: AGS cells
were co-cultured with the isogenic H. pylori strains at a multiplicity of infection (MOI) of 100:1 for 24 h; cell
morphology was observed using microscopy. Red arrows indicate needle-like hummingbird cells. Panel B:
The mean (+SD) proportion of hummingbird cells induced by the H. pylori isogenic cagA mutants during co-
culture. *:p<0.05, Student’s T-test. Panel C: AGS cells were co-cultured with the isogenic H. pylori strains at
a multiplicity of infection (MOI) of 100:1. The media were sampled at 24 h, centrifuged at 16,000 g, and
supernatants collected. AGS cell IL-8 secretion (Mean+SD) was measured by an enzyme-linked
immunosorbent assay. *:p<0.05, Student’s T-test.
doi:10.1371/journal.ppat.1004621.g007
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 13 / 25

co-culture with the isogenic H. pylori cagA variants induced significantly higher IL-8 levels.
Under these conditions, the isogenic cagA mutant containing the EPIYA B-TPM induced
significantly more IL-8 induction than the mutant with the EPIYT B-TPM (Fig. 7C), a finding
indicating differential EPIYA- and EPIYT-B TPM protein functions. The isogenic mutant with
an EPIAT B-TPM had significantly decreased IL-8 induction (Fig. 7C). These results confirm
that CagA can induce AGS cell IL-8 production via its B-domain TPM, and indicate the differ-
ential EPIYT and EPIYA functions.
Discussion
A key host interaction factor of H. pylori, the CagA protein, has multiple polymorphisms
which differ in their affinities to host interaction partners and in their regulation of gastric cell
signaling cascades. EPIYA TPMs are critically important for CagA regulation of host signaling
pathways [28,55], and the four types (A, B, C and D) have different host interaction partners
[26] and/or varying affinities to the same partners [31,46], suggesting differential roles in regu-
lation of host signaling pathways.
Matsunari et al. first reported there are three most common types of EPIYA sequences in-
cluding the EPIYA, EPIYT and ESIYA, and that EPIYT of B-TPM is more predominant in
Western CagAs [56]. In this study, we further reveal the A/T polymorphism that specifically
occurs within the Western type B-domain, and we provide evidence for the first time that this
polymorphism significantly affects CagA functions in host cells. Indeed, the B-domain poly-
morphisms of the Western strains differed in their correlation with upper GI tract diseases
(Table 4), suggesting that a single SNP in a major bacterial interactive factor could decide dis-
ease outcome. The isogenic H. pylori cagA mutants expressing from the native genetic locus
created for the present investigations may be valuable for further studies.
Through its B-domain, CagA interacts with host partners including the Shp2 phosphatase,
Csk and PI3-kinases, as well as adaptor proteins Grb2 and Crk and the Shp1phosphatase, all of
which carry SH2-domains [28]. Among these, Shp1 and Shp2 also interact with the A- or C-
domains, Csk with the A-domain, Grb2 with C-domain, while PI3-kinase and CrkII only with
the B-domain in a tyrosine-phosphorylation-dependent manner [26,28]. Different TPMs have
both shared and specific host interaction partners suggesting that different EPIYA motifs could
have specific roles in regulating host signaling pathways. Moreover, other motifs aside from
the EPIYA TPMs could also be involved in CagA interactions with these host factors. CagA ac-
tivation of PI3-kinase/AKT appears dependent on the CRPIA sequences [24], but activation of
PI3-kinase/AKT also may be CagA-independent [37]. Our finding that H. pylori CagA with
functional EPIYA or EPIYT B-domains binds with PI3-kinase confirms and extends the obser-
vations by Selbach et al.[28]. Recently, Lind et al. developed a novel strategy to systematically
analyse phosphotyrosine antibodies recognizing single phosphorylated CagA EPIYA-motifs
utilizing synthesized phospho- and non-phosphopeptides [57]. With this strategy, by generat-
ing and analyzing a novel phospho-specific CagA B-motif antibody (anti-pCagA-EPIYT-918)
and isogenic CagA mutants with abolished TPMs, we further confirmed the CagA EPIYT-B
domain tyrosine-phosphorylation status during H. pylori co-culture with host cells and re-
vealed that tyrosine phosphorylation of the B-domain is necessary for the interaction between
CagA and PI3-kinase. We observed that the EPIYT B-domain has higher affinity to PI3-kinase
and greater AKT activation than the EPIYA B-domain. Our analysis by structural modeling of
the CagA EPIYA and EPIYT B-motifs interacting with the SH2 domain of PI3-kinase further
revealed the nature of differential interaction effects caused by the A/T polymorphism.
During gastric colonization, the host tyrosine kinases Src and Abl phosphorylate H. pylori
CagA EPIYA motifs [21–23,25], which differ from the classical consensus phosphorylation
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 14 / 25

sites in eukaryotic target factors (E-E-I-Y-E/G-X-F and I/V/L-Y-X-X-P/F of two tyrosine ki-
nases, respectively) [58]. This suggests that the CagA EPIYA motif phosphorylation level in
host cells may not be maximal. In that case, we propose that the A/T polymorphism/switch in
the EPIYA motif could be important in regulation of the TPM phosphorylation efficiency
and stability.
Strain-specific CagA sequence variation involves both conserved and non-conserved re-
gions. CagA
147C
used in our studies and CagA
26695
used by Suzuki et al. share only 87.1% iden-
tify and have numerous SNPs flanking each EPIYA/T TPM as well as differing tagging.
Considering the complex interactions between CagA with host protein partners as well as the
complex signaling network, use of transfected protein-expressing systems and both technical
and structural differences may affect signaling. The number of H. pylori CagA molecules within
host cells in different assays (e.g. CagA transfection and co-culture with H. pylori with native
expressing CagA) could markedly vary with differing kinetics of CagA phosphorylation, lead-
ing to different outcomes. Increased AKT activation and decreased IL-8 secretion of AGS cells
[59], and PI3-kinase/AKT pathway repression of IL-8 production during Salmonella co-culture
with intestinal epithelial cells [60] have been described. Strain- and time-dependent H. pylori
CagA-mediated IL-8 induction in AGS cells occurs through the Erk and NF-κB pathways
[49,61–64]. CagA-mediated PI3-kinase/AKT activation attenuates IL-8 induction by repressing
Erk/NF-κB including through the Shp-2/Erk pathway [49], the Shp2-independent Ras/Raf/
Mek/Erk pathway [65], and by Ras-independent Erk activation [66]. Inactivating B-TPM abol-
ished PI3-kinase/AKT activation, but decreased IL-8 secretion. Through B-TPM, CagA also
may interact with multiple other proteins in a site-competition and/or time-dependent man-
ner. For example, CagA TPMs interact with Shp2 through phosphorylated EPIYAs [28,34] en-
hancing Shp2 activity and Erk phosphorylation [31]. The B-TPM could positively regulate IL-8
production through activating the Shp2/Erk/ NF-κB pathway [49]. Abolishing the isogenic
single B-TPM inactivated the IL-8-repressing PI3-kinase/AKT, but also inactivated IL-8-
stimulating Shp2/Erk. Repression of IL-8 production by the B-TPM-mediated PI3-kinase/AKT
effect reflects cross-talk between the PI3-kinase/AKT and Shp2/Erk pathways. Consistent with
the overall differential signaling, Western cagA+ H. pylori strains with EPIYT or EPIYA B-
TPMs are associated with different patterns of clinical outcomes (Table 4 and Fig. 8), an obser-
vation that needs to be confirmed. The clinical outcome of H. pylori colonization results from
long-term processes, and therefore, how the B-TPM-mediated PI3-kinase/AKT effect alters
host gastric cancer development in long-term H. pylori colonization deserves further investiga-
tion. The PI3-kinase/AKT signaling pathway controls many of the hallmarks of cancer, and
many tumor tissues have enhanced PI3-kinase/AKT activities [67,68]. However, PI3-kinase/
AKT and their effectors are pleiotropic and have complex crosstalk and feedback behaviors in
the signaling network, which are not fully known [67,68]. We studied the regulation of CagA
on PI3-kinase/AKT pathway in vitro at an early time of bacterial interaction with host cells,
while long-term studies in mice or observations in patients at risk for gastric cancer will help
resolving the clinical significance of the polymorphism.
H. pylori colonization induces AGS cell scattering and elongation (hummingbird pheno-
type) through multiple CagA-related mechanisms; CagA binds to Csk through its A- or B-
TPMs, inhibiting SFK activity, and binds to Shp2 through its A-, B- or C-TPMs, leading to
FAK dephosphorylation [35,69]. Attenuated hummingbird phenotype induction present in the
cagA mutant with the EPIYT B-TPM (vs. EPIYA) reflects differential B-domain functional
roles, possibly through modulating direct interactions with Csk or Shp2. Inhibition of AKT ac-
tivation by the PI3-kinase inhibitor LY294002 has no effect on the hummingbird phenotype
[36]. However, LY294002 inhibits PI3-kinase catalysis by competing for ATP binding [70], but
does not directly affect the p85 binding activity with tyrosine-phosphorylated motifs such as
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 15 / 25

the CagA B-TPM. Such findings suggest that hummingbird induction by the CagA B-TPM re-
lates to the competition between PI3-kinase and Csk or Shp2 for binding at the B-TPM, but is
not directly related to PI3-kinase activity.
The A- and B-domains have unique host interacting partners, such as Csk, which do not in-
teract with C- or D-TPMs. These domains could possibly attenuate the C-domain-Shp2-inter-
action by binding with Csk to inactivate SFK members [71]. In this model, the C- and D-TPMs
serve as the primary phosphorylation motifs interacting with host signaling partners, and the
A- and B-TPMs serve as secondary sites, phosphorylated after C- or D-TPMs [25], suggesting
a potential regulatory role through competition between different TPMs. The EPIYT/EPIYA
B-TPM polymorphism that we studied provides a new level of complexity in H. pylori coloni-
zation and pathophysiology.
Materials and Methods
Bacterial strains, media and growth conditions
H. pylori strain 26695 was used to construct a series of isogenic cagA mutants, which express
CagA variants from the native CagA genetic locus [72]. H. pylori strains 147C and 147A, a pair
of naturally occurring isogenic cagA strains with EPIYA ABC and AB motifs, respectively [49],
were used as isogenic cagA sequence templates. H. pylori CagA-expressing strains P227, Oki-
61, P341, 2002-370 and 26695, also were used for AGS co-culture and evaluation of CagA
phosphorylation [73]. The H. pylori strains were grown at 37°C in 5% CO
2
on trypticase soy
agar (TSA) plates with 5% sheep blood (TSA, BBL Microbiology Systems, Cockeysville MD) or
Brucella agar plates (BA, Difco Laboratories, Detroit MI) supplemented with 10% newborn calf
serum (NBCS; Serologicals Corporation, Norcross GA) and suitable antibiotics [74]. Antibiot-
ic-resistant isogenic H. pylori strains were selected with kanamycin (Km; 10 μg/mL) or chlor-
amphenicol (Cm; 30 μg/mL), as appropriate. Alternatively, H. pylori strains were grown in
thin layers on horse serum GC agar plates supplemented with vancomycin (10 μg/mL), nysta-
tin (1 μg/mL), and trimethoprim (5 μg/mL), and for defined mutants with Cm (6 μg/mL) and/
or Km (8 μg/mL) at 37°C for 2 days in an anaerobic jar containing a Campygen gas mixture of
5% O2, 10% CO2, and 85% N2 (Oxoid, Wesel, Germany) [75]. E. coli DH5αwas grown in
Figure 8. Proposed model showing that CagA may regulate activation of the PI3-kinase/AKT pathway
through B-domain alternative TPM sequences. CagA proteins possessing a B-domain TPM with an
EPIYT sequence may have more chance of tyrosine phosphorylation by host kinases. The different
phosphorylation levels regulate CagA-tyrosine phosphorylation-dependent PI3-kinase interaction and AKT
activation. Activated AKT further regulates AGS cell cytoskeleton rearrangements to affect CagA-induced
hummingbird cell formation and inflammatory cytokine IL-8 secretion.
doi:10.1371/journal.ppat.1004621.g008
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 16 / 25

Luria-Bertani (LB) medium at 37°C [76]. Ampicillin (Ap; 100 μg/mL), Cm (30 μg/mL) or Km
(50 μg/mL) were used for selecting vectors or the constructs in E. coli during cloning.
Construction of isogenic H. pylori strains
Western type H. pylori strain 147C cagA has an EPIYT B-TPM, as well as one EPIYA -A and
-C TPM (cagA
147C B:EPIYT A&C:Y
)[49]. To evaluate B-TPM A/T polymorphism effects on CagA
functions, the threonine site of EPIYT B-TPM was replaced with alanine by recombination-
PCR mediated site-directed mutagenesis, leading to the generation of the isogenic cagA,
cagA
147C B:EPIYA A&C:Y
. To evaluate B-TPM A/T polymorphism effects on tyrosine phosphory-
lation of CagA B-TPM, the two tyrosine phosphorylation sites, A- and C-TPMs of the wild-
type cagA (cagA
147C B:EPIYT A&C:Y
) and the isogenic cagA (cagA
147C B:EPIYA A&C:Y
), were re-
placed with alanine, leaving the B-TPM as the only functional tyrosine phosphorylation site.
This resulted in two isogenic cagAs: cagA
147C B:EPIYT A&C:Y>A
and cagA
147C B:EPIYA A&C:Y>A
.
For controls, the tyrosine of B-TPMs of the wild-type and the isogenic cagAs were further
replaced with alanine to abolish B-TPM tyrosine phosphorylation function, leading to
cagA
147C B:EPIAT A&C:Y
and cagA
147C B:EPIAT A&C:Y>A
. Each of the wild-type and isogenic cagA
genes was first fused at the 3’end to the hemagglutinin (HA) tag and then linked to a 617 bp
cagA downstream region sequence based on the 26695 genomic sequence [72]. An aphA
(Km
R
) cassette [77] was inserted between the cagA-HA fusion gene and the cagA downstream
region sequence as a selection marker for the H. pylori mutant construction. Each construc-
tion was cloned into the vector pGEM-T easy (Promega, Madison WI), creating plasmids
pXZ476, pXZ465, pXZ468, pXZ471, pXZ472, and pXZ475, which carry cagA
147C B:EPIYT A&C:Y
,
cagA
147C B:EPIYA A&C:Y
,cagA
147C B:EPIAT A&C:Y
,cagA
147C B:EPIYT A&C:Y>A
,cagA
147C B:EPIYA A&C:Y>A
,
and cagA
147C B:EPIAT A&C:Y>A
, respectively (S2 Table in S1 Text). To replace the cagA
26695
se-
quence of H. pylori strain 26695 with the isogenic cagA
147C
sequences, a truncated cagA
147CN
(2445 bp) lacking C-terminal A- B- or C-TPMs was cloned and linked with a cat (Cm
R
) cas-
sette [77] and then with the 617 bp cagA downstream region sequence based on the 26695 se-
quence using pGEM-T easy, creating pXZ478 (S2 Table in S1 Text). To construct the H. pylori
ΔcagA control, the cagA upstream (839 bp) and downstream (1076) region sequences of H.
pylori 26695 were linked and inserted with an intervening sacB-cat (Cm
R
) cassette to replace
the entire cagA
26695
ORF on the same vector, creating plasmid pXZ083 (S2 Table in S1 Text).
To express the series of mutant cagA genes from the cagA native genetic locus in the 26695
genetic background, we first replaced the cagA
26695
sequence on the 26695genome with a
cagA
147C
s via homologous recombination (S1 Fig. in S1 Text). The wild-type H. pylori strain
26695 was transformed by plasmid pXZ478 to Cm
R
to create strain HPXZ1043 with isogenic
cagA
147CN
replacing native cagA
26695
. DNA sequencing confirmed the replacement of
cagA
26695
with the cagA
147CN
sequence in the 26695-derived Cm
R
/Km
S
strain HPXZ1043,
and western blot confirmed the expression of the truncated CagA
147CN
protein from the
native locus and the CagA promoter. Strain HPXZ1043 Cm
R
/Km
S
was then transformed
to Km
R
/Cm
S
with plasmids, pXZ476, pXZ465, pXZ468, pXZ471, pXZ472, or pXZ475, to
create mutants HPXZ1061 (cagA
147C B:EPIYT A&C:Y
), HPXZ1062 (cagA
147C B:EPIYA A&C:Y
),
HPXZ1065 (cagA
147C B:EPIAT A&C:Y
), HPXZ1066 (cagA
147C B:EPIYT A&C:Y>A
), HPXZ1067
(cagA
147C B:EPIYA A&C:Y>A
) and HPXZ1070 (cagA
147C B:EPIAT A&C:Y>A
), respectively. The wild-
type H. pylori strain 26695 was transformed to Cm
R
with pXZ083 to create the cagA-negative
mutant HPXZ1146 (ΔcagA::sacB-cat) (S2 Table in S1 Text). To confirm each construction,
sequencing of related regions was performed at Macrogen (Rockville MD), and all sequence
analysis was performed using Sequencher (Gene Codes, Ann Arbor MI).
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 17 / 25

Cloning, complementation and site-directed mutagenesis of CagA using
shuttle vector pHel3
To analyse the EPIYA- and EPIYT-motifs in the CagA protein, the complete cagA gene of H.
pylori strain 26695 (accession number: AAD07614) containing its promoter was amplified by
PCR, cloned into the pCR2.1 vector (Invitrogen) and sequenced [73]. For construction of a
complementation vector, this cagA fragment was cloned in the E. coli/H. pylori shuttle vector
pHel3 containing the oriT of RP4 and a kanamycin resistance gene cassette (Aph-A3) as a se-
lectable marker, resulting in vector pSB19 [73]. Site-directed mutagenesis of tyrosines Y-899,
Y-918 and Y-972 in the CagA sequence was done using the Sculptor mutagenesis kit, as
directed (Amersham Pharmacia Biotech) and resulting plasmids were transformed into H.
pylori isogenic ΔcagA mutant [78], as described [79].
Synthesis of phospho- and non-phospho CagA TPM peptides
The C-STEPIYAKVNK (EPIYA-A), C-STEPI(pY)AKVNK (phospho-EPIYA-A), C-PEE-
PIYTQVAK (EPIYT-B), C-PEEPI(pY)TQVAK (phospho-EPIYT-B), C-PEEPIYAQVAK
(EPIYA-B), C-PEEPI(pY)AQVAK (phospho-EPIYA-B), C-SPEPIYATIDD (EPIYA-C) and
C-SPEPI(pY)ATIDD (phospho-EPIYA-C) amino acid sequences were synthesized by Jerini
AG (Berlin, Germany). These 11-mer peptides were chosen because prior studies have shown
that α-phosphotyrosine antibodies typically recognize short phosphopeptides, and 11-mer and
9-mer sequences are both necessary and sufficient [57]. Commonly, 11-mer peptides also are
used for immunizations to generate phospho-specific antibodies, which then recognize the cor-
responding phosphopeptides bound to affinity columns and in ELISA (Biogenes, Berlin, Ger-
many). All above EPIYA and EPIYT peptides were purified by HPLC, and full-length synthesis
as well as purity of each peptide was confirmed by mass spectrometry by Jerini AG. The pep-
tides were resolved at a concentration of 1 mg/mL in DMSO and stored at −20°C.
Dotblot analysis
Twenty μg of each CagA peptide were mixed in 1 mL of TBST blotting buffer (140 mM NaCl;
25 mM Tris-HCl, pH 7.4; 0.1% Tween-20). These peptide samples were spotted onto Immobi-
lon-P membrane (Merck Millipore, Darmstadt, Germany) using the BioDot SF apparatus (Bio-
Rad, Munich, Germany). The resulting Dotblots were dried and subjected to antibody detec-
tion as described below for Western blots [57].
AGS culture and co-culture assays
Human gastric epithelial (AGS; ATCC CRL 1739) cells (obtained from American Type Culture
Collection) were cultured at 37°C in a humidified atmosphere with 5% CO2 in RPMI 1640
(Invitrogen, Carlsbad CA) with 10% fetal bovine serum (FBS; Invitrogen) with antibiotic-anti-
mycotic mixture (1X; Life Technologies, Grand Island NY) [80]. Before co-culture experi-
ments, AGS cells (2×10
5
cells/well) were transferred to a new 6-well plate and incubated in
fresh RPMI 1640 with 10% FBS and antibiotic-antimycotic for 24 h. The attached AGS cells
were washed and incubated in the RPMI 1640 media without serum or antibiotic for 16 h. The
AGS cells were then co-cultured with PBS-prewashed H. pylori cells, which were collected from
24-h TSA plates at a multiplicity of infection (MOI) of 100:1 and from fresh serum- and antibi-
otic-free RPMI 1640 for 8–24 h.
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 18 / 25

Cell elongation assays
AGS cultures and the co-culture of AGS and H. pylori were grown on coverglass (Fisher Scien-
tific, Pittsburgh PA) in 6-well plates [81]. After 24-h co-culture, 10 random fields of view for
each coverglass were examined at a magnification of ×200 using a Leica DMI 6000B micro-
scope. Alternatively, the AGS cells were co-cultured with H. pylori cells at MOI of 50 for 6 h,
when the cells were harvested in ice-cold PBS containing 1 mmol/L Na
3
VO
4
(Sigma-Aldrich).
Elongated AGS cells in each experiment were quantitated in 10 different 0.25-mm
2
fields using
an Olympus IX50 phase contrast microscope [82]. All experiments were performed
in triplicate.
IL-8 assay
After 24-h incubation, co-culture media were sampled and centrifuged at 16,000 g, and super-
natants collected. AGS cell IL-8 secretion was measured by an enzyme-linked immunosorbent
assay using the Human IL-8 ELISA Kit II (BD Biosciences, San Jose CA), in accordance with
the manufacturer’s instructions.
Generation of phospho-specific and non-phospho-specific α-EPIYT
antibodies
Phospho-specific and non-phospho polyclonal rabbit CagA antibodies were raised against
peptides corresponding to the following amino acid residues derived from the B-TPM motif
of strain 26695: C-PEEPIYTQVAK (non-phospho-EPIYT-B) and C-PEEPI(pY)TQVAK
(phospho-EPIYT-B). For this purpose, both peptides were conjugated to Limulus polyphemus
haemocyanin carrier protein and two rabbits each were immunized by Biogenes GmbH
(Berlin, Germany), according to standard protocols. The resulting phospho-specific antibodies
(α-pCagA-EPIYT-918) were affinity-purified against the corresponding non-phospho peptide
bound to a column. The resulting non-phospho antibodies (α-CagA-EPIYT-918) were affini-
ty-purified against the corresponding phospho-peptide. Both antibodies were prepared and
purified by Biogenes GmbH (Berlin, Germany). Their specificity was confirmed by dot blotting
against the phospho- and non-phospho peptides (S2 Fig. in S1 Text).
Immunoblotting, immunoprecipitation, and antibodies
To prepare whole cell extracts for immunoblotting, media were removed after 24-h incubation
and AGS cells were washed with ice-cold PBS 5 times to remove H. pylori cells. The whole cell
lysates for western blotting were prepared with RIPA lysis buffer (Thermo Scientific Pierce,
Rockford IL) with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific
Pierce). Lysates were separated by SDS-PAGE (Expedeon Inc. San Diego CA) and transferred
to Immobolin-P PVDF Transer Membrane (Fisher Scientific). Membranes were blocked in
TBST with 3% BSA or 5% skim milk for 1 or 2 h at room temperature. Membranes were incu-
bated with the following antibodies according to the instructions of the manufacturer. Immu-
nodetection of CagA peptides and the various proteins of interest were performed using
horseradish peroxidase–conjugated anti-mouse or anti-rabbit polyvalent sheep immunoglobu-
lin secondary antibodies and using chemiluminescence reagents, West Femto Chemilumines-
cent Substrate (Thermo Scientific Pierce) or Amersham ECL Western Blotting Detection
Reagents (GE Healthcare, Piscataway NJ) in accordance with the manufacturers’instructions
[83]. After exposure to X-ray film, the target band intensities were quantified using ImageJ
software (NIH, Bethesda MD). For immunoprecipitation, the whole cell lysates were prepared
with IP Lysis/Wash buffer (Thermo Scientific Pierce, Rockford IL) with Halt Protease and
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 19 / 25

Phosphatase Inhibitor Cocktail (Thermo Scientific Pierce). Immune complexes were prepared
using Pierce Crosslink Immunoprecipitation kit (Thermo Scientific Pierce) in accordance with
the manufacturers’instructions. The immunoprecipitates were subjected to SDS-PAGE, as de-
scribed above. Anti-actin, anti-AKT, anti-phospho-AKT (pThr308), and anti-phospho-AKT
(pSer473) ePI3-kinase were obtained from Cell Signaling Technology (Danvers MA). Anti-
HA, anti-Shp2, anti-Crk II, and anti-phospho-CrkII (pTyr221) were obtained from Thermo
Scientific Pierce, anti-phosphotyrosine was obtained from EMD Millipore Inc. (Billerica MA),
anti-Csk, anti-phospho-Csk (pSer364), anti-GAPDH and pan-phosphotyrosine antibody pY-
99 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz CA), and anti-CagA was
produced as described [49]. Anti-PI3-kinase (p85) antibodies were obtained from Cell Signal-
ing Technology (Danvers MA) or Santa Cruz Biotechnology, Inc. (Santa Cruz CA). Rabbit
polyclonal and mouse monoclonal α-CagA antibodies were from Austral Biologicals, or from
Emd Millipore Corporation (Billerica MA).
The phosphorylation status of CagA and bound PI3-kinase also was verified by immuno-
precipitation experiments as described [73,79]. Briefly, co-cultured or control AGS cells were
washed with cold PBS and lysed for 30 min at 4°C in lysis buffer (20 mM Tris pH 7.2, 150 mM
NaCl, 5 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM Na3VO4, COMPLETE™inhibitor
mix from Roche). Lysates were pre-cleared with protein G-Sepharose (Pharmacia, Uppsala,
Sweden) for 2 h at 4°C. Two micrograms of the monoclonal α-CagA antibody (Austral Biologi-
cals, San Ramon CA) or polyclonal antibody against the p85 subunit of PI3-kinase (Santa Cruz
Biotechnology, Dallas TX) were added to the supernatant and incubated overnight at 4°C on a
shaker. Immune complexes were precipitated by addition of protein G-sepharose for 2 h,
washed three times in 0.5× PBS and then mixed with equal amounts of 2× SDS-PAGE buffer.
Precipitates were analyzed by SDS-PAGE and immunoblotting.
Quantification of Dotblot and Western blot signals
Spot or band intensities on blots probed with the different α-phosphotyrosine antibodies were
quantitated with the Lumi-Imager F1 (Roche Diagnostics, Mannheim, Germany). Densitomet-
ric measurement of signal intensities revealed the percentage of phosphorylation per sample
[84].
3D-Modeling
The CagA-PI3-kinase interaction was modeled using the crystal structure of the N-terminal
PI3-kinase SH2-domain in complex with a C-kit phosphotyrosyl peptide (PDB: 2IUH) as a
template [85]. Modeling of the Cag-A B-TPM motif was performed using SwissModel [86] and
included the sequence stretches “EPIYTQVA”or “EPIYAQVA”. Structural analysis and visual-
ization was performed using RasMol [87].
Assessment of CagA EPIYA motif polymorphisms
A total of 2561 H. pylori complete or partial CagA protein sequences available at GenBank on
August 8
th
2013 were collected. The cagA EPIYA A-, B-, C-, and D-TPM types were defined as
described [46]. The numbers of each type of EPIYA TPMs and the polymorphisms within the
five specified amino acids were tabulated independently three times. A Chi-square test was per-
formed to evaluate the variance in the representation of each EPIYA TPM.
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 20 / 25

Supporting Information
S1 Text. The supporting Information file includes S1, S2, and S3 Fig., and S1 and S2 Tables.
(DOCX)
Acknowledgments
We thank Drs. Edward Skolnik, Haiping Zhou, and Yingfei Ma for advice and
technical assistance.
Author Contributions
Conceived and designed the experiments: MJB SB XSZ NT LT GPP. Performed the experi-
ments: XSZ NT LT GPP SJ HS. Analyzed the data: XSZ NT LT GPP SJ HS SB MJB. Contribut-
ed reagents/materials/analysis tools: XSZ NT LT GPP SJ HS. Wrote the paper: XSZ MJB NT
SB HS.
References
1. Murata-Kamiya N (2011) Pathophysiological functions of the CagA oncoprotein during infection by
Helicobacter pylori. Microbes Infect 13: 799–807. doi: 10.1016/j.micinf.2011.03.011 PMID: 21477660
2. Marshall BJ, Warren JR (1984) Unidentified curved bacilli in the stomach of patients with gastritis and
peptic ulceration. Lancet 1: 1311–1315. PMID: 6145023
3. Linz B, Balloux F, Moodley Y, Manica A, Liu H, et al. (2007) An African origin for the intimate association
between humans and Helicobacter pylori. Nature 445: 915–918. doi: 10.1038/nature05562 PMID:
17287725
4. Achtman M, Azuma T, Berg DE, Ito Y, Morelli G, et al. (1999) Recombination and clonal groupings
within Helicobacter pylori from different geographical regions. Mol Microbiol 32: 459–470. PMID:
10320570
5. Wroblewski LE, Peek RM Jr., Wilson KT (2010) Helicobacter pylori and gastric cancer: factors that
modulate disease risk. Clin Microbiol Rev 23: 713–739. doi: 10.1128/CMR.00011-10 PMID: 20930071
6. Parsonnet J, Hansen S, Rodriguez L, Gelb AB, Warnke RA, et al. (1994) Helicobacter pylori infection
and gastric lymphoma. N Engl J Med 330: 1267–1271. PMID: 8145781
7. el-Serag HB, Sonnenberg A (1998) Opposing time trends of peptic ulcer and reflux disease. Gut 43:
327–333. PMID: 9863476
8. Wiseman EF, Ang YS (2011) Risk factors for neoplastic progression in Barrett’s esophagus. World J
Gastroenterol 17: 3672–3683. doi: 10.3748/wjg.v17.i32.3672 PMID: 21990948
9. Eucker TP, Samuelson DR, Hunzicker-Dunn M, Konkel ME (2014) The focal complex of epithelial cells
provides a signaling platform for interleukin-8 induction in response to bacterial pathogens. Cell
Microbiol.
10. Oertli M, Muller A (2012) Helicobacter pylori targets dendritic cells to induce immune tolerance, pro-
mote persistence and confer protection against allergic asthma. Gut Microbes 3: 566–571. doi: 10.
4161/gmic.21750 PMID: 22895083
11. Blaser MJ, Perez-Perez GI, Kleanthous H, Cover TL, Peek RM, et al. (1995) Infection with Helicobacter
pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of
the stomach. Cancer Res 55: 2111–2115. PMID: 7743510
12. Hatakeyama M (2014) Helicobacter pylori CagA and Gastric Cancer: A Paradigm for Hit-and-Run Car-
cinogenesis. Cell Host Microbe 15: 306–316. doi: 10.1016/j.chom.2014.02.008 PMID: 24629337
13. Jurik A, Hausser E, Kutter S, Pattis I, Prassl S, et al. (2010) The coupling protein Cag{beta} and its inter-
action partner CagZ are required for type IV secretion of the Helicobacter pylori CagA protein. Infect
Immun.
14. Tummuru MK, Cover TL, Blaser MJ (1993) Cloning and expression of a high-molecular-mass major an-
tigen of Helicobacter pylori: evidence of linkage to cytotoxin production. Infect Immun 61: 1799–1809.
PMID: 8478069
15. Tegtmeyer N, Wessler S, Backert S (2011) Role of the cag-pathogenicity island encoded type IV secre-
tion system in Helicobacter pylori pathogenesis. Febs J.
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 21 / 25

16. 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. Gastroenter-
ology 117: 342–349. PMID: 10419915
17. Azuma T, Yamakawa A, Yamazaki S, Fukuta K, Ohtani M, et al. (2002) Correlation between variation of
the 3’region of the cagA gene in Helicobacter pylori and disease outcome in Japan. J Infect Dis 186:
1621–1630. PMID: 12447739
18. Furuta Y, Yahara K, Hatakeyama M, Kobayashi I (2011) Evolution of cagA Oncogene of Helicobacter
pylori through Recombination. PLoS One 6: e23499. doi: 10.1371/journal.pone.0023499 PMID:
21853141
19. Higashi H, Yokoyama K, Fujii Y, Ren S, Yuasa H, et al. (2005) EPIYA motif is a membrane-targeting
signal of Helicobacter pylori virulence factor CagA in mammalian cells. J Biol Chem 280: 23130–
23137. PMID: 15831497
20. Nagase L, Murata-Kamiya N, Hatakeyama M (2011) Potentiation of Helicobacter pylori CagA virulence
through homodimerization. J Biol Chem.
21. Backert S, Feller SM, Wessler S (2008) Emerging roles of Abl family tyrosine kinases in microbial path-
ogenesis. Trends Biochem Sci 33: 80–90. doi: 10.1016/j.tibs.2007.10.006 PMID: 18182299
22. Stein M, Bagnoli F, Halenbeck R, Rappuoli R, Fantl WJ, et al. (2002) c-Src/Lyn kinases activate Helico-
bacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs. Mol Microbiol 43: 971–980.
PMID: 11929545
23. 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.
PMID: 17408661
24. Suzuki M, Mimuro H, Kiga K, Fukumatsu M, Ishijima N, et al. (2009) Helicobacter pylori CagA phos-
phorylation-independent function in epithelial proliferation and inflammation. Cell Host Microbe 5: 23–
34. doi: 10.1016/j.chom.2008.11.010 PMID: 19154985
25. Muller A (2012) Multistep activation of the Helicobacter pylori effector CagA. J Clin Invest 122: 1192–
1195. doi: 10.1172/JCI61578 PMID: 22378039
26. Backert S, Tegtmeyer N, Selbach M (2010) The Versatility of Helicobacter pylori CagA Effector Protein
Functions: The Master Key Hypothesis. Helicobacter 15: 163–176. doi: 10.1111/j.1523-5378.2010.
00759.x PMID: 20557357
27. Hayashi T, Senda M, Morohashi H, Higashi H, Horio M, et al. (2012) Tertiary structure-function analysis
reveals the pathogenic signaling potentiation mechanism of Helicobacter pylori oncogenic effector
CagA. Cell Host Microbe 12: 20–33. doi: 10.1016/j.chom.2012.05.010 PMID: 22817985
28. 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. doi: 10.1016/j.chom.2009.03.004
PMID: 19380118
29. Ne Sbreve Ic D, Miller MC, Quinkert ZT, Stein M, Chait BT, et al. (2009) Helicobacter pylori CagA inhib-
its PAR1-MARK family kinases by mimicking host substrates. Nat Struct Mol Biol.
30. Nesic D, Buti L, Lu X, Stebbins CE (2014) Structure of the Helicobacter pylori CagA oncoprotein bound
to the human tumor suppressor ASPP2. Proc Natl Acad Sci U S A 111: 1562–1567. doi: 10.1073/pnas.
1320631111 PMID: 24474782
31. Lee IO, Kim JH, Choi YJ, Pillinger MH, Kim SY, et al. (2010) Helicobacter pylori CagA phosphorylation
status determines the gp130-activated SHP2/ERK and JAK/STAT signal transduction pathways in gas-
tric epithelial cells. J Biol Chem 285: 16042–16050. doi: 10.1074/jbc.M110.111054 PMID: 20348091
32. Neel BG, Gu H, Pao L (2003) The ‘Shp’ing news: SH2 domain-containing tyrosine phosphatases in cell
signaling. Trends Biochem Sci 28: 284–293. PMID: 12826400
33. Saadat I, Higashi H, Obuse C, Umeda M, Murata-Kamiya N, et al. (2007) Helicobacter pylori CagA tar-
gets PAR1/MARK kinase to disrupt epithelial cell polarity. Nature 447: 330–333. PMID: 17507984
34. Higashi H, Tsutsumi R, Muto S, Sugiyama T, Azuma T, et al. (2002) SHP-2 tyrosine phosphatase as an
intracellular target of Helicobacter pylori CagA protein. Science 295: 683–686. PMID: 11743164
35. 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. PMID: 10588744
36. Higashi H, Nakaya A, Tsutsumi R, Yokoyama K, Fujii Y, et al. (2004) Helicobacter pylori CagA induces
Ras-independent morphogenetic response through SHP-2 recruitment and activation. J Biol Chem
279: 17205–17216. PMID: 14963045
37. Nagy TA, Frey MR, Yan F, Israel DA, Polk DB, et al. (2009) Helicobacter pylori regulates cellular migra-
tion and apoptosis by activation of phosphatidylinositol 3-kinase signaling. J Infect Dis 199: 641–651.
doi: 10.1086/596660 PMID: 19199544
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 22 / 25

38. Ohnishi N, Yuasa H, Tanaka S, Sawa H, Miura M, et al. (2008) Transgenic expression of Helicobacter
pylori CagA induces gastrointestinal and hematopoietic neoplasms in mouse. Proc Natl Acad Sci U S A
105: 1003–1008. doi: 10.1073/pnas.0711183105 PMID: 18192401
39. Ren S, Higashi H, Lu H, Azuma T, Hatakeyama M (2006) Structural basis and functional consequence
of Helicobacter pylori CagA multimerization in cells. J Biol Chem 281: 32344–32352. PMID: 16954210
40. Suzuki R, Shiota S, Yamaoka Y (2013) Molecular epidemiology, population genetics, and pathogenic
role of Helicobacter pylori. Infect Genet Evol 12: 203–213. doi: 10.1016/j.meegid.2011.12.002 PMID:
22197766
41. Logan RP, Berg DE (1996) Genetic diversity of Helicobacter pylori. Lancet 348: 1462–1463. PMID:
8942769
42. Alm RA, Trust TJ (1999) Analysis of the genetic diversity of Helicobacter pylori: the tale of two ge-
nomes. J Mol Med 77: 834–846. PMID: 10682319
43. Falush D, Wirth T, Linz B, Pritchard JK, Stephens M, et al. (2003) Traces of human migrations in
Helicobacter pylori populations. Science 299: 1582–1585. PMID: 12624269
44. Aras RA, Lee Y, Kim SK, Israel D, Peek RM Jr., et al. (2003) Natural variation in populations of persis-
tently colonizing bacteria affect human host cell phenotype. J Infect Dis 188: 486–496. PMID:
12898434
45. Azuma T, Yamazaki S, Yamakawa A, Ohtani M, Muramatsu A, et al. (2004) Association between diver-
sity in the Src homology 2 domain—containing tyrosine phosphatase binding site of Helicobacter pylori
CagA protein and gastric atrophy and cancer. J Infect Dis 189: 820–827. PMID: 14976598
46. Hatakeyama M (2004) Oncogenic mechanisms of the Helicobacter pylori CagA protein. Nat Rev Can-
cer 4: 688–694. PMID: 15343275
47. Higashi H, Tsutsumi R, Fujita A, Yamazaki S, Asaka M, et al. (2002) Biological activity of the
Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation
sites. Proc Natl Acad Sci U S A 99: 14428–14433. doi: 10.1073/pnas.222375399 PMID: 12391297
48. Jones KR, Joo YM, Jang S, Yoo YJ, Lee HS, et al. (2009) Polymorphism in the CagA EPIYA Motif Im-
pacts Development of Gastric Cancer. J Clin Microbiol.
49. Kim SY, Lee YC, Kim HK, Blaser MJ (2006) Helicobacter pylori CagA transfection of gastric epithelial
cells induces interleukin-8. Cell Microbiol 8: 97–106. PMID: 16367869
50. Huang H, Li L, Wu C, Schibli D, Colwill K, et al. (2008) Defining the specificity space of the human SRC
homology 2 domain. Mol Cell Proteomics 7: 768–784. PMID: 17956856
51. Suzuki M, Mimuro H, Suzuki T, Park M, Yamamoto T, et al. (2005) Interaction of CagA with Crk plays
an important role in Helicobacter pylori-induced loss of gastric epithelial cell adhesion. J Exp Med 202:
1235–1247. doi: 10.1084/jem.20051027 PMID: 16275761
52. Crabtree JE, Wyatt JI, Trejdosiewicz LK, Peichl P, Nichols PH, et al. (1994) Interleukin-8 expression in
Helicobacter pylori infected, normal, and neoplastic gastroduodenal mucosa. J Clin Pathol 47: 61–66.
PMID: 8132812
53. Beswick EJ, Reyes VE (2008) Macrophage migration inhibitory factor and interleukin-8 produced by
gastric epithelial cells during Helicobacter pylori exposure induce expression and activation of the epi-
dermal growth factor receptor. Infect Immun 76: 3233–3240. doi: 10.1128/IAI.01534-07 PMID:
18474653
54. Argent RH, Hale JL, El-Omar EM, Atherton JC (2008) Differences in Helicobacter pylori CagA tyrosine
phosphorylation motif patterns between western and East Asian strains, and influences on interleukin-8
secretion. J Med Microbiol 57: 1062–1067. doi: 10.1099/jmm.0.2008/001818-0 PMID: 18719174
55. Takata S, Ito M, Wada Y, Yoshihara M, Tanaka S, et al. (2009) Pathogenetic role of the tyrosine-phos-
phorylated CagA EPIYA sequence of Helicobacter pylori in histological gastritis in Japanese patients. J
Gastroenterol.
56. 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: 876–883.
doi: 10.1128/JCM.05562-11 PMID: 22189111
57. Lind J, Backert S, Pfleiderer K, Berg DE, Yamaoka Y, et al. (2014) Systematic analysis of phosphotyro-
sine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of Western-type Helicobacter
pylori strains. PLoS One 9: e96488. doi: 10.1371/journal.pone.0096488 PMID: 24800748
58. Ubersax JA, Ferrell JE Jr. (2007) Mechanisms of specificity in protein phosphorylation. Nat Rev Mol
Cell Biol 8: 530–541. PMID: 17585314
59. Li SP, Chen XJ, Sun AH, Zhao JF, Yan J (2010) CagA(+)H.pylori Induces Akt1 Phosphorylation and
Inhibits Transcription of p21(WAF1/CIP1) and p27(KIP1) via PI3K/Akt1 Pathway. Biomed Environ Sci
23: 273–278. doi: 10.1016/S0895-3988(10)60063-3 PMID: 20934114
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 23 / 25

60. Huang F-C, Li Q, Cherayil BJ (2005) A phosphatidyl-inositol-3-kinase-dependent anti-inflammatory
pathway activated by Salmonella in epithelial cells. FEMS Microbiology Letters 243: 265–270. PMID:
15668028
61. Schneider N, Krishna U, Romero-Gallo J, Israel DA, Piazuelo MB, et al. (2009) Role of Helicobacter
pylori CagA Molecular Variations in Induction of Host Phenotypes with Carcinogenic Potential. J Infect
Dis 199: 1218–1221. doi: 10.1086/597416 PMID: 19278338
62. Zhang Y, Takeuchi H, Nishioka M, Morimoto N, Kamioka M, et al. (2009) Relationship of IL-8 production
and the CagA status in AGS cells infected with Helicobacter pylori exposed to low pH and activating
transcription factor 3 (ATF3). Microbiol Res 164: 180–190. PMID: 17449233
63. Kodaman N, Pazos A, Schneider BG, Piazuelo MB, Mera R, et al. (2014) Human and Helicobacter py-
lori coevolution shapes the risk of gastric disease. Proc Natl Acad Sci U S A 111: 1455–1460. doi: 10.
1073/pnas.1318093111 PMID: 24474772
64. Ando T, Peek RM Jr., Lee YC, Krishna U, Kusugami K, et al. (2002) Host cell responses to genotypical-
ly similar Helicobacter pylori isolates from United States and Japan. Clin Diagn Lab Immunol 9: 167–
175. doi: 10.1128/CDLI.9.1.167-175.2002 PMID: 11777849
65. 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. doi: 10.1073/pnas.0409873102 PMID: 15972330
66. Zhu Y, Zhong X, Zheng S, Du Q, Xu W (2005) Transformed immortalized gastric epithelial cells by viru-
lence factor CagA of Helicobacter pylori through Erk mitogen-activated protein kinase pathway. Onco-
gene 24: 3886–3895. PMID: 15856031
67. Fruman DA, Rommel C (2014) PI3K and cancer: lessons, challenges and opportunities. Nat Rev Drug
Discov 13: 140–156. doi: 10.1038/nrd4204 PMID: 24481312
68. Yuan TL, Cantley LC (2008) PI3K pathway alterations in cancer: variations on a theme. Oncogene 27:
5497–5510. doi: 10.1038/onc.2008.245 PMID: 18794884
69. Tsutsumi R, Takahashi A, Azuma T, Higashi H, Hatakeyama M (2006) Focal adhesion kinase is a sub-
strate and downstream effector of SHP-2 complexed with Helicobacter pylori CagA. Mol Cell Biol 26:
261–276. doi: 10.1128/MCB.26.1.261-276.2006 PMID: 16354697
70. Vlahos CJ, Matter WF, Hui KY, Brown RF (1994) A specific inhibitor of phosphatidylinositol 3-kinase, 2-
(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 269: 5241–5248. PMID:
8106507
71. Tsutsumi R, Higashi H, Higuchi M, Okada M, Hatakeyama M (2003) Attenuation of Helicobacter pylori
CagA × SHP-2 signaling by interaction between CagA and C-terminal Src kinase. J Biol Chem 278:
3664–3670. PMID: 12446738
72. Tomb JF, White O, Kerlavage AR, Clayton RA, Sutton GG, et al. (1997) The complete genome se-
quence of the gastric pathogen Helicobacter pylori. Nature 388: 539–547. PMID: 9252185
73. Mueller D, Tegtmeyer N, Brandt S, Yamaoka Y, De Poire E, et al. (2012) c-Src and c-Abl kinases con-
trol hierarchic phosphorylation and function of the CagA effector protein in Western and East Asian
Helicobacter pylori strains. J Clin Invest 122: 1553–1566. doi: 10.1172/JCI61143 PMID: 22378042
74. Zhang XS, Blaser MJ (2012) DprB facilitates inter- and intragenomic recombination in Helicobacter py-
lori. J Bacteriol 194: 3891–3903. doi: 10.1128/JB.00346-12 PMID: 22609923
75. 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. doi: 10.1371/journal.pone.0071220 PMID: 23940723
76. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning, A Laboratory Manual. Cold Spring Har-
bor, NY: Cold Spring Harbor Laboratory Press.
77. Zhang XS, Blaser MJ (2012) Natural transformation of an engineered Helicobacter pylori strain defi-
cient in type II restriction endonucleases. J Bacteriol 194: 3407–3416. doi: 10.1128/JB.00113-12
PMID: 22522893
78. Tegtmeyer N, Lind J, Schmid B, Backert S (2014) Helicobacter pylori CagL Y58/E59 mutation turns-off
type IV secretion-dependent delivery of CagA into host cells. PLoS One 9: e97782. doi: 10.1371/
journal.pone.0097782 PMID: 24893039
79. Tegtmeyer N, Wittelsberger R, Hartig R, Wessler S, Martinez-Quiles N, et al. (2011) Serine Phosphory-
lation of Cortactin Controls Focal Adhesion Kinase Activity and Cell Scattering Induced by Helicobacter
pylori. Cell Host Microbe 9: 520–531. doi: 10.1016/j.chom.2011.05.007 PMID: 21669400
80. Pillinger MH, Marjanovic N, Kim SY, Scher JU, Izmirly P, et al. (2005) Matrix metalloproteinase secre-
tion by gastric epithelial cells is regulated by E prostaglandins and MAPKs. J Biol Chem 280: 9973–
9979. PMID: 15640153
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 24 / 25

81. Bourzac KM, Botham CM, Guillemin K (2007) Helicobacter pylori CagA induces AGS cell elongation
through a cell retraction defect that is independent of Cdc42, Rac1, and Arp2/3. Infect Immun 75:
1203–1213. doi: 10.1128/IAI.01702-06 PMID: 17194805
82. 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.
83. 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. doi: 10.1007/
s00535-012-0618-8 PMID: 22722905
84. Krause-Gruszczynska M, Boehm M, Rohde M, Tegtmeyer N, Takahashi S, et al. (2011) The signaling
pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine
kinases and guanine exchange factor Vav2. Cell Commun Signal 9: 32. doi: 10.1186/1478-811X-9-32
PMID: 22204307
85. Nolte RT, Eck MJ, Schlessinger J, Shoelson SE, Harrison SC (1996) Crystal structure of the PI 3-ki-
nase p85 amino-terminal SH2 domain and its phosphopeptide complexes. Nat Struct Biol 3: 364–374.
PMID: 8599763
86. Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for compara-
tive protein modeling. Electrophoresis 18: 2714–2723. PMID: 9504803
87. Sayle RA, Milner-White EJ (1995) RASMOL: biomolecular graphics for all. Trends Biochem Sci 20:
374. PMID: 7482707
CagA B-Motif A/T Polymorphism Regulates Cell Interactions
PLOS Pathogens | DOI:10.1371/journal.ppat.1004621 February 3, 2015 25 / 25