Pharmacology & Pharmacy, 2013, 4, 13-18
doi:10.4236/pp.2013.45A003 Published Online August 2013 (http://www.scirp.org/journal/pp)
13
Analysis of the 3' Variable Region of Cytotoxin-Associated
Gene A (cagA) in Helicobacter pylori Isolates in China
Boqing Li*#, Donglong Du*, Wanju Sun, Qizhi Cao, Zhen Zhang, Zhenzhen Du
Department of Pathogen Biology, Binzhou Medical College, Binzhou, China.
Email: #sdliboqing@163.com
Received May 14th, 2013; revised June 25th, 2013; accepted July 4th, 2013
Copyright © 2013 Boqing Li et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Infection by the bacterium Helicobacter pylori is a putative cause of various gastric disorders, including gastric adeno-
carcinoma. Incident rates are associated with variants of the H. pylori virulence factor cytotoxin-associated gene A pro-
tein (CagA), encoded by the gene cagA. However, these variants have not been characterized in China, where gastric
cancer is common. We investigated the diversity of CagA variants in H. pylori strains isolated from a Chinese popula-
tion. The 3' variable region of cagA genes from 66 clinical isolates in China were amplified by polymerase chain reac-
tion, sequenced, aligned, and analyzed. All 66 H. pylori strains were CagA-positive, of which 93.9% were East Asian
type and the tyrosine phosphorylation motifs (TPMs) were EPIYA-ABD. The remainder was Western type, in which
TPMs were EPIYA-ABC. Interestingly, two of sixty-two strains (3.2%) of the East Asian type were mutated into
ESIYA-B, whereas all four Western type (100%) strains were mutated into EPIYT-B. Both of the two strains with
Western-type CagA obtained from gastric cancer patients contained a distinguished mutation on the first residue fol-
lowing the EPIYA site in the EPIYA-A motif. The predominant CagA type in these H. pylori strains isolated from Chi-
nese patients in China was East Asian, with TPMs EPIYA-ABD, and there existed mutations in both the East Asian and
Western type CagA.
Keywords: Helicobacter pylori; CagA; 3' Variable Region; China; Gastritis; Gastric Cancer
1. Introduction
Helicobacter pylori, a spiral microaerophilic gram-nega-
tive bacterium, colonizes the stomachs of 50% or more
people worldwide [1]. It is the causative agent of many
peptic disorders, including gastritis, gastric and duodenal
ulcers, and gastric mucosa-associated lymphoid tissue
lymphoma. H. pylori infection is also a recognized risk
factor for gastric adenocarcinoma [2,3]. However, not all
people with H. pylori infections will suffer these dis-
eases—about 30% of the population of Western countries
is infected but the rate of gastric cancer is only 0.1% -
1%. In East Asian countries such as China, Japan, and
Korea, the prevalence of H. pylori infection is 60% -
88%, and H. pylori-associated gastric cancers occur more
frequently than in Western countries [4-6]. Many epide-
miological studies have suggested that progression from
H. pylori infection to adverse effects is controlled by
combinations of genetic variants in the host, environ-
mental factors, and H. pyl o ri gene polymorphisms [7-9].
An H. pylori virulence marker found in most isolates
in high-risk areas of the world is a 40-kb cytotoxin-as-
sociated gene (cag) pathogenicity island, which forms a
type IV secretion system for delivering bacterial gene
products into host gastric epithelial cells. One of these
products is the oncoprotein CagA (encoded by the gene
cagA), putatively responsible for most malignancies as-
sociated with the bacterium [10]. CagA has a variable C-
terminus containing structural tyrosine phosphorylation
motifs (TPMs) within repetitive amino acid sequences of
Glu-Pro-Ile-Tyr-Ala (EPIYA) [11]. There are four known
EPIYA motifs (EPIYA-A, EPIYA-B, EPIYA-C and EPI
YA-D), differentiated by specific flanking amino acids
[12,13].
Two types of CagA, Western and East Asian, have
been described. Western CagA has EPIYA-A and EPI
YA-B segments followed by (1 to 3) EPIYA-C segments.
In the East Asian form, a EPIYA-D segment substitutes
for the EPIYA-C segments of the Western CagA. When
a CagA molecule is injected into the host cell, its tyrosi-
nes are phosphorylated and it binds to the N- and C-SH2
*Contributed equally.
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Analysis of the 3' Variable Region of Cytotoxin-Associated Gene A (cagA) in Helicobacter pylori Isolates in China
14
domains of a SHP-2 tyrosine phosphatase of the host,
initiating a cascade of abnormal enzymatic responses and
changes in cellular phenotype. The greater binding affin-
ity of East Asian CagA to SHP-2 makes East Asian
CagA more virulent than the Western type; the degree of
pathogenicity of Western CagA is associated with the
number of EPIYA-C motifs [12-15].
Variations in the TPMs of the 3’ region of CagA have
been reported in Western countries, and these variations
are associated with different peptic disorders. Yet to the
best of our knowledge, there have been no systematic
studies of CagA variants in China or their association
with diseases, although China is representative of the
East Asian area and gastric cancer is common [16], with
incidence rates in Shanghai (the latest statistics available)
reported at 34.2 and 17.3 per 100,000 men and women,
respectively [17]. It is not known if the EPIYA motifs of
CagA in Chinese H. pylori strains are East Asian, or
whether TPM types in China are associated with gastric
cancer.
In this study, we investigated the diversity of CagA
found in H. pylori strains isolated from a Chinese popu-
lation. We amplified the 3' region of cagA genes from 66
H. pylori clinical isolates, and then sequenced, aligned
and compared the amino acid sequences of correspond-
ing regions.
2. Materials and Methods
2.1. Patients
Gastric tissue biopsy samples from the antrum were ob-
tained via endoscopy of 66 patients (46 men and 20
women; mean age 49.8 y, range 20 - 82 y) at three hos-
pitals in Shandong, China. These patients were diagnosed
with gastritis (n = 31), gastric ulcer (n = 11), duodenal
ulcer (n = 6), or gastric cancer (n = 18), respectively.
2.2. Strain Culture and DNA Extraction
To examine the gastric biopsy specimens for the pres-
ence of H. pylori, the tissues were ground and then cul-
tured on Campylobacter agar base plates containing 10%
sheep blood and vancomycin (6 μg/mL), trimethoprim (5
μg/mL), polymyxin B (4 μg/mL), amphotericin B (2.5
μg/mL) under microaerophilic conditions (5% O2, 10%
CO2, 85% N2) at 37˚C for 72 h. The bacteria were har-
vested and genomic DNA was extracted using a QIAamp
DNA mini kit (Qiagen, Hilden, Germany) in accordance
with the instructions of the manufacturer.
2.3. Detection of CagA and EPIYA Motifs by
PCR
The 3' variable region and TPMs of cagA were amplified
by PCR using the primers shown in Table 1. All the
primers were designed using “Primer Premier 5.0” soft-
Table 1. PCR primers and the expected size of PCR prod-
ucts.
Primer Sequence (5’-3’) Size (bp)
cagF CCTAGTCGGTAATGGGTTAT
cagRa TTAATGCGTGTGTGGCTG 659
cagAa CTTGTCCTGTTTTCTTTTTATT 172
cagBa GCATTTACCTTTTTAGCAACT 224
cagCa CCGAGATCATCAATCGTAG 380
cagDa TGCTTGATTTGCCTCATC 396
aWith the forward primer cagF.
ware based on the homologous nucleotide sequence of
cagA gene among strains ATCC26695 and F32, repre-
sentative strains of Western and East Asian types, pub-
lished on the PubMed. Primers cagF and cagR were used
to amplify the full-length of the 3' variable region; prim-
ers cagA, cagB, cagC and cagD together with cagF were
used to detect the EPIYA-A, -B, -C and -D motifs, re-
spectively.
A reaction mixture contained 2.5 μL of each primer, 5
μL of genomic DNA, 25 μL 2 × Taq PCR MasterMix,
and ddH2O to a total volume of 50 μL. PCR conditions
consisted of pre-denaturation for 5 min at 95˚C; 30 cy-
cles of 30 s at 94˚C, 30 s at 52˚C, and 1 min at 72˚C; and
a final extension for 5 min at 72˚C. Five μL of each PCR
product was then resolved via electrophoresis on a 1%
agarose gel and observed under UV light after staining
with ethidium bromide.
2.4. Alignment and Analysis of the Amino Acid
Sequences
The PCR products of the cagF and cagR primers were
sequenced by Shanghai Majorbio. Alignment and analy-
sis of the sequences in the 3' variable region were carried
out using DNAStar 5.0 software.
3. Results
The gene amplification and sequencing protocols were
successful, and all the H. pylori strains detected in this
study were cagA-positive (cagA+). Representative PCR
products are shown in Figures 1(a) and (b).
The prevalence of East Asian CagA (62/66, 93.9%,)
was significantly higher than that of Western CagA (4/66,
6.1%; Table 2). Among the strains isolated from gastric
cancer patients specifically, 88.9% (16/18) were East
Asian CagA, and 11.1% (2/18) were Western CagA,
while the strains from the remaining patients were 95.8%
(46/48) East Asian and 4.2% (2/48) Western. All the
TPMs in East Asian-type strains were EPIYA-ABD,
while all the Western-type were EPIYA-ABC. No
EPIYA-ABCC or -ABCCC TPMs were found. Interest-
ingly, 3.2% of the East Asian-type strains (2/62, gastritis
Copyright © 2013 SciRes. PP
Analysis of the 3' Variable Region of Cytotoxin-Associated Gene A (cagA) in Helicobacter pylori Isolates in China 15
(a)
(b)
Figure 1. (a) Representative PCR products using the prim-
ers cagF and cagR. Lanes: M, molecular ladder (100 bp
stepwise ladder; range 100 - 1500 bp); 1 to 6, 3’ variable
regions of East Asian-type strains; 7 to 9, 3’ variable re-
gions of Western-type strains; (b) Representative PCR
products detected by multiple primer pairs. Lanes: M, mo-
lecular ladder (100 bp stepwise ladder; range 100 - 1500 bp);
W: 3’ variable region of Western CagA; E: 3’ variable re-
gion of East Asian CagA; A: EPIYA-A motif amplified by
primers cagF and cagA; B: EPIYA-B motif amplified by
primers cagF and cagB; C: EPIYA-C motif amplified by
primers cagF and cagC; D: EPIYA-D motif amplified by
primers cagF and cagD.
Table 2. H. pylori CagA types detected in 66 isolates.
Group East Asian Western Total
Gastric cancer 16 2 18
Gastritis 30 1 31
Gastric ulcer 11 0 11
Duodenal ulcer 5 1 6
Total 62 4 66
patients) were mutated into ESIYA-B, in which the P
(proline) residue is replaced by S (serine), whereas all 4
of the Western-type strains (100%, 2 gastric cancers, 1
gastritis, and 1 duodenal ulcer) were mutated into EPIYT-
B, in which the A (alanine) residue is replaced by T
(threonine). Intriguingly, both of the gastric cancer pa-
tients with the Western CagA type harbored stains with a
unique mutation, from K (lysine) to E (glutamate), on the
first residue following the EPIYA site in the EPIYA-A
motif (Table 3).
The alignment of the amino acid sequences in the 3’-
variable region of CagA is shown in Figure 2.
Table 3. Diversity of TPMs in 3' variable region of CagA.
TPMs East Asian Western Total
ABD 60 0 60
AB’D* 2 0 0
AB”C* 0 4 4
Total 62 4 66
*B’ refers to ESIYA-B, in which the P residue is replaced by S; B” refers to
EPIYT-B, in which the A residue is replaced by T.
4. Discussion
CagA is the most thoroughly studied virulence factor of
H. pylori. Patients infected with CagA+ strains of H. py-
lori have significantly increased levels of inflammation
of gastric epithelial cells and gastritis severity [18-21].
The East Asian CagA is more virulent than the Western,
because it has more affinity to bind SHP-2 [12,13,15].
The N- and C-SH2 domains of SHP-2 were highly re-
lated to its binding activity; the complement binding se-
quences for the two domains are pY-(S/T/A/V/I)-X-(V/
I/L)-X-(W/F). Intriguingly, the consensus sequence of
East Asian CagA, pY-A-T-I-D-F, perfectly matches the
binding sequence, while in Western CagA the sequence
is pY-A-T-I-D-D. Once the bacterium adheres to gastric
epithelium, CagA is continually translocated into the host
cells, establishing a sustained and deregulated stimula-
tion to signal pathways, and finally leading to cell dys-
function and even carcinogenesis [22-24].
In the 66 gastric biopsy tissues obtained from Chinese
patients in China in the present study, all contained H.
pylori and all were CagA+. We found that the prevalence
of East Asian CagA (93.9%, 62 of 66) was significantly
higher than that of the Western type (6.1%, 4 of 66). We
confirmed our sequence data by using multiple reverse
primers to individually amplify EPIYA-A, -B, -C and -D
motifs. Although China is an East Asian country, four
Western CagA strains were found in our investigation.
Taking the patients’ age, gender and other demographic
factors into account, we considered that this is probably
own to that with the developing of the world economy, it
was more fascinate and frequently for the exchange of
the Eastern and Western countries, those patients who
were diagnosed as Western type CagA might have been
gone abroad and been infected with Western type H. py-
lori.
Yamazaki et al. [25] reported finding four types of
mutations in the TPMs of the 3’ variable region of CagA.
Shunfu et al. [26] analyzed 539 cagA genes of 539 H.
pylori isolates and found 14 mutations; the most common
of which can be described as EPIYT-B and ESIYA-B.
Even an EPTYA-B was reported by Reyes-Leon et al.
[27]. According to the results of our present research, all
the TPMs of the East Asian strains were EPIYA-ABD,
including 2 strains mutated into ESIYA-B. Of the West-
Copyright © 2013 SciRes. PP
Analysis of the 3' Variable Region of Cytotoxin-Associated Gene A (cagA) in Helicobacter pylori Isolates in China
Copyright © 2013 SciRes. PP
16
Figure 2. Alignment of the amino acid sequences in the 3' variable region of CagA.
ern CagA, all were EPIYA-ABC. No EPIYA-ABCC or
EPIYA-ABCCC strains were found in our study. Inter-
estingly, all 4 strains of the Western type were mutated
into EPIYT-B.
In the present study, the amino acid sequence of the
EPIYA-A motif, EPIYA (K/Q) VNKKK (A/T) GQ, was
confirmed based on the classification defined by Higashi
et al. [12] and Panayotopoulou et al. [28]. There were 2
Western-type strains isolated from the gastric cancer pa-
tients, and both contained a remarkable mutation, from
lysine to glutamic acid, on the first residue following the
EPIYA-A site. The isoelectric points of the basic amino
acid lysine and the acidic glutamic acid are 9.74 and 3.22,
respectively, and we speculate that differences in the
isoelectric point values of the two residues cause a spatial
configuration change in the mutated CagA protein mole-
cule, which in turn strengthens the pathogenicity of
Western-type strains. This is the first report of an in-
crease in pathogenicity caused by a single amino acid
change, and the mechanism of this phenomenon is still
under investigation.
Of the 18 patients with gastric cancer in our study, 16
(88.9%) carried H. pylori with the East Asian CagA and
the remaining 2 (11.1%) had the Western type. Those
strains isolated from non-gastric cancer patients were
95.8% (46 of 48) East Asian CagA and 4.2% (2 of 48)
were Western. The CagA type did not appear to influ-
ence clinical outcome in this study. Although many epi-
demiological studies have shown that CagA is associated
with gastric cancer, these findings have been contra-
dicted by other researchers. Yamaoka et al. [29] reported
no differences between the levels of CagA antibody in
the serum of gastric cancer patients and the non-cancer
group. Other studies in Japan [30-32] also suggested that
there was no association between the presence of H. py-
lori and gastric cancer. However, it may be that the gas-
tric mucosa of gastric cancer patients could no longer
support colonization by H. pylori, and while the bacte-
rium may have had a causative role it was no longer de-
tectable. What is more, if the genes which encoded cag-
PAI were mutated or the type IV secretion system was
disabled, the protein would not be translocated into host
cells to achieve its biological function even the strains
were CagA+. Most important of all, gastric cancer is a
process that is not only influenced by polygenic varia-
tions of the pathogen, but also factors associated with the
host and environment.
Strain diversity among H. pylori isolates has been
studied at the gene level. Studies have shown that differ-
ent clinical outcomes could be linked with differences in
H. pylori virulence factors, which play essential roles in
pathogenesis [33,34]. Another study of Chinese, Indian,
and Malay patients showed that the gene structure of H.
pylori was related to the isolate’s geographic location
[35]. Studies of the relatedness of geography, disease,
and H. pylori polymorphisms will contribute to the un-
derstanding of strain-host interaction and the pathogenic-
ity level of each strain.
In summary, in this study conducted in China, we de-
tected strains of H. pylori in all the gastric biopsy sam-
Analysis of the 3' Variable Region of Cytotoxin-Associated Gene A (cagA) in Helicobacter pylori Isolates in China 17
ples, all were CagA positive, and the prevalence of the
East Asian CagA was considerably higher than that of
the Western CagA. There was not sufficient evidence to
indicate that the type of TPM was related to different
clinical outcomes. EPIYA-ABD and EPIYA-ABC were
the most common types found, and mutations existed in
both the East Asian- and Western-type CagA.
5. Acknowledgements
This report is based on work supported in part by grants
from the NSFC (81072429), Program for New Century
Excellent Talents in University (NCET-11-1026), and
grants-in-aid for Shandong Province Outstanding Young
Scientist Research Award Fund (2010BSB140), Colleges
and universities in Shandong province science and tech-
nology projects (J10LF21) and Yantai Municipal Science
and Technology Development Program (2010172).
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