Correspondence: Takeshi Azuma, Department of Gastroenterology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Tel.: +81 78 382 5774; fax: +81 78 382 6039; e-mail: firstname.lastname@example.org
Helicobacter pylori CagA protein is considered a major virulence factor associated with gastric cancer. There are two major types of CagA proteins: the Western and East Asian CagA. The East Asian CagA-positive H. pylori infection is more closely associated with gastric cancer. The prevalence of gastric cancer is quite low in the Philippines, although Philippine populations are considered to originate from an East Asia source. This study investigates the characteristics of the cagA gene and CagA protein in Philippine H. pylori strains and compares them with previously characterized reference strains worldwide. The full-length cagA gene was sequenced from 19 Philippine isolates and phylogenetic relationships between the Philippine and 40 reference strains were analyzed. All Philippine strains examined were cagA positive, and 73.7% (14/19) strains were Western CagA-positive. The phylogenetic tree based on the deduced amino acid sequence of CagA indicated that the Philippine strains were classified into the two major groups of CagA protein: the Western and the East Asian group. These findings suggest that the modern Western influence may have resulted in more Western type H. pylori strains in the Philippines. Therefore, H. pylori-infected Filipinos can be considered to be at a low risk of developing gastric cancer.
Helicobacter pylori is a Gram-negative bacterium that infects about 50% of the world's population. Infection with H. pylori can result in chronic active gastritis and is a risk factor for peptic ulcers, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma (Parsonnet et al., 1991; The EUROGAST Study Group, 1993; Uemura et al., 2001; Parsonnet & Isaacson, 2004). Helicobacter pylori has been implicated in gastric carcinogenesis on the basis of various epidemiological studies. A Working Group of the World Health Organization International Agency for Research on Cancer concluded that H. pylori is a group I carcinogen in humans (International Agency for Research on Cancer Working Group, 1994). The prevalence of H. pylori infection varies in different parts of the world and recent studies reported that humans actually acquired H. pylori in the early days of their history, long before the migration of modern humans out of Africa, and the diverse distribution of H. pylori today is associated with waves of human migration in the past (Yamaoka et al., 2002, 2008; Falush et al., 2003; Linz et al., 2007; Moodley et al., 2009). The rate of H. pylori infection is high in Africa, East Asia and South Asia; however, the incidence of gastric cancer is high in East Asia, but not in South Asia or Africa; this may be explained partly by the diversity of H. pylori strains in these regions (Yamaoka et al., 2008).
CagA is one of the most studied virulence factors of H. pylori, and the cagA gene is one of the genes in the cag pathogenicity island (PAI). cagPAI contains about 30 genes and six of the cag genes are thought to encode a putative type IV secretion system that specializes in the transfer of a variety of multimolecular complexes across the bacterial membrane to the extracellular space or into other cells (Covacci et al., 1999). Recently, it was shown that CagA is directly injected into epithelial cells by means of the bacterial type IV secretion system like a needle, where it undergoes tyrosine phosphorylation by Src and Ab1 kinases (Selbach et al., 2002; Stein et al., 2002; Tammer et al., 2007). Tyrosine-phosphorylated CagA then forms a physical complex with SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase), which is known to play a positive role in mitogenic signal transduction, and stimulates phosphatase activity (Higashi et al., 2002b). Consequently, the CagA–SHP-2 complex activates the multiplication stimulus continuously within the cell, which allows permeation of the CagA protein, and is thought to cause cells to deviate from their normal multiplication control mechanism, leading to gastric cancer (Higashi et al., 2002a; Yamazaki et al., 2003; Azuma et al., 2004b). It is possible that the deregulation of SHP-2 by the translocation of CagA plays a role in the acquisition of a cellular-transformed phenotype at a relatively early stage in the carcinogenesis of gastric carcinoma (Higashi et al., 2002b; Azuma et al., 2004b). A recent study (Ohnishi et al., 2008) on generating CagA in transgenic mice has provided the first direct evidence of the role of CagA as a bacterium-derived oncoprotein that acts in mammals and further indicates the importance of tyrosine phosphorylation, which enables CagA to deregulate SHP-2, in the development of H. pylori-associated neoplasms. Based on the characteristics of the phosphorylation and binding to SHP-2, the H. pylori CagA protein could be divided into a Western type and an East Asian type (Higashi et al., 2002a; Azuma et al., 2004b). The East Asian CagA protein exhibits stronger SHP-2-binding activity and so is more pathogenic than the Western CagA protein in H. pylori-infected patients (Higashi et al., 2002a). Persistent active inflammation, atrophic gastritis, and a higher risk of gastric cancer were more closely related to the East Asian type of CagA than the Western type, based on clinical data from East Asia, Japan, and South Korea(Azuma et al., 2004a, b; Satomi et al., 2006; Jones et al., 2009). The characteristics of H. pylori strains, especially the cagA gene and the CagA protein, can assist in determining which H. pylori-infected patients are at a high risk of developing gastric cancer.
The Philippines is a developing country located in South East Asia. The incidence of gastric cancer in the Philippines is quite low, with rates of 7.9/100 000 and 5.4/100 000 for males and females (Curado et al., 2007), respectively, although the prevalence of H. pylori infection has been reported to be as high as 60% (Destura et al., 2004). The present study reports the diverse characteristics of the cagA gene and classification of the CagA protein in H. pylori-infected patients from the Philippines, based on the full genomic cagA sequences in comparison with previously reported H. pylori strains worldwide.
Materials and methods
Helicobacter pylori strains
One hundred and eighty nine patients with abdominal symptoms who underwent nonemergent gastroduodenal endoscopy at the St. Luke's Medical Center, Philippines, from 2005 to 2009, were included in the study. All patients were unrelated and Filipino in origin. Patients who had received nonsteroidal anti-inflammatory drugs were excluded from the study, and none of the patients had recently been prescribed antibiotics. Four biopsy specimens were obtained from each patient: two from the gastric antrum and two from the gastric body. One specimen each from the antrum and body was fixed in buffered formalin and was used in histological analysis. One specimen each from the antrum and body was used for culture of H. pylori. Only specimens that were positive for H. pylori culture were included.
This study was approved by the hospital ethics committee and a signed informed consent was obtained from each patient before enrollment.
Biopsy specimens were fixed and stained with hematoxylin–eosin and Giemsa. The specimens were examined for the clinical diagnosis by a pathologist with no knowledge of the endoscopic findings.
Helicobacter pylori culture conditions
Gastric biopsy specimens from each patient were inoculated onto a Mueller–Hinton agar (with 7% horse blood) plate and cultured at 37 °C in an anaerobic jar with a Campypak gas generator. After 3 days, the plates were observed for colony growth, and incubated further for up to 7 days. Gram stain and biochemical tests for the presence of urease, catalase, and oxidase were performed using a single colony from the plate to confirm the presence of H. pylori. If it is positive for all three enzymes, a single colony was picked from each primary culture plate, inoculated onto a fresh Mueller–Hinton (with Skirrows) agar plate (with 7% horse blood), and cultured under the same conditions described above. After 3–7 days, the plate was flooded with 1 mL Brucella broth and all colonies were scraped off. A part of this bacterial suspension was placed in a freezing medium (800 μL H. pylori culture in Brucella broth, 100 μL dimethyl sulfoxide, 100 μL fetal bovine serum) and stored at −80 °C. DNA from the H. pylori isolate was extracted using the QIAamp DNA Mini Kit (Qiagen), following the manufacturer's instructions, and stored at 4 °C until PCR amplification was performed.
Nucleotide sequence of cagA
The full product of the cagA gene was determined by PCR using the primers cagA L2(+) and cagA L2(−) (Table 1) (Yamazaki et al., 2005) in a 100 μL reaction mixture containing the following: TaKaRA ExTaq polymerase (5 U mL−1), 10 × ExTaq buffer, dNTP mixture (2.5 mM each), sterile distilled water, and 1 μL of the sample DNA. The regions containing full-length cagA were amplified by PCR under the following conditions: 94 °C for 1 min; 25 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 3.45 min; followed by 72 °C for 10 min. PCR products were run on a 1.5% agarose gel (Agarose S) that was stained with ethidium bromide and examined under UV. The PCR products of samples that were cagA+ were purified using Amicon Centricon centrifugal filter devices YM 100MW (Millipore) or the High Pure PCR Product Purification Kit (Roche), according to the manufacturer's instructions. DNA direct sequencing was performed using a Big Dye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems) (3 μL of the purified PCR product in a 20 μL total reaction mixture containing the following: Big Dye, primer, and sterile distilled water). The primers used and their sequences are listed in Table 1 (Yamazaki et al., 2005). The sequencing PCR products were then purified using the Dye Ex 2.0 Spin Kit (Qiagen), according to the manufacturer's instructions. The purified sequencing PCR products were processed for sequencing performed on the ABI PRISM 3100-Avant genetic analyzer (Applied Biosystems). DNA sequences were analyzed using genetyx v. 7 (Software Development, Tokyo, Japan).
It has been demonstrated previously that CagA can be divided into Western and East Asian types by the kind of amino acid at a tyrosine phosphorylation site (Higashi et al., 2002a). Strains that possess WSS (Western CagA-specific, SHP-2-binding sequence) are classified as Western type CagA, whereas strains that possess ESS (East Asian CagA-specific, SHP-2-binding sequence) are classified as East Asian type CagA (Higashi et al., 2002a). Tyrosine phosphorylation of CagA occurs at unique Glu–Pro–Ile–Try–Ala (EPIYA) motifs repeated several times in the C-terminal region. These EPIYA motifs are involved in the interaction of CagA with SHP-2. The first and second EPIYA motifs (designated as ‘EPIYA-A’ and ‘EPIYA-B’, respectively) are present in almost all Western and East Asian CagA proteins, although the subsequent amino acid sequence is quite different between Western and East Asian type CagA. The third EPIYA motifs included in WSS or ESS were designated as ‘EPIYA-C’ or ‘EPIYA-D’ (Higashi et al., 2002a), respectively.
Characteristic diversity of CagA
A total of 19 H. pylori strains from 19 patients was used in this study: eight patients with gastritis, three patients with duodenal ulcer, six with gastric ulcer, and two with gastric cancer. There were ten males and nine females, with a mean age of 52.89±11.55 years (range from 30 to 67 years).
All Philippine strains examined were cagA-positive and the CagA genotypes of the 19 Philippine strains are shown in Table 2. The Philippine strains can be divided into East Asian (five strains) or Western (14 strains) types. Sequencing of the cagA gene showed a variable size of 3504–3651 bp full-length encoding region, and the predicted size of CagA in 19 strains ranged from 1168 to 1217 amino acids.
Table 2. Genetic characteristics of cagA in 19 Philippine Helicobacter pylori isolates
Size of the full-length cagA gene (bp)
Size of the predicted protein (aa)
% G+C content
GenBank accession number
C′, variable EPIYA-C region.
B′, EPIYT – alanine (A) residue is replaced by threonine (T).
The alignment of the deduced amino acid sequence in the 3′ region of the cagA gene among the Philippine strains is shown in Fig. 1a and b. Reference Western strain 26695 (accession number: AE000511) has a single WSS and is thus classified as the ‘A-B′-C’ type, and the reference East Asian strain F32 (accession number: AF202972) has a single ESS and is thus classified as the ‘A-B-D’ type. These references were used for a comparison of the amino acid sequence alignment in the 3′ region. Among the Philippine East Asian CagA strains, there was a conserved sequence of 58 amino acids, indicated by letters in the box (Fig. 1a), which had only a single variation in strain PHL10. The Philippine Western CagA strains showed much more variation between the EPIYA-A and the EPIYA-B motifs, as well as between the EPIYA-B and the EPIYA-C motifs (Fig. 1b).
The homology of the nucleotide and amino acid sequences was determined (data not shown). In the East Asian group, the highest degrees of homology were 97.24% and 95.89%, and the lowest were 95.97% and 93.09%, for the full nucleotide and amino acid sequences, respectively. Among the Western CagA strains, the highest degrees of homology were 99.77% and 99.41%, and the lowest were 93.55% and 90.65%, for the full nucleotide and amino acid sequences, respectively. The Japanese representative strain for East Asian type CagA, F32, and the Western representative strain, 26695, were included for comparison with the Philippine strains. The highest degrees of homology of F32 and 26695 with the Philippine strains were 97.10% and 95.60% for the nucleotide sequences, and 96.16% and 92.96% for the amino acid sequences, respectively. The lowest degrees of homology were 86.53% and 87.35% for the nucleotide sequences, and 78.40% and 77.60% for the amino acid sequences, respectively.
Phylogenetic analysis of the cagA sequences
The phylogenetic tree of the complete amino acid sequences demonstrated the genetic relationship among the 19 Philippine strains, as well as 40 references (Fig. 2). There were two major types: an East Asian and a Western type. In addition, there was a Japanese subtype in Western CagA type (J-Western CagA subtype) (Truong et al., 2009) composed of Okinawa strains. All East Asian CagA-positive Philippine strains based on the EPIYA motif were included in the East Asian cluster. In contrast, all Western CagA-positive Philippine, Thailand, and Vietnam strains based on the EPIYA motif were included in the major Western cluster, not in the J-Western CagA subtype.
CagA is considered to be a major virulence factor associated with gastric cancer. We have reported that the grades of inflammation, activity of gastritis, and atrophy are significantly higher in gastritis patients infected with the East Asian CagA-positive strain than in gastritis patients infected with the CagA-negative or the Western CagA-positive strain (Azuma et al., 2004b). The prevalence of the East Asian CagA-positive strain is associated with the mortality rate from gastric cancer in Asia (Azuma, 2004). Endemic circulation of H. pylori populations carrying more biologically active CagA proteins in East Asian countries, where the mortality rates from gastric cancer are among the highest in the world, may be involved in increasing the risk of gastric cancer in these populations (Azuma, 2004). The present study is the first, to our knowledge, that has investigated the full sequences of the cagA gene and CagA protein from Philippine H. pylori strains. In this study, all Philippine strains examined were CagA-positive; however, 73.7% of the strains were Western CagA-positive. This observation supports the notion that H. pylori-infected Filipinos can be considered to be at a low risk of developing gastric cancer. Although the statistical analysis of the association between the CagA diversity and the clinical outcome could not be applied to the small number of patients evaluated in this study, it is interesting to point out that one of two gastric cancer strains was East Asian CagA-positive (ABD), and the other strain was Western type CagA, which had two repeats of the EPIYA-C motif (ABCC). It has been reported that the presence of strains with multiple repeats of the EPIYA motif was associated with gastritis with atrophy and gastric cancer (Hatakeyama & Higashi, 2005). The increasing number of EPIYA-C motifs has been reported to increase the risk of gastric cancer (Basso et al., 2008). They concluded that for gastric cancer risk, the most important factor is the number of CagA EPIYA-C segments among Western strains. The present data were consistent with these previous reports.
In the phylogenetic analysis of the deduced full amino acid sequence of CagA, all East Asian CagA-positive Philippine strains based on the EPIYA motif comprised the East Asian cluster. In contrast, we reported previously the presence of a Japanese subtype in the Western CagA type (J-Western CagA subtype) (Truong et al., 2009). All Western CagA-positive Philippine, Thailand, and Vietnam strains based on the EPIYA motif were included in the major Western cluster, not in the J-Western CagA subtype. These findings support that the origin of J-Western CagA-positive strains isolated in Okinawa is different from Western CagA-positive strains isolated in Southeast, South, and Central Asia. It has been reported that the diverse distribution of H. pylori is now associated with waves of migration in the past (Falush et al., 2003; Linz et al., 2007; Moodley et al., 2009). Thus, Africans are infected by H. pylori populations hpAfrica1 and hpAfrica2, Asians are infected by hpAsia2 and hpEastAsia, and Europeans are infected by hpEurope (Falush et al., 2003; Linz et al., 2007; Moodley et al., 2009). Because the Philippines is an Asian country, Filipinos would therefore be infected mostly by hpAsia2 and hpEastAsia. Recently, it was reported that two prehistoric migrations peopled the Pacific, and that these migrations were accompanied by two distinct populations of H. pylori: hpSahul and hspMaori (Moodley et al., 2009). The hspMaori is a subpopulation of hpEastAsia, which was isolated from Polynesians in New Zealand and three individuals from the Philippines and Japan (Moodley et al., 2009). They suggested that hspMaori is a marker for the entire Austronesian expansions rather than only for Polynesians and their findings point to Taiwan as the source of the Austronesian expansions. They determined that hspMaori was widespread among aboriginal Taiwanese tribes and their phylogenetic analysis also showed that the genetic diversity was significantly higher in Taiwanese hspMaori than in non-Taiwanese hspMaori. The non-Taiwanese hspMaori haplotypes formed a single clade, the Pacific clade, which originates from one of several clades among indigenous Taiwanese haplotypes. Polynesians, Melanesians, and Filipinos were included in this Pacific clade. This might explain the presence of East Asian type H. pylori strains in Philippines; however, the majority of CagA type was Western type. It is possible that the intermarriages of the various races and nationalities with the indigenous ethnic groups and the strong Western influence and culture in the Philippines have resulted in more Western-type H. pylori strains in the country. hpEurope is common in Europe and countries colonized by Europeans (Yamaoka et al., 2008). The Philippines was a former colony of Spain (333 years), and it has also extensive relations and communications with Western countries. Compared with other East or Southeast Asian countries, the incidence of gastric cancer in the Philippines is quite low. This may be a reflection of the mostly Western CagA type of Philippine H. pylori strains; however, gastric cancer is a multifactorial disease (Hatakeyama, 2009) and incidence cannot be solely attributed to the type of bacteria or bacterial virulence factor. Investigations on a greater number of H. pylori strains isolated from Philippine patients need to be carried out.
In conclusion, the present study found that cagA is present all H. pylori strains examined from the Philippines. Philippine populations are considered to originate from Austronesian expansions; however, the major type of CagA in the Philippines is the Western type. These findings support that the modern Western influence has resulted in more Western-type H. pylori strains in the Philippines, which may explain the low incidence of gastric cancer, and H. pylori-infected Filipinos can be considered to be at a low risk of developing gastric cancer. In addition, J-Western strains are unique in Okinawa and different from other Western CagA-positive strains in Asian countries such as the Philippines, Thailand, and Vietnam.
We thank Ms Kumiko Sueyoshi for her technical assistance. This work was partly supported by funds from the Japan Society for the Promotion of Science.