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Second Department of Internal Medicine, Faculty of Medical Sciences, University of Fukui, Fukui, Japan
Department of Endoscopic Medicine, Faculty of Medical Sciences, University of Fukui, Fukui, Japan
Division of Internal Medicine, Okinawa Chubu Hospital, Okinawa, Japan
Division of Molecular Oncology, Institute for Genetic Medicine and Graduate School of Science, Hokkaido University, Sapporo, Japan
Frontier Medical Science in Gastroenterology, International Center for Medical Research and Treatment, Kobe University School of Medicine, Kobe, Japan
ABSTRACT
Colonization of the stomach mucosa by Helicobacter pylori is a major cause of acute and chronic gastric pathologies in humans. Several H. pylori virulence genes that may play a role in its pathogenicity have been identified. The most important determinants are vacA and cagA in the cag pathogenicity island (cagPAI) genes. In the present study, to consider the association of molecular genetics between vacA and the cagPAI regarding clinical outcome, we selected H. pylori strains with various genotypes of vacA in Japan and sequenced full-length vacA, cagA, and cagE genes. Sequencing of vacA and cagA genes revealed variable size, whereas the cagE gene was well conserved among strains. Each of the phylogenetic trees based on the deduced amino acid sequences of VacA, CagA, and CagE indicated that all three proteins were divided into two major groups, a Western group and an East Asian group, and the distributions of isolates exhibited similar patterns among the three proteins. The strains with s2 and s1a/m1a vacA genotypes and the Western-type 3' region cagA genotype were classified into the Western group, and the strains with the s1c/m1b vacA genotype and the East Asian-type 3' cagA genotype were included in the East Asian group. In addition, the prevalence of infection with the Western group strain was significantly higher in patients with peptic ulcer (90.0%, 9/10) than in patients with chronic gastritis (22.7%, 5/22) (2 = 12.64, P = 0.00057). These data suggest that the molecular genetics of vacA and cagPAI are associated and that the Western group with vacA and cagPAI genes is associated with peptic ulcer disease.
INTRODUCTION
Helicobacter pylori, a spiral, gram-negative, microaerophilic bacterium, colonizes at least half of the world's population and is recognized as a major cause of chronic gastritis and peptic ulcer and as an important risk factor for gastric cancer (24, 30, 33). On the basis of various epidemiological studies, H. pylori was classified as a class I carcinogen in humans by a working group of the World Health Organization International Agency for Research on Cancer (19).
Several H. pylori virulence genes that may play a role in its pathogenicity have been identified. Of these, the most important determinants are vacA and cagA genes. H. pylori strains have been divided into two broad families, type I and type II, which are based on whether or not they possess the vacA and cagA genes. Type I strains have the ability to produce VacA and CagA, while type II strains lack that ability (36). Type I strains are regarded as having greater pathogenicity and potential to cause development of disease. It is also known that H. pylori can be divided into distinct populations with different geographical distributions (13, 15).
VacA protein induces the formation of intracellular vacuoles in eukaryotic cells in vitro. The vacA gene contains at least two variable parts. Two sequence families, called s1 and s2, have been identified in different isolates. Among type s1 strains, subtypes s1a, s1b, and s1c have been identified. The m-region (middle) occurs as the m1 or m2 allelic type. Among type m1 strains, subtypes m1a and m1b have been identified (5, 20, 34, 36). Production of vacuolating cytotoxin is related to the mosaic structure of vacA. In general, type s1/m1 and s1/m2 strains produce high and moderate levels of toxin, respectively, whereas s2/m2 strains produce little or no toxin (5).
CagA protein, which is encoded by the cagA gene, is a highly immunogenic protein. It is thought that H. pylori strains possessing cagA are associated with significantly increased risk for the development of atrophic gastritis and gastric cancer (11, 18, 26). The cagA gene is located at one end of a 40-kb DNA insertion called the cag pathogenicity island (cagPAI) and may have originated from a non-Helicobacter source (12). The cagPAI contains 31 putative genes, 6 of which are thought to encode components of a bacterial type IV secretion system (2, 13). Recent studies have indicated that CagA is directly injected into epithelial cells via the type IV secretion system and undergoes tyrosine phosphorylation in the host cells (4, 9, 23, 27, 28). Furthermore, it was recently confirmed (16) that phosphorylated CagA 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 (39), and deregulates its enzymatic activity. Deregulation of SHP-2 by CagA may induce abnormal proliferation and movement of gastric epithelial cells. On the basis of the sequence constituting the SHP-2 binding site, CagA proteins can be subclassified into Western and East Asian types. The East Asian-type CagA possesses stronger SHP-2 binding and transforming activities than the Western-type CagA (17). It has also been reported that large sequence differences distinguish the cagA gene fragments from Asian strains and other strains (1, 37).
It is thought that there is a genetic linkage between vacA and cagA allelic geographic variations (35), although these genes are located separately on the genome (3, 31). Therefore, in this study, to consider the association of the molecular genetics of vacA and the cagPAI regarding clinical outcome, we selected H. pylori strains according to various genotypes of vacA and sequenced full-length vacA, cagA, and cagE, which is located near cagA in the cagPAI. The cagE gene product is a component of the type IV secretion system (13). Furthermore, we analyzed the phylogenetic relationship among the entire VacA, CagA, and CagE amino acid sequences.
MATERIALS AND METHODS
H. pylori strains. Two different areas of Japan were selected as sources for strains. Fukui is a typical rural prefecture located on the central Japanese mainland (Honshu), while Okinawa consists of islands in the southwestern part of Japan. These two areas are separated by more than 1,300 km. The clinical outcomes of H. pylori infections are quite different between Fukui and Okinawa. The prevalence of atrophic gastritis, a precursor lesion of gastric cancer, is more frequent in Fukui, and the death rate from gastric cancer is more than 2.4 times higher in Fukui than in Okinawa. In contrast, the prevalence of duodenal ulcer is more frequent in Okinawa, and the ratio of incidence of duodenal ulcer and gastric ulcer is more than 1.8 times higher in Okinawa than in Fukui. A total of 220 H. pylori clinical isolates (115 Fukui strains and 105 Okinawa strains) were obtained during upper gastroduodenal endoscopy at the Second Department of Internal Medicine, Faculty of Medical Sciences, University of Fukui, and Okinawa Chubu Hospital, Okinawa, respectively. The 115 patients in Fukui consisted of 41 patients with chronic gastritis (22 men and 19 women; mean age, 57.7 years), 26 with gastric cancer (14 men and 12 women; mean age, 60.2 years), 26 with duodenal ulcer (17 men and 9 women; mean age, 49.9 years), 18 with gastric ulcer (10 men and 8 women; mean age, 55.3 years), and 4 with gastroduodenal ulcer (4 men; mean age, 58.5 years). The 105 patients in Okinawa consisted of 58 patients with chronic gastritis (20 men and 38 women; mean age, 59.6 years), 4 with gastric cancer (3 men and 1 woman; mean age, 70.3 years), 24 with duodenal ulcer (20 men and 4 women; mean age, 53.8 years), 18 with gastric ulcer (11 men and 7 women; mean age, 59.2 years), and 1 with gastroduodenal ulcer (1 man; age, 40 years).
H. pylori culture conditions. Gastric biopsy specimens from each patient were inoculated onto a trypticase soy agar II-5% sheep blood plate and cultured for 3 to 5 days at 37°C under microaerobic conditions (5% O2, 5% CO2, 90% N2). A single colony was picked from each primary culture plate, inoculated onto a fresh trypticase soy agar II plate, and cultured under the conditions described above. H. pylori cells were harvested from each plate, transferred into a brucella broth liquid culture medium containing 10% fetal calf serum, and cultured for 24 h with agitation under the same conditions described above. A part of the bacterial suspension was stored at –80°C in 0.01 M phosphate-buffered saline containing 20% glycerol. DNA from each H. pylori isolate was extracted from the pellet of the bacterial suspension by use of the protease/phenol-chloroform method, suspended in TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA), and stored at 4°C until PCR amplification was performed.
PCR-based typing of vacA. Genotyping of the vacA gene was performed by PCR amplification in the s-region (the signal peptide-encoding region) and the m-region (middle region) described previously (5, 20, 34) using the primers shown Table 1. PCR products were separated by 2% agarose gel electrophoresis and examined under UV illumination.
Nucleotide sequencing of the entire vacA, cagA, and cagE genes. We selected 33 H. pylori strains (13 Fukui and 20 Okinawa strains) according to the vacA genotypes. The 13 patients in Fukui consisted of 10 with chronic gastritis, 1 with duodenal ulcer, 1 with gastric ulcer, and 1 with gastric cancer. The 20 patients in Okinawa consisted of 12 with chronic gastritis and 8 with duodenal ulcer. The 11 strains (F16, F17, F28, F32, F79, F80, OK101, OK107, OK109, OK112, and OK129) for which the entire cagPAI sequences were previously reported were included in this study (8). Primers for PCR amplification and direct sequencing of the entire coding regions of vacA, cagA, and cagE are shown in Table 2. The region containing full-length vacA was amplified by PCR under the following conditions: 95°C for 5 min; 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 4.5 min; followed by 72°C for 7 min. The regions containing full-length cagA and cagE were amplified under the same conditions except for the extension time (72°C for 4.5 min): in this case, cagA for 4 min and cagE for 3.5 min. PCR products were then purified with Centricon-100 Concentrator columns (Amicon, Beverly, Massachusetts). DNA direct sequencing was performed using a BigDye Terminator v.3.1 cycle sequencing kit (Applied Biosystems, Foster City, California) and an ABI PRISM 3100-Avant genetic analyzer (Applied Biosystems) according to the manufacturer's recommendations. The full-length amino acid sequences of each gene were constructed and translocated from the nucleotide sequence and aligned and analyzed by use of GENETYX-Mac version 11.2.3 (Software Development, Tokyo, Japan).
Phylogenetic analysis. To clarify the phylogenetic relationships between Japanese isolates and previously characterized H. pylori strains from the West, sequences of VacA, CagA, and CagE were aligned (GENETYX-Mac). Phylogenetic trees were constructed using the unweighted pair group method using the same software. The previously published gene sequences of strain 26695 (GenBank accession number AE000598, vacA [HP0887]; AE000569, cagA [HP0547]; and AE000568, cagE [HP0544]), strain J99 (AE001511, vacA [jhp0819]; AE001483, cagA [jhp0495]; and AE001482, cagE [jhp0492]), NCTC11638 (HPU07145, vacA; AF282853, cagA and cagE), and NCTC11637 (AF049653, vacA; AF202973, cagA) were included in the analysis.
Statistical analysis. The associations between the diversity of cagA and vacA genes and clinical outcome were analyzed with the chi-square test and Fisher's exact probability test.
Nucleotide sequence accession numbers. The DNA sequences of cagA, cagE, and vacA of each strain characterized here were deposited in the GenBank database. Accession numbers are shown in Table 3.
RESULTS
Diversity of vacA. The list of vacA genotypes is shown in Table 4. We previously reported that the predominant vacA genotype in Japan was s1c/m1b (40). In Fukui, strains with the s1c/m1b genotype account for 81.7% of all strains, whereas in Okinawa, there are various vacA genotypes, although the predominant vacA genotype is also s1c/m1b (71.4%) (Table 5). In the present study, we selected 33 H. pylori strains according to the vacA genotypes. However, the s1b/m2 and s1c/m1a genotypes and hybrid m1/m2 genes were not present, although we checked a total of 220 strains (115 Fukui and 105 Okinawa strains) (Table 5).
Sequencing of vacA genes revealed a variably sized 3,867- to 3,981-bp open reading frame (ORF), encoding proteins of 1,289 to 1,327 amino acids. The homology in the signal sequence region among F37 (s1a), F80 (s1b), F32 (s1c), and OK155 (s2) is shown in Fig. 1a and Table 6 (values above the dashes). The homology in the m-region (PCR fragment for genotyping of vacA) among F37 (m1a), F32 (m1b), and OK155 (m2) is shown in Fig. 1b and Table 6 (values below the dashes). In addition, the homologies of total amino acid residues and nucleotide sequences among F37, F80, F32, and OK155 are shown in Table 7. Furthermore, Atherton et al. (5) compared the VacA sequences between strain 60190 (m1) and strain Tx30a (m2) and found that there was only 59.0% amino acid identity in the middle of VacA (strain 60190, 244-amino-acid region, residues 509 to 752; strain Tx30a, 253-amino-acid region, residues 522 to 774). Therefore, we checked the homology of F32 (m1b) to OK210 (m2) in the m-region (F32, 244-amino-acid region, residues 512 to 755; OK210, 253-amino-acid region, residues 533 to 785), and it was found to be 58.9% (Fig. 2). Also, the homology of F32 (m1b) to F37 (m1a, 244-amino-acid region, residues 517 to 760) was 85.3%.
Diversity of CagA. The alignment of the deduced amino acid sequence in the 3' region of the cagA gene (CagA repeat domain) for strains 26695 and F32, which have typical Western and East Asian CagA proteins, respectively, is shown in Fig. 3. We previously demonstrated that CagA can be divided into Western and East Asian types (6, 17). Briefly, tyrosine phosphorylation of CagA occurs at the unique Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs present several times in the C-terminal region (10, 16, 29). These EPIYA motifs are involved in the interaction of CagA with SHP-2. The first and second EPIYA motifs (designated EPIYA-A and EPIYA-B, respectively) are present in almost all Western and East Asian CagA proteins, although the subsequent amino acid sequences are quite different between Western and East Asian CagA. The Western-type CagA possesses WSS (Western CagA-specific SHP-2 binding sequence), while the East Asian-type CagA possesses a distinct sequence, ESS (East Asian CagA-specific SHP-2 binding sequence), in the corresponding region. The EPIYA motifs included in WSS or ESS were designated EPIYA-C or EPIYA-D, respectively. Strain 26695 has a single WSS and is thus classified as the "A-B'-C" type, and F32 has a single ESS and is thus classified as the "A-B-D" type.
On the basis of the above typing, the list of CagA genotypes is shown in Table 4. "B'" means EPIYT-B, in which the A (alanine) residue is replaced by T (threonine); "B"" means ESIYA-B, in which the P (proline) residue is replaced by S (serine); "B'''" means ESIYT-B, in which the P and A residues are replaced by S and T; and "A'" means NPIYA-A, in which the E (glutamic acid) residue is replaced by N (asparagine). Although the number of repeats of the EPIYA motif was different among strains, all strains can be divided into the Western (12 strains) or East Asian (20 strains) types, except for the OK181 strain, which had neither the WSS nor the ESS sequence and was classified as "A-B'." B' was characteristic of the Western type because it appeared in all strains which possessed Western-type CagA, except for NCTC11637, NCTC11638, OK155, and OK160 in the present study. OK155 and OK160 had B''' instead of B'. B" appeared in the four East Asian-type CagA-possessing strains (F26, F55, OK159, and OK204). A' appeared in the F37 strain only. Sequencing of cagA genes revealed a variable size of 3,444- to 3,825-bp ORFs encoding proteins of 1,148 to 1,275 amino acids. The homology of OK130 (Western type) to F32 (East Asian type) in total amino acid residues was found to be 80.3%, and in the CagA repeat domain (Fig. 3) (OK130, 109-amino-acid region, residues 884 to 992; F32, 114-amino-acid region, residues 870 to 983) it was calculated to be 53.0%.
The F80 and OK204 strains had a mixed-type CagA (Fig. 4). Insertion of a 13-amino-acid (residues 823 to 835 of 26695) region, which exists forward of the CagA repeat domain, is characteristic of the Western type. Both strains had an insert region, but they also had the ESS (F80 was classified into the A-B'-D-D type, and OK204 was classified into the A-B"-D type). OK204 had the complete ESS, while in the F80 ESS, the first half of the region was the same as the WSS sequence, and the following EPIYA-D region was the ESS sequence. So, F80 CagA may be closer to the Western type than to OK204 CagA.
Diversity of cagE. The cagE gene was well conserved among strains. In all isolates except for the F36 strain, sequencing of cagE genes revealed a 2,949-bp ORF encoding proteins of 983 amino acids, which was the same as the previously published strains 26695, J99, and NCTC11637. The F36 strain possessed a 2,943-bp ORF, which encoded 981 amino acids because of a deletion of the first 2 amino acids and was the same as the previously published NCTC11638. The homology between each isolate in amino acids was very high, at over 98% (Table 4).
Phylogenetic analysis of the VacA, CagA, and CagE sequences. The phylogenetic tree of the VacA, CagA, and CagE proteins demonstrated a genetic relationship among 33 clinical isolates and 4 previously published strains from the West: 26695, J99, NCTC11638, and NCTC11637 (Fig. 5). All three proteins were divided into two major groups, a Western group and an East Asian group (Table 4). The distribution of the isolates was almost the same among each protein, with several exceptions.
For CagA, OK181, which was the A-B' type, was included in a Western group. F80 and OK204 were also contained a Western group, although they possessed ESS (Fig. 4). Since their VacA and CagE also contained a Western group, both strains may have originated from a Western area rather than an East Asian area. In the other strains, the EPIYA type accorded with the geographic type based on the phylogenetic tree.
In VacA, the phylogenetic analysis indicated a clear separation between the m1 and m2 sequences. All m2 strains are classified into the Western cluster. Furthermore, in the m1 cluster, m1a and m1b clusters were clearly distinguished. All m1a and m1b strains are classified into the Western and East Asian clusters, respectively. Moreover, in the m1a and m1b clusters, s1a or s1c and s1b clusters were divided. Subtypes of s1a and s1c were not separated. F37 and OK129 were included in a Western group, although both of their CagA types were East Asian. This resulted from their subtypes in the m region: F37 was m1a and OK129 was m2. On the other hand, OK111 (vacA genotype, s1b/m1b) was included in an East Asian group, although the CagA type was the Western type, resulting from the subtypes of m1b.
For CagE, F79, OK155, OK187, and OK210 were included in the East Asian group although the CagA type was the Western type. In the other strains, except the above seven strains (F37, F79, OK111, OK129, OK155, OK187, and OK210) (78.8%, 26/33), the geographic types corresponded among the three proteins.
Interestingly, the prevalence of infection with the Western cluster strain in both CagA and VacA was significantly higher in patients with peptic ulcer (90.0%, 9/10) than in patients with chronic gastritis (22.7%, 5/22) (2 = 12.64, P = 0.00057).
DISCUSSION
It is known that in East Asian countries, such as Japan and Korea, the incidence of gastric carcinoma is among the highest in the world. There are also indications of significant geographic differences among H. pylori strains (13, 15). H. pylori strains have been classified into type I and type II, depending on the presence of VacA and CagA (36), and type I strains are thought to be more virulent than type II strains. It has been confirmed that almost all Japanese isolates are type I strains (20). It is also thought that there are correlations between the vacA and cagA allelic geographic variations, and both genes are classified as virulence markers (18, 35). In the present study, we examined the molecular genetic relationships among vacA, cagA, and cagE in Okinawa and Fukui, Japan. We selected 33 H. pylori isolates according to the genotypes of vacA in most Okinawa isolates, since there are various vacA genotypes; although the s1c/m1b type is predominant in Okinawa (71.4%), almost all isolates in Fukui (81.7%) express the s1c/m1b type (40). Since Okinawa is an area which has had active international communication with the West historically and had a relatively large American presence in the last half-century, different types of H. pylori may be present. On the other hand, the low genetic diversity in vacA genes of H. pylori isolates in Fukui may be due to the typical Japanese traits. Japan is a country consisting of a relatively homogeneous population and is geographically isolated. Therefore, the opportunity for the transfer of DNA between strains of different genotypes may be lower than that in Western countries. Van Doorn et al. also reported that the subtype s1c was prevalent in East Asia but appeared to be very rare in other parts of the world (35).
The phylogenetic analysis of VacA demonstrated that all m2 strains are classified into the Western group. Ji et al. also investigated the phylogenetic analysis of a set of eight Chinese and six Western vacA genes and reported that while the m-region of m1 alleles has an evolutionary topology similar to the conserved regions of the genes, the m-region of m2 alleles has an evolutionary history independent of the rest of the gene. They suggested that the m2 m-region spread after the separation of Chinese and Western strains (22). In addition, s1a/m1a, s1b/m1a, and s1b/m1b strains, which appear to be very rare in Japan, are also involved in the Western group. In the present study, we calculated the homology in the s-region and m-region among s1a, s1b, s1c, and s2 or m1a, m1b, and m2 subtypes. In the s-region, there was more than 80% homology among s1 subtypes, and the correlation of s1a and s1c subtypes was higher than that of s1b, whereas there was only about 60% homology between the s1 and s2 subtypes. In the m-region, we had similar results; the correlation of m1a and m1b subtypes was higher, but that of the m1 and m2 subtypes was lower. Atherton et al. also compared the VacA sequences between strain 60190 (m1) and strain Tx30a (m2) and found that there was only 59.0% amino acid identity in the middle of VacA. The differences between s1 and s2 and m1 and m2 may be linked to strong and weak toxicities (5). Whereas different rates of evolution in different regions of the same gene might be expected, dramatically different patterns of evolution between different regions of the same set of genes are best explained by recombination. Hence, it is likely that the mosaicism in the vacA gene is maintained in the population by genetic recombination, and since there is a single copy of the vacA gene in H. pylori, this implies horizontal transfer of DNA (22).
It has been considered that cagA-positive H. pylori strains are associated with increased risk for the development of atrophic gastritis and gastric carcinoma (11, 18, 26), and large sequence differences distinguish the cagA gene fragments from East Asian and Western strains (1, 37). Recently, it was demonstrated by using in vitro transfection experiments of a human gastric cancer cell line (AGS) that the East Asian-specific sequence of CagA protein confers stronger SHP-2 binding and transforming activities than does the Western-specific sequence (17), meaning that the potential of East Asian CagA to disturb host cell functions as a virulence factor may be higher than that of Western CagA. In clinical cases, we have reported that the CagA-SHP-2 complex is found in in vivo human gastric mucosa (38), and the grades of inflammation, activity of gastritis, and atrophy are significantly higher in patients with gastritis infected with the East Asian CagA-positive H. pylori strains than in patients with gastritis infected with the cagA-negative or Western CagA-positive strains (6). In this study, we observed two major subtypes, Western and East Asian CagA, according to the EPIYA region sequence constituting the SHP-2 binding site as previously reported (6, 7, 40). The EPIYA type corresponds with the cluster of the phylogenetic tree of CagA in most strains. We also examined the homology in the CagA repeat domain between Western and East Asian strains, and there is less homology (only 53%) when comparing the total sequence, indicating that large differences in the sequences may affect the strength or weakness of the toxicity. Furthermore, there were two strains possessing a mixed-type CagA in the study. Although it is possible that interstrain gene transfer and recombination occurred in the strains, they may be derived from a Western area, because both VacA and CagE were included in the Western type (as described below).
cagE, which is a homolog of the ptlC and virB4 genes of Bordetella pertussis and Agrobacterium tumefaciens, respectively (12), is located in upper proximity to cagA in the cagPAI, and the product is a component of the type IV secretion system (32). It has been reported that CagE is involved in the induction of interleukin-8 expression in gastric epithelial cells (32). In this study, we showed that the cagE gene is well conserved among strains. Although we could not find the Western or East Asian group-specific profile in the full-length amino acid sequences, the protein is clearly divided into two groups in the phylogenetic tree.
The phylogenetic analysis of the three proteins demonstrates the genetic relationship. The distributions of strains exhibited almost the same patterns among the three proteins, indicating that there is a genetic linkage between vacA and the cagPAI, although there is substantial distance between vacA loci and cag genes on the bacterial genome (3, 31). Thus, the selection of a vacA/cagPAI genotype may have a functional basis, perhaps in counterbalancing proinflammatory and anti-inflammatory characteristics of strains to facilitate long-term equilibrium in the human gastric mucosa. Interestingly, peptic ulcer strains were associated with the Western cluster in both CagA and VacA in this study. The strain diversity observed in Okinawa may affect the difference in the prevalence of diseases related to H. pylori infection between Fukui and Okinawa. Further analysis of strain diversity and clinical relevance is needed to clarify the pathogenicity of H. pylori.
ACKNOWLEDGMENTS
This work was supported by a grant-in-aid for Scientific Research (B) from the Japan Society for the Promotion of Science.
We thank Yusuke Kashiwazaki for technical assistance.
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