Shiho Yamazaki

Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites.

Helicobacter pylori is a causative agent of gastritis and peptic ulcer. cagA+H. pylori strains are more virulent than cagA− strains and are associated with gastric carcinoma. The cagA gene product, CagA, is injected by the bacterium into gastric epithelial cells and subsequently undergoes tyrosine phosphorylation. The phosphorylated CagA specifically binds SHP-2 phosphatase, activates the phosphatase activity, and thereby induces morphological transformation of cells. CagA proteins of most Western H. pylori isolates have a 34-amino acid sequence that variably repeats among different strains. Here, we show that the repeat sequence contains a tyrosine phosphorylation site. CagA proteins having more repeats were found to undergo greater tyrosine phosphorylation, to exhibit increased SHP-2 binding, and to induce greater morphological changes. In contrast, predominant CagA proteins specified by H. pylori strains isolated in East Asia, where gastric carcinoma is prevalent, had a distinct tyrosine phosphorylation sequence at the region corresponding to the repeat sequence of Western CagA. This East Asian-specific sequence conferred stronger SHP-2 binding and morphologically transforming activities to Western CagA. Finally, a critical amino acid residue that determines SHP-2 binding activity among different CagA proteins was identified. Our results indicate that the potential of individual CagA to perturb host-cell functions is determined by the degree of SHP-2 binding activity, which depends in turn on the number and sequences of tyrosine phosphorylation sites. The presence of distinctly structured CagA proteins in Western and East Asian H. pylori isolates may underlie the strikingly different incidences of gastric carcinoma in these two geographic areas.

Association between diversity in the Src homology 2 domain--containing tyrosine phosphatase binding site of Helicobacter pylori CagA protein and gastric atrophy and cancer.

We investigated the relationship between the diversity of Helicobacter pylori CagA protein and clinical outcome. The cagA gene was sequenced in 115 clinical isolates. The binding affinity of CagA to Src homology 2 domain-containing tyrosine phosphatase (SHP-2) was examined by in vitro infection. Two major CagA subtypes were observed--the East Asian and the Western type. The grades of inflammation, activity of gastritis, and atrophy were significantly higher in patients with gastritis infected with the East Asian CagA-positive strain than in patients with gastritis infected with cagA-negative or Western CagA-positive strains. All strains isolated from patients with gastric cancer were East Asian CagA positive. East Asian CagA exhibited stronger SHP-2-binding activity than did Western CagA. These findings suggest that infection with East Asian CagA-positive H. pylori is associated with atrophic gastritis and gastric cancer and that persistent active inflammation induced by the East Asian CagA-positive strain may play a role in the pathogenesis of disease.

The CagA Protein of Helicobacter pylori Is Translocated into Epithelial Cells and Binds to SHP-2 in Human Gastric Mucosa

Recent experiments have indicated that CagA of Helicobacter pylori is injected into epithelial cells via the type IV secretion system and undergoes tyrosine phosphorylation in cells and that translocated CagA binds the SRC homology 2 domain-containing tyrosine phosphatase (SHP-2). We investigated these phenomena in in vivo human gastric mucosa. Tyrosine-phosphorylated CagA and CagA-coimmunoprecipitated SHP-2 were detected in gastric mucosa from H. pylori-positive patients with atrophic gastritis and in noncancerous tissues from H. pylori-positive patients with early gastric cancer. In contrast, CagA was not detected in gastric mucosa with either intestinal metaplasia or cancer. Our results provide the first evidence that CagA is translocated into the gastric epithelial cells, receives tyrosine phosphorylation, and binds SHP-2 in in vivo human gastric mucosa. Deregulation of SHP-2 by CagA may play a role in the acquisition of a cellular-transformed phenotype at a relatively early stage of multistep gastric carcinogenesis.

Distinct Diversity of vacA, cagA, and cagE Genes of Helicobacter pylori Associated with Peptic Ulcer in 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) (χ2 = 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.

Distinct Diversity of the cag Pathogenicity Island among Helicobacter pylori Strains in Japan

ABSTRACT The severity of Helicobacter pylori-related disease is correlated with the presence of a cag pathogenicity island (PAI). Genetic diversity within the cag PAI may have a modifying effect on the pathogenic potential of the infecting strain. We analyzed the complete cag PAI sequences of 11 representative Japanese strains according to their vacA genotypes and clinical effects and examined the relationship between the diversity of the cag PAI and clinical features. The cag PAI genes were divided into two major groups, a Western and a Japanese group, by phylogenetic analysis based on the entire cag PAI sequences. The predominant Japanese strains formed a Japanese cluster which was different from the cluster formed by Western strains. The diversity of the cag PAI was associated with the vacA and cagA genotypes. All strains with the s1c vacA genotype were in the Japanese cluster. In addition, all strains with the East Asian-type cagA genotype were also in the Japanese cluster. Patients infected with the Japanese-cluster strain had high-grade gastric mucosal atrophy. These results suggest that a distinct diversity of the cag PAI of H. pylori is present among Japanese strains and that this diversity may be involved in the development of atrophic gastritis and may increase the risk for gastric cancer.

The diversity of vacA and cagA genes of Helicobacter pylori in East Asia

It has been reported that Helicobacter pylori infection with the type I strain, which expresses the VacA and CagA antigens, is associated with duodenal ulcer. We examined the diversity of vacA and cagA genes in 143 isolates obtained from patients with duodenal ulcer or chronic gastritis in East Asia (two different areas of Japan, Fukui and Okinawa, and also in Hangzhou, China) by polymerase chain reaction (PCR) and sequence analysis. Diversities of cagA and vacA genes were detected in East Asia. The prevalence of cagA-positive H. pylori was significantly different between Fukui and Okinawa (P=0.0032). The prevalence of Western type CagA was significantly higher in Okinawa than in Fukui (P<0.0001). However, there was no significant association between the genotype of cagA and clinical outcome. In Japan, the predominant vacA genotype was s1c/m1b. In contrast, in Hangzhou, the predominant vacA genotype was s1c/m2, and they were all East Asian CagA-positive. These findings suggest that a distinct distribution of the vacA and cagA genotypes is present in East Asia, regardless of clinical outcome.

Cytotoxicity and recognition of receptor-like protein tyrosine phosphatases, RPTPα and RPTPβ, by Helicobacter pylori m2VacA

Helicobacter pylori vacuolating cytotoxin, VacA, induces vacuolation in mammalian cell lines. Sequence differences in the middle of VacA molecules define two families, termed m1VacA and m2VacA, which differ in cell specificity. Similar to m1VacA, m2VacA is activated by acid or alkali, which enhances its binding to cells. Immunoprecipitation experiments showed that, in AZ‐521 cells, activated m2VacA, similar to m1VacA, binds to two receptor‐like protein tyrosine phosphatases, RPTPα and RPTPβ suggesting that activated m2VacA as well as m1VacA may contribute to gastrointestinal disease following H. pylori infection. G401 cells express RPTPα, not RPTPβ, and responded to both m1VacA and m2VacA. HeLa cells likewise expressed RPTPα, not RPTPβ, but, in contrast to other cell lines, responded poorly to m2VacA. m1VacA associated with RPTPα of HeLa cells to an extent similar to that in other toxin‐sensitive cells, whereas activated m2VacA bound HeLa cell RPTPα less well, consistent with its low vacuolating activity against these cells. The molecular mass of RPTPα from HeLa cells is less than that of the protein from G401 cells, although their extracellular amino acid sequences are virtually identical, with only two amino acid differences noted. Different post‐translational modifications of RPTPα in HeLa cells may be responsible for the reduced susceptibility to m2VacA.

Diversity of vacA and cagA genes of Helicobacter pylori in Japanese children

Background : Helicobacter pylori infection is generally acquired in childhood and persists as an asymptomatic infection for decades in most infected individuals. Only a minority develops a clinical outcome even in childhood, such as peptic ulcer. It has been reported that H. pylori infection with the type I strain, which expresses the VacA and CagA antigen, is associated with peptic ulcer.

Association of the HLA-DRB1 gene locus with gastric adenocarcinoma in Japan

BACKGROUND AND AIM Helicobacter pylori infection is associated with gastric adenocarcinoma, however, the odds ratio is relatively low. The aim of the present study was to investigate host genetic factors that increase the risk of gastric adenocarcinoma among H. pylori-infected individuals. METHODS A total of 70 patients with early gastric adenocarcinoma and 121 unrelated healthy controls were examined for H. pylori infection and HLA-DRB1 genotyping. The frequencies of HLA-DRB1 alleles were compared among groups. RESULTS The allele frequency of DRB1*04051 was significantly higher in patients with gastric adenocarcinoma (17.9%) than in controls (7.9%) (P(correct) = 0.045). The odds ratio of gastric adenocarcinoma associated with the presence of the HLA-DRB1*04051 allele compared with its absence was 2.55 (95% confidence limits, 1.35-4.83). This genetic risk was not associated with H. pylori infection. There was no significant difference in the HLA-DRB1 allele frequency between H. pylori-positive and H. pylori-negative controls. The frequency of genotypes that possessed the DRB1*04051 allele in gastric adenocarcinoma patients (34.3%) was significantly higher than that in H. pylori-negative controls (11.9%) (p = 0.0089) and H. pylori-positive controls (15.2%) (p = 0.0066). CONCLUSION These findings suggest that immunogenetic factors for susceptibility to gastric adenocarcinoma are present in the host, the HLA-DRB1*04051 allele is a host genetic risk factor for gastric adenocarcinoma, and that this genetic risk is independent of H. pylori infection.

Helicobacter pylori‐dependent NF‐kappa B activation in newly established Mongolian gerbil gastric cancer cell lines

Mongolian gerbils are an ideal animal model to explore the role of H. pylori on cancer development. However, there have been no established adenocarcinoma cell lines from this model animal. In the present study, we have established cancer cell lines from a primary gastric cancer tissue of a Mongolian gerbil. The derived cells could be stably attached with H. pylori, revealed under a scanning electron microscope, and easily transplanted to the nude mice. Rapid phosphorylation of IκB, Erk1/2, and AKT of these cells was observed by Interleukin‐1 beta stimulation, and luciferase reporter gene assay on transcriptional activation of Nuclear Factor kappa B after challenging with either H. pylori NCTC11637 or its isogenic cagE‐knockout mutant, H. pylori revealed the cagE‐dependent NF‐κB transcriptional activation. The newly established cancer cell lines from the in vivo gastric carcinogenesis model animal, the Mongolian gerbil, can be used to develop effective therapeutic strategies against gastric cancer, especially in exploring the effect of H. pylori, and thus might greatly contribute to gastric cancer prevention and treatment in humans. (Cancer Sci 2005; 96: 170–175)