Dynamic Expansion and Contraction of cagA Copy Number in Helicobacter pylori Impact Development of Gastric Disease.

Infection with Helicobacter pylori is a major risk factor for development of gastric disease, including gastric cancer. Patients infected with H. pylori strains that express CagA are at even greater risk of gastric carcinoma. Given the importance of CagA, this report describes a new molecular mechanism by which the cagA copy number dynamically expands and contracts in H. pylori Analysis of strain PMSS1 revealed a heterogeneous population in terms of numbers of cagA copies; strains carried from zero to four copies of cagA that were arranged as direct repeats within the chromosome. Each of the multiple copies of cagA was expressed and encoded functional CagA; strains with more cagA repeats exhibited higher levels of CagA expression and increased levels of delivery and phosphorylation of CagA within host cells. This concomitantly resulted in more virulent phenotypes as measured by cell elongation and interleukin-8 (IL-8) induction. Sequence analysis of the repeat region revealed three cagA homologous areas (CHAs) within the cagA repeats. Of these, CHA-ud flanked each of the cagA copies and is likely important for the dynamic variation of cagA copy numbers. Analysis of a large panel of clinical isolates showed that 7.5% of H. pylori strains isolated in the United States harbored multiple cagA repeats, while none of the tested Korean isolates carried more than one copy of cagA Finally, H. pylori strains carrying multiple cagA copies were differentially associated with gastric disease. Thus, the dynamic expansion and contraction of cagA copy numbers may serve as a novel mechanism by which H. pylori modulates gastric disease development.IMPORTANCE Severity of H. pylori-associated disease is directly associated with carriage of the CagA toxin. Though the sequences of the CagA protein can differ across strains, previous analyses showed that virtually all H. pylori strains carry one or no copies of cagA This study showed that H. pylori can carry multiple tandem copies of cagA that can change dynamically. Isolates harboring more cagA copies produced more CagA, thus enhancing toxicity to host cells. Analysis of 314 H. pylori clinical strains isolated from patients in South Korea and the United States showed that 7.5% of clinical strains in the United States carried multiple cagA copies whereas none of the South Korean strains did. This study demonstrated a novel molecular mechanism by which H. pylori dynamically modulates cagA copy number, which affects CagA expression and activity and may impact downstream development of gastric disease.

Helicobacter pylori-Induced HB-EGF Upregulates Gastrin Expression via the EGF Receptor, C-Raf, Mek1, and Erk2 in the MAPK Pathway.

Helicobacter pylori is associated with hypergastrinemia, which has been linked to the development of gastric diseases. Although the molecular mechanism is not fully understood, H. pylori is known to modulate the Erk pathway for induction of gastrin expression. Herein we found that an epidermal growth factor (EGF) receptor kinase inhibitor significantly blocked H. pylori-induced gastrin promoter activity, suggesting involvement of EGF receptor ligands. Indeed, H. pylori induced mRNA expression of EGF family members such as amphiregulin, EGF, heparin-binding EGF-like growth factor (HB-EGF), and transforming growth factor-α. Of these, specific siRNA targeting of HB-EGF significantly blocked H. pylori-induced gastrin expression. Moreover, H. pylori induced HB-EGF ectodomain shedding, which we found to be a critical process for H. pylori-induced gastrin expression. Thus, we demonstrate a novel role for human mature HB-EGF in stimulating gastrin promoter activity during H. pylori infection. Further investigation using specific siRNAs targeting each isoform of Raf, Mek, and Erk elucidated that the mechanism underlying H. pylori-induced gastrin expression can be delineated as the sequential activation of HB-EGF, the EGF receptor, C-Raf, Mek1, and the Erk2 molecules in the MAPK pathway. Surprisingly, whereas Erk2 acts as a potent activator of gastrin expression, siRNA knockdown of Erk1 induced gastrin promoter activity, suggesting that Erk1 typically acts as a repressor of gastrin expression. Elucidation of the mechanism of gastrin modulation by HB-EGF-mediated EGF receptor transactivation should facilitate the development of therapeutic strategies against H. pylori-related hypergastrinemia and consequently gastric disease development, including gastric cancers.

Helicobacter pylori outer membrane protein, HomC, shows geographic dependent polymorphism that is influenced by the Bab family.

The array of outer membrane proteins (OMPs) found in Helicobacter pylori provides a crucial component for persistent colonization within the gastric niche. Not only does H. pylori harbor a wide number of OMPs, but these OMPs often vary across strains; this likely contributes to immune evasion, adaptation during long term colonization, and potentially differential disease progression. Previous work from our group described OMP differences among the Bab family (babA, babB, and babC) and Hom family (homA and homB) from 80 American H. pylori clinical isolates (AH) and 80 South Korean H. pylori clinical isolates (KH). In the current study, we expanded our investigation to include the less well characterized Hom family member, HomC.Overall, we identified and genotyped three homC variants: homC S , homC L , and homC M , in both populations. Similar to other polymorphic genes, the KH group showed less overall diversity, with 97.5% of strains harboring homC L . In contrast, a more heterogeneous profile was observed in strains derived from an American population; we found nearly equal distribution of homC S and homC L . Further analysis of the AH group identified associations between homC polymorphism and bab genotype; in AH strains, there was a significant association between homC L and carriage of babA at locus A. Since babA is an important virulence factor for the development of severe gastric disease, these data may suggest that homC polymorphism plays a role in H. pylori pathogenesis.

Effect of Areca Nut on Helicobacter pylori-Induced Gastric Diseases in Mice.

Areca nut (AN) chewing is a habit in many countries in Central, Southern, and Southeast Asia. It is strongly associated with the occurrence of oral, pharyngeal, and esophageal cancer as well as systemic inflammation. However, the association between AN intake and the development of gastric lesions has not yet been identified. The aim of this study was to investigate the effect of AN on gastric diseases using a mouse model for Helicobacter pylori infection. We studied four groups of mice: those fed a normal diet (ND), those fed a diet containing 2.5% AN (AD), those fed ND and infected with H. pylori PMSS1 strain (ND/HP), and those fed AD and infected with H. pylori PMSS1 strain (AD/HP). Food intake and body weight were monitored weekly during the experiments. At 10 weeks, the mice were sacrificed, and the stomach weight, H. pylori colonization, and gastric inflammation were evaluated. The stomach weight had increased significantly in the ND/HP and AD/HP groups along with increases in H. pylori colonization; however, there was no significant difference between these two groups with respect to stomach weight and colonization. On histological grading, mononuclear cell infiltration was severer in the AD/HP group than in the ND/HP group. These data suggest that chronic gastric inflammation was aggravated by AN treatment in the mice with H. pylori-induced gastric lesions. Furthermore, as previously suggested, this animal model is useful to determine the effect of potential carcinogens on gastric lesions induced by H. pylori infection.