AIM: The cag pathogenicity island (PAI) is one of potential virulence determinants of Helicobacter pylori. The Mongolian gerbil is a suitable experimental animal for the screening of virulence factors of H pylori.

METHODS: Five-week-old Mongolian gerbils were inoculated with a standard H pylori strain (ATCC 43504) possessing the cag PAI or a clinical isolate lacking the genes’ cluster (OHPC-0002). The animals were killed at 2, 4, 8, 24 and 48 wk after inoculation (n = 5 each), and macroscopic and histopathological findings in the stomachs were compared.

RESULTS: In gerbils infected with ATCC 43504, a more severe degree of infiltration of polynuclear and mononuclear cells and lymphoid follicles was observed from 4 wk after inoculation compared to gerbils infected with OHPC-0002 especially in the antrum and transitional zone from the fundic to pyloric gland area. In addition, glandular atrophy, intestinal metaplasia, gastric ulcer and hyperplastic polyps were noted in gerbils infected with ATCC 43504, whereas only mild gastric erosions occurred in those infected with OHPC-0002.

CONCLUSION: Our results indicate that the cag PAI could be directly involved in gastric immune and inflammatory responses in the Mongolian gerbils, leading to a more advanced gastric disease.

Helicobacter pylori (H pylori) is a Gram-negative, spiral-shaped, microaerophilic bacterium that colonizes the human gastric mucosa[1-3]. The prevalence of H pylori infection is still high in Asian countries including Japan, and this organism is now recognized as a major cause of chronic active gastritis[1-4], peptic ulcers[2-6] and gastric malignancies[4,5,7,8]. However, the clinical manifestation of H pylori infection varies widely in infected individuals and most persons are asymptomatic all their life[9]. The pathogenic mechanism of this pleomorphism is not completely understood, but might be explained, in part, by strain diversity[10-13].

Several factors have been proposed as possible virulence determinants[14-18]. Among them, the cag pathogenicity island (cag PAI), a 40-kb cluster of -30 genes of supposedly extraneous origin[19], is reportedly associated with advanced gastroduodenal diseases including gastric cancer[11-14].

In an effort to develop a model that would parallel as close as possible the course of infection as it occurs in humans, Hirayama et al[20] established an experimental model of inbred Mongolian gerbils with persistent H pylori infection. Infection of Mongolian gerbils with H pylori results in chronic active gastritis, gastric ulcer, intestinal metaplasia and gastric cancer[21-24]. The Mongolian gerbil model may be valuable not only in elucidating H pylori-related neoplasia but also in evaluating virulence factors[11,12]. We sought to investigate the chronological and spatial changes of gastric inflammation after inoculation of H pylori strains possessing or lacking the cag PAI.

Various animals have been used for studying experimental H pylori infection including monkeys, dogs, piglets, domestic cats, rats and nude mice[28-31]. These animal models had disadvantages such as low infection rates, instability, immunodeficiency and high costs[11,32]. Infection of conventional mice with H pylori strain SS1 overcame many of the above problems, but gastritis in mice induced by this infection is less intense than in humans[33]. In 1996, Hirayama et al[20] reported the development of a successful Mongolian gerbil model with persistent H pylori infection. Several investigators confirmed successively the progression from chronic active gastritis to atrophic gastritis and then to intestinal metaplasia and the occurrence of gastric ulcer in the Mongolian gerbil model[22,23]. In addition, long-term H pylori infection by itself resulted even in the development of gastric adenocarcinoma[24]. Thus, the gerbil seems to be the best experimental model for H pylori infection, and we employed the Mongolian gerbil to unravel the impact of cag PAI status, an important virulence determinant, on gastric pathogenesis in vivo.

In Mongolian gerbils infected with ATCC 43504 with total cag PAI, mild infiltration of neutrophils and mononuclear cells appeared in the antrum at 2 wk after inoculation. From 4 wk, moderate to severe chronic active gastritis, often accompanied by lymphoid follicle formation, was noted predominantly in the antrum and transitional zone. In the fundic mucosa, the severity of gastritis worsened to moderate from 8 wk. Such chronological changes of histological gastritis and spatial shift from antral to corporeal mucosa observed in gerbils infected with ATCC 43504 were consistent with previous results in Mongolian gerbils inoculated with other standard or clinical strains possessing intact cag PAI[11,12,22,23].

The major finding of the present study is that the strain possessing the cag PAI induced more severe degree of gastritis in Mongolian gerbils than clinical strain lacking the genes’ cluster[25]. Because there were no differences in the susceptibility and bacterial density between the two strains, ATCC 43504 and OHPC-0002, the deletion did not change the ability of bacterial colonization or proliferation but affected the inflammatory reactions in the stomach of Mongolian gerbils. Akanuma et al[12] cited similar results; a genetically handled mutant lacking total cag PAI or knockout strain of cagE, one of the functional genes in cag PAI, ameliorated histopathological gastritis in this animal. In addition, glandular atrophy, incomplete intestinal metaplasia, gastric ulcer and hyperplastic polyps were noted in gerbils infected with the cag PAI-positive strains, whereas only mild gastric erosions concomitant with slight infiltration of inflammatory cells occurred in animals infected with the cag PAI-negative strains. These findings indicate that intact cag PAI could be directly involved in the host immune and inflammatory processes and cause more advanced gastric diseases.

Deletions of the cag PAI and several cag insertion mutations reportedly block the induction of proinflammatory cytokine, interleukin-8, in gastric epithelial cell lines[19,34]. The expression of interleukin-8 gene is up-regulated by nuclear factor kappa B (NF-κB), which is involved in various inflammatory conditions such as inflammatory bowel diseases, rheumatoid arthritis and H pylori-associated gastritis through the regulation of a plethora of genes that encode bioactive molecules that mediate various immune and inflammatory responses[35-37]. In fact, accumulating evidence indicates that intact cag PAI is a prerequisite for the activation of NF-κB[34]. Considered together, these results suggest that the cag PAI-mediated activation of NF-κB plays an important role in the pathogenesis of gastritis in Mongolian gerbils.

The VacA protein, which causes vacuolation in cultured cells[17,18], is responsible for the virulence of H pylori and ulcer formation in the stomach and duodenum[10,38]. However, since the VacA-positive strains often have intact cag PAI, the involvement of VacA in inducing gastritis is still controversial[11,39]. In this regard, the VacA-disrupted H pylori strain could cause inflammatory changes in the stomach of Mongolian gerbils to a degree similar to those seen in Mongolian gerbils infected with the wild strain[11], indicating that VacA is not crucial for gastric inflammation. Several other candidate genes outside the cag PAI[15,16] should be validated in this reliable rodent model for the screening of determinants of virulence of H pylori.