ABSTRACTcagA, a gene that codes for an immunodominant antigen, is present only in Helicobacter pylori strains that are associated with severe forms of gastroduodenal disease (type I strains). We found that the genetic locus that contains cagA (cag) is part of a 40-kb DNA insertion that likely was acquired horizontally and integrated into the chromosomal glutamate racemase gene. This pathogenicity island is f lanked by direct repeats of 31 bp. In some strains, cag is split into a right segment (cagI) and a left segment (cagII) by a novel insertion sequence (IS605). In a minority of H. pylori strains, cagI and cagII are separated by an intervening chromosomal sequence. Nucleotide sequencing of the 23,508 base pairs that form the cagI region and the extreme 3 end of the cagII region reveals the presence of 19 ORFs that code for proteins predicted to be mostly membrane associated with one gene (cagE), which is similar to the toxin-secretion gene of Bordetella pertussis, ptlC, and the transport systems required for plasmid transfer, including the virB4 gene of Agrobacterium tumefaciens. Transposon inactivation of several of the cagI genes abolishes induction of IL-8 expression in gastric epithelial cell lines. Thus, we believe the cag region may encode a novel H. pylori secretion system for the export of virulence determinants.

The gram negative, microaerophilic bacterium Helicobacter I~Iori colonizes the human gastric mucosa and establishes a chronic infection that is tightly associated with atrophic gastritis, peptic ulcer, and gastric carcinoma. Cloning of the H. pylori cytotoxin gene shows that the protein is synthesized as a 140-kD precursor that is processed to a 94-kD fully active toxin. Oral administration to mice of the purified 94-kD protein caused ulceration and gastric lesions that bear some similarities to the pathology observed in humans. The cloning of the cytotoxin gene and the development of a mouse model of human gastric disease will provide the basis for the understanding of H. ~lori pathogenesis and the development of therapeutics and vaccines. The recently discovered, gram negative, microaerophilic bacterium Helicobacter pylori colonizes the human gastric mucosa and establishes a chronic infection that is tightly associated with atrophic gastritis, peptic ulcer, and gastric carcinoma (1-5). H. pylori infection is a worldwide problem, since in developing countries it affects over 80% of the population older than 20. Also in developed countries the infection is present in 20% of the population by the age of 30 rising to over 50% by the age of 60. Clinical isolates of H. pylori can be classified into two groups based on the presence or absence of the vacuolating cytotoxin (6, 7) whose expression is linked to a surface exposed immunodominant antigen (CagA) (8, 9). Since high titers of serum antibodies to the CagA protein are detected in all patients with duodenal ulcer (8) and most of those with gastric carcinoma (10, 11), it has been proposed that disease development requires infection with cytotoxin-producing strains.The cytotoxin causes massive vacuolation in several mammalian cell lines (6), and similar vacuoles have also been observed in the gastric epithelia of patients with active chronic gastritis associated with H. pylori infection (12), indicating that the cytotoxin can contribute significantly to the pathogenesis of gastritis. Cell vacuolation in vitro can be blocked and reversed by inhibitors of V-type ATPases and potentiated by inhibitors of the Na+-K + ATPase (13,14), suggesting that the mechanism of action of the toxin is due to aberrant cation transport within the target cells. The purified toxin has been described as a protein of ~87 kD that is found in the bacterial culture supernatants, and the sequence of the NH2-terminal 23 amino acids has been determined (7).Despite the epidemiological correlation between infection with cytotoxic strains and disease (8) and the in vitro evidence for the presence of a cytotoxin, the in vivo roles of infection and cytotoxin have not been established due to the lack of a suitable animal model. H. ~lori does not colonize the gastric mucosa of mice or other small laboratory animals.To overcome this limitation, we administered H. pylori extracts and purified cytotoxin orally to mice. Using this model, extracts from cytotoxic H. Ioylori strains and purified cytotoxin ind...

The human pathogen Helicobacter pylori is associated with gastritis, peptic ulcer disease, and gastric cancer. The pathogenesis of H. pylori infection in vivo was studied by adapting fresh clinical isolates of bacteria to colonize the stomachs of mice. A gastric pathology resembling human disease was observed in infections with cytotoxin-producing strains but not with noncytotoxic strains. Oral immunization with purified H. pylori antigens protected mice from bacterial infection. This mouse model will allow the development of therapeutic agents and vaccines against H. pylori infection in humans.

To elucidate the pathogenesis of Helicobacter pylori-associated gastritis, we studied immune responses of C57BL/6J wild-type (WT), SCID, and gene deficient (IFN-γ−/− and IL-4−/−) mice following infection with a pathogenic isolate of H. pylori (SPM326). During early infection in WT mice, mononuclear and polymorphonuclear cells accumulated in the gastric lamina propria, and the numbers of cells in the inflamed mucosa expressing IFN-γ, but not IL-4, mRNA rose significantly (p < 0.005), consistent with a local Th1 response. Splenic T cells from the same infected WT mice produced high levels of IFN-γ, no detectable IL-4, and low amounts of IL-10 following in vitro H. pylori urease stimulation, reflecting a systemic Th1 response. Infected C57BL/6J SCID mice did not develop gastric inflammation despite colonization by many bacteria. Infected C57BL/10J and BALB/c mice also did not develop gastric inflammation and displayed a mixed Th1/Th2 splenic cytokine profile. These data imply a major role for the Th1 cytokine IFN-γ in H. pylori-associated gastric inflammation in C57BL/6J mice. Compared with WT animals, infected IL-4−/− animals had more severe gastritis and higher levels of IFN-γ production by urease-stimulated splenocytes (p < 0.01), whereas IFN-γ−/− mice exhibited no gastric inflammation and higher levels of IL-4 production by stimulated splenocytes. These findings establish C57BL/6J mice as an important model for H. pylori infection and demonstrate that up-regulated production of IFN-γ, in the absence of the opposing effects of IL-4 (and possibly IL-10), plays a pivotal role in promoting H. pylori-induced mucosal inflammation.

Helicobacter pylori is involved in gastritis, gastric and duodenal ulcers, gastric adenocarcinoma, and mucosaassociated lymphoid tissue lymphoma. Earlier studies already suggested a role for autoimmune phenomena in H. pylori-linked disease. We now report that lipopolysaccharides (LPS) of H. pylori express Lewis y, Lewis x, and H type I blood group structures similar to those commonly occurring in gastric mucosa. Immunization of mice and rabbits with H. pylori cells or purified LPS induced an anti-Lewis x or y or anti-H type I response, yielding antibodies that bound human and murine gastric glandular tissue, granulocytes, adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma cells. Experimental oral infections in mice or natural infection in humans yielded anti-Lewis antibodies also. The ␤ chain of gastric H ؉ ,K ؉ -ATPase, the parietal cell proton pump involved in acid secretion, contained Lewis y epitopes; gastric mucin contained Lewis x and y antigenic determinants. Growth in mice of a hybridoma that secretes H. pylori-induced anti-Lewis y monoclonal antibodies resulted in histopathological evidence of gastritis, which indicates a direct pathogenic role for anti-Lewis antibodies. In conclusion, our observations demonstrate that molecular mimicry between H. pylori LPS and the host, based on Lewis antigens, and provide understanding of an autoimmune mechanism for H. pylori-associated type B gastritis.

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Killer strains of Kluyveromyces lactis secrete a toxin which presumably is processed during secretion from a larger precursor. Analysis of the sequence of the K. lactis killer toxin gene predicts that the first 16 amino acids at the amino terminus of the protein should represent its leader peptide. We have tested the capability of this leader peptide to direct secretion of a protein fused to it by inserting a synthetic oligonucleotide identical to the sequence of the putative leader peptide into a yeast expression vector. Subsequently, the cDNA coding for the secreted active portion of the human interleukin 1 beta (IL‐1 beta) was fused to the leader peptide sequence of the killer toxin. This construction in Saccharomyces cerevisiae is capable of directing synthesis and secretion of correctly processed IL‐1 beta into the culture medium.