Helicobacter pylori is a curved Gram-negative bacterium that is now recognized as the predominant cause of chronic gastritis in humans. Although most infected persons remain asymptomatic, infection with this pathogen is a significant risk factor for the development of peptic ulcer disease, and possibly gastric carcinoma. The long-term objective of this project is to determine pathogenic mechanisms whereby H. pylori causes human disease. One potential virulence determinant of H pylori is a cytotoxin that induces vacuolation of eukaryotic cells. Based upon detection in a cell culture assay, 50-60% of H pylori isolates produce the vacuolating cytotoxin in vitro. The prevalence of infection with cytotoxin-producing strains is higher among H pylori-infected patients with duodenal ulceration than among infected patients with gastritis alone. Neutralizing antibodies to the cytotoxin are present in sera from H pylori- infected humans, which confirms that the cytotoxin is produced in vivo. An 89 Kda protein that induces cell vacuolation has now been purified from H pylori broth culture supernatants. Antiserum raised against the 89 Kda protein neutralizes cytotoxic activity. In addition, high-titer cytotoxin- neutralizing activity in sera from H pylori-infected humans is associated with recognition of the 89 Kda protein in immunoblotting studies. The hypothesis of this study is that the vacuolating cytotoxin activity of H pylori is mediated by an 89 Kda protein, and is an important virulence factor.
The specific aims are 1) to determine the relationship between cytotoxin production by H pylori in vivo and in vitro, 2) to identify and analyze the sequence of the gene for the 89 Kda protein of H pylori, and 3) to determine whether the recombinant 89 Kda protein has vacuolating cytotoxic activity, and to construct isogenic H pylori strains that differ only in vacuolating cytotoxin production. To accomplish the first objective, the serologic response to the cytotoxin will be used as an indicator of in vivo cytotoxin production, and will be correlated with production of cytotoxin in vitro. H pylori isolates from patients with peptic ulceration or gastritis alone will be tested for cytotoxin production, and the serologic response to the cytotoxin in these groups of patients will be compared. The gene for the 89 Kda protein will then be cloned and its sequence analyzed. Whether this gene is present in both tox+ and tox H pylori isolates will be determined by probe hybridization. To determine whether the cloned protein mediates cytotoxic activity, recombinant E. coli will be tested for cytotoxin production; if necessary, the gene will be mobilized into c. jejuni via a shuttle vector. The construction of isogenic H pylori strains differing only in cytotoxin production will enable the eventual evaluation of this virulence determinant in an animal model. Understanding the genetic basis whereby H pylori produces the vacuolating cytotoxin may aid in the development of strategies to decrease the morbidity associated with complications of H pylori infection.
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