Gastritis and peptic ulcer disease are widespread medical problems in the U.S. today costing a billion dollars lost in wages, absenteeism, and health care costs each year. It is now clearly established that Helicobacter pylori, a gram- negative microaerophilic spiral bacterium is an etiologic agent of these diseases. Gastric carcinoma is a deadly sequela to type 13 gastritis and gastric ulcer and may be triggered by chronic H. pylori infection. This bacterium has a colonization range restricted to the human gastric mucosa and produces a number of protein products including urease, cytotoxin, flagella, CagA protein, and adherence factors that contribute to its ability to colonize, avoid host defenses, and inflict damage to the host. Urease is clearly central to the virulence of H. pylori. This high molecular weight nickel-metalloenzyme hydrolyzes urea to ammonia and carbon dioxide, allows survival of the bacterium in the gastric mucosa, contributes to cellular damage, is a prominent antigenic component of the organism, and is useful as a diagnostic indicator of infection. The H. pylori urease gene cluster spans 8.3 kb of chromosomal DNA and is comprised of nine genes. Two of the genes, ureA and ureB, encode the structural subunits of the enzyme itself. A subset of the remaining seven genes are necessary for incorporation of nickel ions into the active site of the enzyme. In addition, the newly described NixA nickel transport protein is also required for full enzymatic activity. A NixA-deficient isogenic mutant was found to retain about half of the urease activity of the parent strain. It is therefore likely that additional, newly described gene products are also necessary for: binding of nickel ions at the surface, entry through the outer membrane, transport across the cytoplasmic membrane, and incorporation into the active site of the apoenzyme. Although much has been learned about the structural features of the urease enzyme, serious gaps in our knowledge exist regarding the activation of urease. To address these issues, the PI proposes: 1) to characterize the NixA nickel transport protein and its corresponding gene sequences; 2) to isolate and characterize new genes associated with production of catalytically active urease; 3) to construct isogenic mutants of H.pylori deficient in both nixA and other urease-enhancing factors; and 4) to determine the involvement of newly described genes in bismuth sensitivity. The long range goals of the project are to understand the mechanism by which an active urease is assembled and therefore to develop targets for possible therapeutic agents.
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