Peptic ulcer disease is a common malady in the United States affecting up to 10% of men and 4% of women over their lifetimes. With manifestations such as abdominal pain, nausea, and vomiting, 500,000 new cases of duodenal ulcer and 100,000 new cases of gastric ulcer are diagnosed each year. Sequelae include gastric cancer. Helicobacter pylori, a gram-negative, microaerophilic spiral-shaped bacterium is the most frequently cited etiologic agent of human gastritis and peptic ulceration. This species, whose niche is highly restricted to the gastric mucosa of humans, has adopted a strategy of survival that includes synthesis of urease as its most abundant cellular protein. Nickel ions are now recognized as a critical cofactor required for catalytic activity of urease and are necessary for induction of certain H. pylori genes and repression of others. We propose that this ion serves as an important environmental cue in the gastric mucosa that up-regulates H. pylori genes needed for colonization of this hostile niche. Nickel acquisition, mediated in part by the 8-transmembrane domain-NixA nickel transport protein, in H. pylori could be viewed as important as is iron acquisition for other bacterial pathogens. In the current proposal, we hypothesize that a network of ion transport, ion-binding, and ion-chaperoning proteins modulate urease activity and virulence of H. pylori. To test this hypothesis, we propose as Specific Aims to use molecular genetic, genomic, proteomic, and bacterial physiological methodology to: 1) Identify genes up-regulated in response to nickel ion limitation in vitro; 2) Determine the transcriptome of H. pylori in vivo and the effect of pH change and nickel limitation; and 3) Correlate gene expression of ion homeostasis genes with urease synthesis, urease activity and nickel insertion into the urease apoenzyme. The transcriptome of H. pylori 26695 and selected mutants, cultured in vitro and in vitro, will be quantified using H. pylori-specific macroarrays. Genomic results will be validated by kinetic RT-PCR. Newly discovered ion homeostasis genes will be subjected to mutagenesis and virulence studies in the Mongolian gerbil model of infection. Alternative methods will include 2-D Fluorescence Difference Gel Electrophoresis. The long-term goals of the study are to understand the mechanism by which H. pylori can colonize the gastric mucosa and to identity more effective targets for therapeutic intervention. ? ?
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