Peptic ulcer disease is a common illness in the United States affecting up to 0.9% of the population over a lifetime. Clinical complaints of abdominal pain, nausea, and vomiting require on the average that 50 individuals per 100,000 population be hospitalized each year; a third of these patients suffer serious complications including hemorrhage, gastric outlet obstruction, perforation, or death. Campylobacter pylori, a spiral-shaped gram-negative microaerophilic rod, is not postulated to be an etiological agent of gastritis and peptic ulcer disease. The organism is sensitive to acidic environments characteristic of the human stomach but can protect itself by generating ammonia and carbon dioxide from urease-catalyzed urea hydrolysis. This remarkably potent urease, which is significantly more active than the ureases of other bacterial pathogens, can completely neutralize stomach HCl. Free ammonia liberated during ureolysis can also directly damage gastric mucosal integrity. Urease is therefore postulated to be an important virulence factor for this organism. In addition, the enzyme serves as a sensitive and specific indicator for the presence of the organism in biopsied tissue and elicits an immune response in patients with gastritis. Other virulence factors have also been postulated for C. pylori such as a cytotoxin, hemolysis, mucinase, and a specific adhesin but, because no system for gene expression or transposon mutagenesis has been developed and because no method for insertion of specific chromosomal mutations has been reported, the contribution of these factors to pathogenesis cannot be assessed. The gnotobiotic piglet is being developed by other investigators as a model for C. pylori infection of the gastric mucosa and will soon be available to test isogenic constructions of C. pylori mutated in specific virulence-associated genes. Until that time it sis essential to characterize urease, a virulence factor in C. pylori, and use acquired information to develop methods for gene manipulation of this newly described human pathogen.
The specific aims of this project are to: 1) purify and characterize the urease of C. pylori; 2) isolate and express urease gene sequences of C. pylori; 3) develop a system for genetic manipulation of C. pylori; and 4) construct a urease-deficient mutant of C. pylori by homologous recombination.
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