Helicobacter pylori infection causes chronic active gastritis that after years of infection can develop into peptic ulceration (10-20%) or gastric adenocarcinoma (0.5-2%). Consequently research into the bacterium’s mechanisms for persistence are of particular importance to understanding how clinical diseases develop. H. pylori is highly adapted to surviving in the harsh conditions of the gastric environment and one key adaptation is the virulence factor urease. Strains that are deficient in this enzyme are unable to establish infection. Although widely postulated, the requirement of urease expression for chronic infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint and permit changes to the urease phenotype during an established infection, we constructed conditional H. pylori urease knockout mutants by adapting the tetracycline inducible expression system to the H. pylori urease operon. In vitro, the urease activity of the conditional knockouts could be repressed below detection limits and importantly was induced to wild-type activity levels upon addition of anhydrotetracycline (ATc). In vivo, these mutants were unable to establish colonization in mice in the absence of ATc. In addition, during active infection, withdrawal of ATc resulted in clearance of the bacteria within seven days. These results demonstrate definitively that urease expression is required for both colonization and maintenance of chronic infection.
Furthermore, isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on H. pylori to continuously express urease so as to maintain chronic infection. Whole genome sequence analysis of escape mutants revealed that only half the escape isolates had mutated the tet repressor while the remainder harboured mutations in other genes including FliA.
Overall this expression system can be expanded to the study of other genes to gain insight into H. pylori mechanisms promoting life-long infection and also allows inquiry into the relative importance of various virulence determinants when the bacterium is put under stress.