Superoxide Dismutase-Deficient Mutants of Helicobacter pylori Are Hypersensitive to Oxidative Stress and Defective in Host Colonization

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Abstract
Superoxide dismutase (SOD) is a nearly ubiquitous enzyme among organisms that are exposed to oxic environments. The single SOD of Helicobacter pylori , encoded by the sodB gene, has been suspected to be a virulence factor for this pathogenic microaerophile, but mutations in this gene have not been reported previously. We have isolated mutants with interruptions in the sodB gene and have characterized them with respect to their response to oxidative stress and ability to colonize the mouse stomach. The sodB mutants are devoid of SOD activity, based on activity staining in nondenaturing gels and quantitative assays of cell extracts. Though wild-type H. pylori is microaerophilic, the mutants are even more sensitive to O 2 for both growth and viability. While the wild-type strain is routinely grown at 12% O 2 , growth of the mutant strains is severely inhibited at above 5 to 6% O 2 . The effect of O 2 on viability was determined by subjecting nongrowing cells to atmospheric levels of O 2 and plating for survivors at 2-h time intervals. Wild-type cell viability dropped by about 1 order of magnitude after 6 h, while viability of the sodB mutant decreased by more than 6 orders of magnitude at the same time point. The mutants are also more sensitive to H 2 O 2 , and this sensitivity is exacerbated by increased O 2 concentrations. Since oxidative stress has been correlated with DNA damage, the frequency of spontaneous mutation to rifampin resistance was studied. The frequency of mutagenesis of an sodB mutant strain is about 15-fold greater than that of the wild-type strain. In the mouse colonization model, only 1 out of 23 mice inoculated with an SOD-deficient mutant of a mouse-adapted strain became H. pylori positive, while 15 out of 17 mice inoculated with the wild-type strain were shown to harbor the organism. Therefore, SOD is a virulence factor which affects the ability of this organism to colonize the mouse stomach and is important for the growth and survival of H. pylori under conditions of oxidative stress.