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

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.

Pathogen DNA as target for host-generated oxidative stress: Role for repair of bacterial DNA damage in Helicobacter pylori colonization

“…Although postulated, oxidative host-generated lethal or premutagenic DNA damage on H. pylori DNA has not been demonstrated (6)(7)(8). Superoxide dismutase-deficient mutants have an impaired ability to colonize the mouse stomach, confirming that this pathogen is exposed to the toxic effect of oxygen-derived products during infection (6). The fact that superoxide dismutase is associated with the bacterial surface is consistent with its action as a primary barrier for exogenous reactive molecules.…”

“…H. pylori induces an oxidative stress that enhances DNA damage in the host's gastric mucosa (2)(3)(4)(5). Although postulated, oxidative host-generated lethal or premutagenic DNA damage on H. pylori DNA has not been demonstrated (6)(7)(8). Superoxide dismutase-deficient mutants have an impaired ability to colonize the mouse stomach, confirming that this pathogen is exposed to the toxic effect of oxygen-derived products during infection (6).…”

Oxidative stress response in Clostridium perfringens

“…However, superoxide-deficient mutants of aerobic organisms [e.g. E. coli (Carlioz & Touati, 1986), Candida albicans (Hwang et al, 2002), Streptococcus agalactiae (Poyart et al, 2001)], or of microaerophilic bacteria exemplified by Helicobacter pylori (Seyler et al, 2001), have been readily obtained. These mutants are generally more sensitive to oxygen or to oxidative stress than the wild-type and display an increased susceptibility to bacterial killing by macrophages.…”

Role of a Bacterial Organic Hydroperoxide Detoxification System in Preventing Catalase Inactivation

“…The construction of other mutant strains (sodB, mdaB, ahpC1, ahpC2, ahpC,napA, and napA) in our laboratory was described previously (13,15,18). An H. pylori ATCC43504 katA,ahpC double mutant was constructed in this study by transforming ahpC:Kan (type I) mutant strain with the plasmid pGEM-katA:Cm.…”

“…NapA, a ferritin-like iron-binding protein, is involved in oxidative stress resistance probably through sequestering free iron in the cells (13,14); also, a NADPH quinone reductase (MdaB) confers oxidative stress resistance by maintaining the quinone pool of the cell in the reduced state (15). The disruption of each individual gene for superoxide dismutase, KatA, AhpC, or MdaB affects severely the ability of the bacterium to colonize the host stomach (15)(16)(17)(18), demonstrating the importance of these enzymes in oxidative stress resistance and host colonization.…”