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

Abstract

In the gastric pathogen Helicobacter pylori, catalase (KatA) and alkyl hydroperoxide reductase (AhpC) are two highly abundant enzymes that are crucial for oxidative stress resistance and survival of the bacterium in the host. Here we report a connection unidentified previously between the two stress resistance enzymes. We observed that the catalase in ahpC mutant cells in comparison with the parent strain is inactivated partially (approximately 50%). The decrease of catalase activity is well correlated with the perturbation of the heme environment in catalase, as detected by electron paramagnetic resonance spectroscopy. To understand the reason for this catalase inactivation, we examined the inhibitory effects of hydroperoxides on H. pylori catalase (either present in cell extracts or added to the purified enzyme) by monitoring the enzyme activity and the EPR signal of catalase. H. pylori catalase is highly resistant to its own substrate, without the loss of enzyme activity by treatment with a molar ratio of 1:3000 H 2 O 2 . However, it is inactivated by lower concentrations of organic hydroperoxides (the substrate of AhpC). Treatment with a molar ratio of 1:400 t-butyl hydroperoxide resulted in an inactivation of catalase by approximately 50%. UV-visible absorption spectra indicated that the catalase inactivation by organic hydroperoxides is caused by the formation of a catalytically incompetent compound II species. To further support the idea that organic hydroperoxides, which accumulate in the ahpC mutant cells, are responsible for the inactivation of catalase, we compared the level of lipid peroxidation found in ahpC mutant cells with that found in wild type cells. The results showed that the total amount of extractable lipid hydroperoxides in the ahpC mutant cells is approximately three times that in the wild type cells. Our findings reveal a novel role of the organic hydroperoxide detoxification system in preventing catalase inactivation.

The antioxidant protein alkylhydroperoxide reductase of Helicobacter pylori switches from a peroxide reductase to a molecular chaperone function

“…Among these, the most abundant one is AhpC. Several functional studies of H. pylori AhpC have shown that the AhpCdeleted mutants have increased sensitivity to oxygen (15), overexpress neutrophil-activating protein (18), and accelerate inactivation of catalase (25). As a member of 2-Cys peroxiredoxins (17,21,31), AhpC has been shown to be Trx-dependent (17,27,32,33).…”

“…To combat the reactive oxygen species released from the host immune system and survive in the gastric mucosa, pathogenic H. pylori is equipped with a number of detoxifying proteins (25,31). Among these, the most abundant one is AhpC.…”

“…However, the electron donor flavoprotein (AhpF) used by most bacteria to reduce AhpC was shown to be inactive for the AhpC of H. pylori (21)(22)(23)(24). In addition, some AhpC-deficient mutant strains have been found to have an increase in the expression of neutrophilactivating protein (NapA) (15,18) or a decrease in the activity of catalase (25). Our previous study has also revealed that the transcription and expression of AhpC in H. pylori isolated from duodenal ulcer patients decrease under long-term oxidative stress (Ͼ8 h in 20% O 2 ) (26).…”

Synergistic Roles of Helicobacter pylori Methionine Sulfoxide Reductase and GroEL in Repairing Oxidant-damaged Catalase

“…The genes were inactivated by inserting antibiotic resistance cassettes (kanamycin and chloramphenicol for Msr and catalase, respectively) as described previously (16,19). H. pylori strains were grown in Brucella agar (BD Biosciences) plates containing 10% defibrinated sheep blood (Quad Five, Ryegate, MT).…”

“…In H. pylori, catalase is one of the most abundant proteins (constitutes ϳ4 -5% of the total proteins, described herein), and it has some unique properties. Its unusual characteristics include a high pI (Ͼ9.0) (18) and high resistance to H 2 O 2 (19). Although an H. pylori catalase mutant (katA) strain is viable in vitro under nonoxidative stress conditions, catalase activity has been shown to be associated with the resistance of the bacterium to professional phagocytes and macrophages (20,21).…”