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Originally published In Press as doi:10.1074/jbc.M408450200 on September 28, 2004

J. Biol. Chem., Vol. 279, Issue 50, 51908-51914, December 10, 2004
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Role of a Bacterial Organic Hydroperoxide Detoxification System in Preventing Catalase Inactivation*

Ge Wang{ddagger}, Richard C. Conover§, Stephane Benoit{ddagger}, Adriana A. Olczak{ddagger}, Jonathan W. Olson{ddagger}||, Michael K. Johnson§, and Robert J. Maier{ddagger}

From the Departments of {ddagger}Microbiology and §Chemistry, University of Georgia, Athens, Georgia 30602

Received for publication, July 26, 2004 , and in revised form, September 22, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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 H2 O2. However, it inactivated is 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The ability of pathogenic bacteria to resist oxidative stress is crucial to their infectiousness and pathogenesis in the host (1, 2). To defend against reactive oxygen species that can cause protein oxidation, lipid peroxidation, and DNA damage, living organisms rely on serial enzymatic machinery. Superoxide dismutase, catalase, and alkyl hydroperoxide reductase (AhpC)1 are virtually ubiquitous enzymes that confer oxidative stress resistance (3–6). Superoxide dismutase dismutates the superoxide anion into hydrogen peroxide and molecular oxygen, and catalase breaks down H2O2 into water and O2. AhpC reduces organic hydroperoxides (ROOH, also extended to include HOOH) into the corresponding non-toxic alcohol (ROH). The elimination of organic hydroperoxides is particularly important for living cells because organic hydroperoxides can initiate a lipid peroxidation chain reaction and consequently propagate free radicals, leading to DNA and membrane damage (7). AhpC is a component of a large family of thiol-specific antioxidant proteins, with roles that are not generally well understood (8, 9).

A highly successful human bacterial pathogen Helicobacter pylori induces a strong inflammatory response within the host, thereby releasing a high level of host-derived toxic oxygen species, but H. pylori can survive and colonize persistently in the harsh conditions of the gastric mucosa (10–12). To account for this capability, H. pylori possesses superoxide dismutase, KatA, and AhpC enzymes (11). In addition, some other factors have been identified that play important roles in oxidative stress resistance. 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 (15–18), demonstrating the importance of these enzymes in oxidative stress resistance and host colonization.

H. pylori expresses abundant levels of catalase and AhpC proteins (19). The genetic and biochemical characterization of H. pylori catalase and AhpC has been performed in different laboratories (13, 20–24). H. pylori catalase is a homotetrameric protein in which each subunit has a molecular mass of 59 kDa. It is a monofunctional catalase without peroxidase activity. A unique property of H. pylori catalase is that it has a pI of >9. Another property of H. pylori catalase, which is distinct from other typical catalases, is its stability at very high concentrations of H2O2 (20). H. pylori cells were shown to be resistant to a high concentration (~100 mM) of H2O2, and this resistance was abolished in katA– mutants (24). H. pylori AhpC is a major component of the AhpC-thioredoxin-thioredoxin reductase-dependent peroxiredoxin system that catalyzes the reduction of hydroperoxides including H2O2 and organic hydroperoxides (22, 25), as well as the reduction of peroxynitrite (26). It was extremely difficult to obtain an ahpC knock-out mutant (21, 22), but the mutant was obtained eventually by screening transformants at a very low O2 (1% partial pressure) condition (13). AhpC mutant cells were shown to exhibit severe growth sensitivity to hydroperoxides and to the superoxide-generating agent, paraquat (13).

We studied the relationship between these two important antioxidant proteins and discovered that the organic hydroperoxide reductase (AhpC) plays a role that was unidentified previously in protecting catalase from inactivation by organic hydroperoxides. For the first time, it is shown that the loss of AhpC function leads to a significant increase of organic hydroperoxides within the cells and that these hydroperoxides are potent inhibitors of catalase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
H. pylori Strains and Growth Conditions—H. pylori strain ATCC43504 or the isogenic mutants were cultured on Brucella agar (Difco) plates (called BA plates) supplemented with 5% fetal bovine serum. Cultures of H. pylori were grown microaerobically at 37 °C in an incubator containing 5% CO2 and 2% oxygen. Chloramphenicol (50 µg/ml) or kanamycin (40 µg/ml) was added in the medium for culturing mutants.

DNA Techniques—All DNA manipulations were performed as described previously (27). Chromosomal DNA was extracted from H. pylori with the Aquapure genomic DNA extraction kit (Bio-Rad). Plasmid DNA preparations were carried out with the QiaPrep Spin mini kit (Qiagen). DNA fragments or PCR products were purified from agarose gels with the Qiaquick gel extraction kit (Qiagen). PCR was performed in a 2400 thermal cycler (PerkinElmer Life Sciences) with Taq or Pfu DNA polymerase (Fisher). Oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA).

Construction of H. pylori Mutants—To construct a katA mutant, a 953-bp fragment containing the H. pylori katA gene with genomic DNA from strain ATCC43504 as a template was amplified by PCR using primers katAF (5'-TCCATAAGAGAACAAGCGCC-3') and katAR (5'-CAACAATGTGATTACGGCCG-3'). The PCR fragment was cloned directly into the pGEM-T vector (Promega), according to the manufacturer's instruction, to generate pGEM-katA. The host strain used for cloning was Escherichia coli DH5{alpha}. Subsequently, a chloramphenicol acetyl transferase cassette was inserted at the unique HindIII site within the katA sequence of pGEM-katA. The recombinant plasmid was then introduced into H. pylori by natural transformation via allelic exchange, and chloramphenicol-resistant colonies were isolated. The disruption of the gene in the genome of the mutant strain (ATCC43504, katA:Cm) was confirmed by PCR showing an increase in the expected size of the PCR product.

With a similar procedure, the ferritin mutant strain (ATCC43504, pfr:Kan) was constructed as follows. A 1213-bp fragment containing the H. pylori pfr gene was amplified by PCR using primers pfrF (5'-TGGCTAGTTTTAAGGGCATG-3') and pfrR (5'-AAGCGCAAAATTTGCAAGCG-3') and cloned into the pGEM-T vector. Subsequently, the 301-bp HindIII fragment within the pfr gene was replaced by a kanamycin resistance cassette.

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.

Cell-free Extract and Membrane Fraction—Plate-grown H. pylori cells were harvested and suspended in phosphate-buffered saline. The cells were collected by centrifugation (10,000 x g for 10 min), resuspended in phosphate-buffered saline, and broken by two passages through a French pressure cell at 18,000 pounds/in2. Crude extracts were then cleared of unbroken cells by centrifugation at 10,000 x g for 10 min. The supernatant (cell-free extract) was then subjected to ultracentrifugation (45,000 x g for 60 min) to obtain the membrane fraction (the pellet).

Protein Concentration Determination and Gel Electrophoresis—Protein concentrations were determined with a bicinchoninic acid protein assay kit (Pierce). For SDS-PAGE, 5 µg of cell extract was placed into an SDS buffer, boiled for 5 min, and applied to a denaturing 12.5% acrylamide gel. Densitometric measurements were made for all the protein bands on the SDS gel, and the portion (percentage) attributed to a specific protein (KatA or AhpC) was calculated. Based on this percentage, the molecular weight of the protein, and the total protein concentration, we estimated the molar concentration of KatA or AhpC in the cell extract.

Purification of Catalase—Native H. pylori catalase was purified following a similar method described by Radcliff et al. (28). Briefly, H. pylori cells grown on plates were harvested by suspension in 0.1 M sodium phosphate buffer (pH 7.5). After centrifugation, the cell pellet was resuspended in the buffer. Cells were disrupted by three cycles through a French pressure cell at 18,000 pounds/in2, and the lysate was centrifuged at 28,000 x g for 10 min to remove the cell debris. The supernatant was then collected and subjected to ultracentrifugation at 100,000 x g for 45 min. The supernatant was applied to a SP-Sepharose cation exchange column (Amersham Biosciences) that had been equilibrated with 25 mM sodium phosphate buffer (pH 7.5), and proteins were eluted by the creation of a gradient with 1 M NaCl in 25 mM sodium phosphate buffer (pH 7.5). Catalase-positive fractions were selected by checking for oxygen-reducing activity in 3% H2O2. The pooled catalase-positive fractions were further purified by gel filtration chromatography using a Sephacryl S-200 column (Amersham Biosciences) and eluted with 25 mM sodium phosphate buffer (pH 7.5). The purified catalase was then filtered to sterilize and stored at 4 °C, protected from light.

Determination of Catalase Activity—The quantitative catalase activity of H. pylori cell extract or purified catalase protein was determined following the method described by Hazell et al. (20). Briefly, catalase activity was measured spectrophotometrically at 25 °C by following the decrease in absorbance at 240 nm ({epsilon}240 nm = 43.48 M–1 cm–1) of 13 mM H2O2 in phosphate-buffered saline. All assays were repeated to give 12 rate determinations for the first minute of reaction. One unit was defined as the amount of enzyme that catalyzes the oxidation of 1 µmol of H2 O2– min 1 under the assay condition.

Determination of Lipid Hydroperoxides—The amount of lipid hydroperoxides within H. pylori cell extracts or the membrane fractions was determined with a lipid hydroperoxide assay kit (Cayman Chemical, Ann Arbor, MI) following the manufacturer's instruction. Briefly, lipid hydroperoxides within a sample were first extracted into chloroform, which eliminates any interference caused by hydrogen peroxide or endogenous ferric ions in the sample. The lipid hydroperoxides in the extracted sample were used directly in the assay by reacting them with ferrous ions. The resulting ferric ions were detected using thiocyanate ions as the chromogen by measuring absorbance at 500 nm ({epsilon}500 nm = 16,667 M–1 cm–1). An ethanolic solution of 13-hydroperoxyoctadecadienoic acid was used as a lipid hydroperoxide standard.

Determination of Organic Peroxide Reductase Activity in Cell Extract—t-Butyl hydroperoxide (t-BOOH) was added to the H. pylori wild type cell extract to a final concentration of 1 mM. At various time points, an aliquot of the sample was removed and assayed for the remaining amount of t-BOOH using the lipid hydroperoxide assay kit as described above. The organic peroxide reductase activity was expressed as the decrease in the amount (nmol) of t-BOOH/min/mg of total protein in the cell extract.

EPR Spectroscopy—In this study, EPR spectroscopy was applied to whole cells, cell extracts, or purified catalase to monitor changes of the catalase heme environment. For whole cell samples, a 5-ml cell suspension in phosphate-buffered saline (A600 nm = 8) was incubated with 20 mM des-ferrioxamine at 37 °C for 15 min. The cells were then centrifuged, washed with cold 20 mM Tris-HCl (pH 7.4), resuspended in a final volume of 0.4 ml of the same buffer, and frozen in 3-mm quartz EPR tubes by immersion in liquid nitrogen. For cell extracts or purified catalase, the samples were incubated (for 30 min at room temperature) with different concentrations of hydroperoxides, as indicated, and frozen immediately in EPR tubes. Samples were stored at –78 °C for EPR spectroscopic analysis.

X band (~9.6 GHz) EPR spectra were recorded on a Bruker ESP-300E EPR spectrometer equipped with an ER-4116 dual mode cavity and an ESR-9 flow cryostat (Oxford Instruments). The intensity of the EPR signals was normalized to the OD of whole cell samples and the protein absorption band of cell-free extracts and purified samples.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Monitoring KatA and AhpC Proteins in H. pylori Cell Extract by SDS-PAGE—Both KatA and AhpC are major proteins expressed in H. pylori cells. Hazell et al. (20) reported that catalase accounts for ~1% of the total protein of the cell in H. pylori. The proteome analysis of Jungblut et al. (19) showed that AhpC (also called TsaA) is the third most abundant protein in H. pylori. The expression level of these proteins in H. pylori cells is visualized easily on SDS-PAGE. We identified the specific protein bands by (a) the expected molecular weight, (b) the loss of the band in the corresponding mutant strain, and (c) direct N-terminal sequencing of the protein band. The regulation mechanisms for the expression of KatA and AhpC in H. pylori are currently unclear, but they may involve regulation at both transcriptional and post-translational levels. Using SDS-PAGE, we measured the net expression level of the proteins. There are some variations in the protein expression levels by different strains (data not shown). The profiles of the total proteins in the parent strain ATCC43504 and the isogenic mutant strains katA, ahpC, or napA are shown in Fig. 1. Compared with the wild type strain, the corresponding protein band of KatA, AhpC, and NapA was missing in the respective mutant strains. As shown previously (13), there are two types of ahpC mutants, with the type I mutant overexpressing NapA. Based on densitometric measurement of the protein bands on the gel, KatA and AhpC each constitute 2–3% of the total proteins in the wild type cell. Compared with other bacteria, for example Campylobacter jejuni (29), the expression levels of KatA and AhpC are much higher in H. pylori.



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  		FIG. 1.
Profiles of total proteins in H. pylori cells detected by SDS-PAGE. Five micrograms of crude extract from the H. pylori wild type strain ATCC43504 and the isogenic mutant of katA, ahpC, or napA were loaded into each lane. Lane M shows the low range markers of molecular mass (indicated on left). The protein bands corresponding to KatA, AhpC, or NapA are marked with arrows (on right). WT, wild type.

 
Decreased Catalase Activity in ahpC Mutant Cells—H2O2 is a general agent of oxidative stress to the cells. To determine the relative contributions of H. pylori superoxide dismutase, KatA, AhpC, NapA, or MdaB in resistance to H2O2, we determined the H2O2 decomposing activity of each mutant strain compared with the wild type strain. As shown in Fig. 2A, wild type H. pylori exhibited a high level of catalase activity (~3000 units/mg of total protein), whereas, knock-out mutants in katA (the catalase gene) resulted in a complete loss of catalase activity. As expected, the disruption of other antioxidant genes such as sodB (the gene for superoxide dismutase) or mdaB (the gene for NADPH quinone reductase) did not affect significantly catalase activity of the cells. To our surprise, however, the catalase activity in gene-targeted ahpC mutant cells was determined to be approximately half of that in the wild type cells (Fig. 2A).



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  		FIG. 2.
Catalase activities and the EPR signals in various H. pylori strains. The cells of the wild type (WT) strain ATCC43504 and the isogenic mutants (at left, ahpC1 and ahpC2 indicate ahpC type I and II strains, respectively) were grown and harvested at late log phase. A, the catalase activities (units/mg protein) were determined from cell-free extract, and the data were obtained from three independent experiments (12 readings in each experiment) with means ± S.D. B, EPR was detected with the whole cells, and a representative example from repeated experiments is shown. The EPR intensity has been normalized based on the optical density of the samples, and the scales for three ahpC mutant strains (ahpC1, ahpC2, and ahpC,napA) are presented as two times (x2) those for other strains. EPR spectra were recorded at 4.2 K with 10-milliwatt microwave power, 0.63 millitesla (mT) modulation amplitude, and a microwave frequency of 9.6 GHz.

 
It was observed in Pseudomonas aeruginosa that catalase activity is affected by the mutation of a separate gene (30); a bfrA mutant of P. aeruginosa had only 47% the KatA activity of wild type strain, despite possessing the wild type expression level of KatA. BfrA, composed of 24 subunits and capable of binding 700 iron atoms, is the major iron storage protein in P. aeruginosa (31). The results of Ma et al. (30) suggest that BfrA is required as a source of iron for the heme prothetic group of KatA. H. pylori possess two iron storage proteins, NapA and Pfr. NapA is a homologue of bacterial DNA-protecting proteins, and its molecular structure has been determined (32, 33). It has a dodecameric structure (12 subunits) that is capable of binding up to 500 iron atoms (32). Pfr is the major iron storage protein in H. pylori (34). As a typical ferritin, Pfr forms a 24-mer structure and binds more than 2000 iron atoms (35). As shown in Fig. 2A, H. pylori KatA activity was not affected significantly by the loss of either NapA or Pfr. Therefore, the observation that H. pylori KatA activity is affected by the loss of AhpC is an unusual phenomenon, unrelated to iron storage, which is different from the modulation of catalase activity by BfrA in P. aeruginosa.

As shown in Fig. 1, the resolution of the KatA protein band and its density relative to the total protein pattern allow it to be monitored unambiguously. A comparison of the protein profiles from crude extracts of the wild type with various mutant strains (Fig. 1) indicated that the knock-out of AhpC did not change significantly the level of catalase protein expression. Hence, a regulatory role for AhpC in catalase expression was ruled out. Therefore, we hypothesized that the catalase in ahpC mutant cells might have undergone certain structural changes leading to its partial inactivation.

Purified H. pylori AhpC was shown to be able to reduce H2O2 in vitro at a rate similar to its t-BOOH reducing activity (22). However, the H2O2 decomposing activity in the katA mutant (AhpC+) cells was undetectable (our results are shown in Fig. 2A) (see Refs. 23 and 24). Because AhpC requires thioredoxin and thioredoxin reductase for its activity, failure to detect the H2O2 decomposing activity of AhpC could be the result of a limited amount of available thioredoxin-thioredoxin reductase proteins. Therefore, we tested whether the wild type cell extract has the activity to reduce organic peroxides. Using t-BOOH as a substrate and without adding reductant, the wild type cell extract showed reductase activity, with a specific activity of 15.6 ± 2.3 nmol (t-BOOH reduced)/min/mg of total protein in the cell extract. If AhpC had a similar rate of H2O2 reducing activity, it would be far below the detection level of the assay system for catalase. Thus, the decrease of catalase activity observed in ahpC mutant cells could not be attributed to the loss of the H2O2 decomposing activity of AhpC but to the perturbation of catalase itself.

A Perturbed Heme Environment Associated with Catalase in ahpC Mutant Cells—Catalase contains a heme-active site, which can be detected by EPR spectroscopy in ferric oxidation states (36). Purified H. pylori catalase exhibits a characteristic, nearly axial high spin ferric EPR signal in which effective g values = 6.36, 5.35, and 1.98 (37). Considering the high abundance of catalase protein in H. pylori and the high transition probability of the low field components of axial high spin ferric resonances, we applied EPR analysis to whole cells to monitor any structural change that could be attributed to the heme group of catalase (Fig. 2B). EPR studies of wild type cells showed features at g values of 6.4 and 5.4, which are characteristic of the low field components of the catalase high spin ferric heme resonance. Confirmation of this assignment was provided by the complete absence of this resonance in the katA gene knock-out strain. No significant change in the catalase EPR signal was observed in the sodB, mdaB, napA, or pfr mutant cells. However, the EPR signals were altered (compared with the parent strain) in the cells of all three mutants of ahpC (strains ahpC type I, ahpC type II, and the double mutant ahpC,napA), with an approximate decrease of 50% in the resonance amplitude where g = 6.4, 5.4, and 2.0 and a concomitant slight increase in a high spin ferric heme resonance where g = 6.8, 5.1, and 2.0. The decrease of the resonance at g values of 6.4, 5.4, and 2.0 correlates well with the decrease in catalase activity (Fig. 2), despite no change in KatA protein expression level (Fig. 1). The intensity of the EPR signals was normalized strictly based on the protein concentration of each sample. These results suggested that the disruption of AhpC in the cells results in a modification of the catalase heme environment, leading to the partial inactivation of the catalase.

At present, the identity of the heme structural perturbation signal corresponding to the resonance g values of 6.8, 5.1, and 2.0 is not clear, but the complete loss of these signals in the katA,ahpC double mutant cells indicated that both resonances arise from catalase but not from other proteins that might have been overexpressed in the ahpC mutant cells. Because similar EPR signals with increased rhombicity have been observed in purified H. pylori catalase samples with formate or azide bound in the distal heme pocket (g value ~ 6.6, 5.4, and 2.0) (37), the heme environment is likely to be modified by the binding of a small molecule in close proximity to the heme iron.

Inactivation of Catalase in Cell-free Extracts by Organic Hydroperoxide—The primary function of AhpC is associated with the detoxification of organic hydroperoxides (8, 9). The active site of the AhpC enzyme can accommodate virtually any ROOH, including HOOH (H2O2) (22, 38). H. pylori AhpC mutant cells exhibit growth sensitivity to hydroperoxides and to the superoxide-generating agent paraquat (13). To test whether any of these oxidative agents is responsible for inactivation of catalase, we treated the cell-free extract of wild type H. pylori with various agents and then measured catalase activity and monitored EPR signals (Fig. 3). Treatment with up to 0.5 M H2O2 did not significantly affect catalase activity and did not change the catalase heme EPR signal, indicating that H. pylori catalase is highly stable in the presence of a high concentration of its own substrate. Similarly, treatment with up to 0.1 M paraquat had no effect (data not shown). Because the whole cells of a sodB mutant strain (Fig. 2) also showed no effect on catalase (on neither the enzyme activity nor the EPR signal), it seems that H. pylori catalase is not sensitive to superoxide.



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  		FIG. 3.
Effect of hydroperoxides on H. pylori catalase in cell-free extract. Wild type strain ATCC43504 cell extract was pretreated with different concentrations of hydroperoxides (H2O2 or t-BOOH) for 30 min at room temperature. Part of each sample was used immediately for determining the catalase activity, and the remaining sample was frozen in EPR tubes for EPR analysis. The catalase activities were determined from three independent experiments (12 readings in each experiment), averaged with means ± S.D., and presented as a percentage compared with the control sample without hydroperoxide treatment. For the EPR results, a representative spectrum from repeated experiments is shown. Note that the EPR scales for three t-BOOH treatment samples are presented as two times (x2) that of the control. The conditions used for EPR measurements are the same as those described in the Fig. 2 legend.

 
To test whether organic hydroperoxides are responsible for the inactivation of catalase, we used t-BOOH, a small molecule of organic hydroperoxide that is soluble in aqueous solution, to treat an H. pylori cell extract. By treatment with various concentrations of t-BOOH, we observed progressive inactivation of catalase, a concomitant decrease in resonance at g = 6.4, 5.4, and 2.0, and a slight increase in the resonance at g = 6.8, 5.1, and 2.0 (Fig. 3). For example, after treatment with 150 mM t-BOOH, approximately 70% of catalase activity was lost and the resonance at g = 6.4, 5.4, and 2.0 was decreased to approximately one-third the intensity in the untreated cell-free extract. Treatment of the wild type cell extract with ~70 mM t-BOOH reduced the catalase activity to one-half the control level, and the EPR signal mimicked that of the whole cells of the ahpC mutant strain (Fig. 2B). As a control, the cell-free extract of the ahpC mutants without treatment (data not shown) gave rise to an EPR signal similar to that of whole cells of ahpC mutant strain.

For the purpose of EPR analysis, the concentration of the cell extract used in these experiments had to be extremely high. Accordingly, high concentrations of hydroperoxides were used to observe the inhibiting effect. Under the experimental condition, the molar ratio of 70 mM t-BOOH to AhpC and to catalase present in the cell extract was estimated (see "Experimental Procedures") to be 20,000:1 and 40,000:1, respectively. The actual amount of t-BOOH that exerted damage on catalase is unknown because part of the added t-BOOH was consumed by AhpC in the cell extract. Nonetheless, under the same experimental condition, the catalase (in the cell extract) was stable with 500 mM H2O2, although it was inhibited by 50% with 70 mM t-BOOH. These results indicated that an organic hydroperoxide is able to induce inactivation and perturbation of the catalase heme environment in cell-free extracts, analogous to those induced by the absence of AhpC in the ahpC mutant cells.

Inactivation of Purified H. pylori Catalase by Organic Hydroperoxide—To examine closely the sensitivity of catalase to hydroperoxides in vitro, H. pylori catalase was purified to near homogeneity (Fig. 4). Compared with the H. pylori catalase used in the study of Loewen et al. (37), which was expressed in E. coli, we purified native H. pylori catalase directly from H. pylori cell extracts (Fig. 4, lane 1) by cation exchange (Fig. 4, lane 3) and gel filtration (Fig. 4, lane 4).



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  		FIG. 4.
Purification of H. pylori catalase. A 12% polyacrylamide gel containing 1% SDS stained with Coomassie Brilliant Blue included the following samples: cell extract from H. pylori strain ATCC43504 (lane 1), flow-through sample from SP-Sepharose cation-exchange column (lane 2), fraction 30 eluted from SP-Sepharose cation-exchange column (lane 3), and fraction 47 eluted from S-200 gel filtration column (lane 4). The protein bands corresponding to KatA as well as AhpC are marked with arrowheads on left. Lane M shows the low range markers of molecular mass (indicated on right).

 
In addition to a high pI (>9), H. pylori catalase has another property distinct from other typical catalases in that it is stable at very high concentrations of H2O2 (20). In the presence of excess H2O2, typical catalases can form an intermediate (compound II (Fe(IV)) as a result of one-electron oxidation; this intermediate does not react with H2O2, and thus the accumulation of compound II leads to the deactivation of catalase (39). In contrast, H. pylori catalase can withstand very high concentrations of H2O2 (molar ratio = 1:3000) without the loss of enzyme activity (Fig. 5). However, purified H. pylori catalase showed sensitivity to organic hydroperoxides, confirming the results observed for cell-free extracts. Treatment with increasing concentrations of t-BOOH resulted in a progressive decrease in the catalase EPR signals (g = 6.4, 5.4, and 2.0) and a concomitant decrease in the catalase activity (Fig. 5). Under these experimental conditions, the pretreatment of purified catalase with t-BOOH in a molar ratio of ~1:400 caused the loss of 50% of catalase activity and an approximate decrease of 50% in the catalase EPR signal.



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  		FIG. 5.
Effect of hydroperoxides on the purified H. pylori catalase. Purified H. pylori catalase was pretreated with hydroperoxides (H2 O2 or t-BOOH) at different molar ratios (catalase:hydroperoxide) for 30 min at room temperature prior to measuring catalase activity and freezing for EPR analysis. The conditions used for catalase assays and for EPR measurements are the same as those described in the Fig. 3 legend.

 
Parallel absorption studies (36) (Fig. 6) indicate that the addition of an 800-fold excess of t-BOOH to purified catalase results in immediate and nearly complete one-electron oxidation to yield the Fe(IV) compound II species as evidenced by the shift in the Soret band maximum from 405 to 430 nm. The Fe(IV) compound II species is EPR silent and is not catalytically competent for dismutation of hydrogen peroxide into water and oxygen. Hence, the inhibition and loss of catalase EPR signal on addition of t-BOOH are attributed to the formation of compound II species.



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  		FIG. 6.
UV-visible absorption spectra of purified H. pylori catalase before (solid line) and after (dashed line) the addition of an 800-fold excess of t-BOOH.

 
Compared with the results observed for the whole cells (Fig. 2) or cell extracts (Fig. 3), EPR signals at g = 6.8, 5.1, and 2.0 were not evident with the purified catalase after t-BOOH treatment. The binding of an organic hydroperoxide in the distal heme pocket is a good candidate for the origin of the EPR signals at g = 6.8, 5.1, and 2.0 in the cells or cell extracts. However, this binding may require one or more additional small molecules that are not present in the purified enzyme sample. We also purified the catalase from H. pylori ahpC mutant cells and examined its sensitivity to hydroperoxides. The results (not shown) were the same as those for the catalase from the wild type strain.

Higher Levels of Lipid Hydroperoxides in ahpC Mutant Cells—Unsaturated fatty acids have been found repeatedly as a constituent of lipids in H. pylori, and the growth of H. pylori displays sensitivity to the addition of unsaturated free fatty acids because of their incorporation into phospholipids and subsequent membrane destruction (40, 41). Thus, under the physiological (oxidative stress) condition, there might be a steady flow of lipid hydroperoxides present within H. pylori cells, a situation that would require an abundant organic peroxide reductase activity to remove the damaging organic hydroperoxides.

The results presented above suggested that the loss of AhpC function leads to the accumulation of organic peroxides, which are responsible for the inactivation of catalase. To further support this notion, we measured the levels of lipid hydroperoxides in wild type and ahpC mutant H. pylori cells (Table I). The total amount of lipid hydroperoxides (in the entire cell extract) in ahpC mutant cells was determined to be approximately three times that of wild type cells. The majority of lipid hydroperoxides within the cells was present in the membrane fraction. When considering only the membrane fraction, the extent of lipid peroxidation in ahpC mutant cells was approximately four times that of wild type cells. This result indicated that organic hydroperoxides indeed accumulated to a significantly greater extent in the ahpC mutant cells compared with the wild type cells.


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  		TABLE I
Total lipid hydroperoxides (nmol/1010 cells) in H. pylori cells

The cell extract or membrane fraction was prepared from H. pylori cells (WT or ahpC mutant), which were grown to late log phase and harvested. The total amount of lipid hydroperoxides was determined and calculated as the amount (nmol) present in the samples from 1010 cells. The data are averaged from three independent experiments with means ± S.D.

 
The above result also suggests that under physiological conditions the major function of H. pylori AhpC is to reduce organic hydroperoxides. Knock-out of AhpC (an abundant protein) to create ahpC mutants enabled us to detect a significant increase of lipid hydroperoxides in the cells. To our knowledge, this is the first direct demonstration of lipid peroxidation in bacterial cells. The detoxification of organic hydroperoxides in H. pylori seems quite different from that in E. coli. It is reported (42) that E. coli lacks the polyunsaturated fatty acids necessary for lipid peroxidation and that the AhpC protein is not as abundant in E. coli as in H. pylori. In accordance with these reports, the recent study by Seaver and Imlay (38) suggested that the major physiological substrate of E. coli AhpC is H2O2 rather than organic hydroperoxides.

We showed that in vitro treatment of purified catalase with t-BOOH in a molar ratio of ~1:400 caused a 50% inactivation of catalase (Fig. 5). To complement this result, we found that the ahpC mutant cells accumulated much more lipid hydroperoxides than the wild type (Table I). However, it is difficult to calculate the molar ratio of catalase to lipid hydroperoxides within the ahpC mutant cells. Also, the lipid hydroperoxides present within H. pylori cells could have different levels of effectiveness in inactivating catalase. Furthermore, the inactivation of catalase by organic hydroperoxides within the cells might take place in a local environment (e.g. periplasm) where the lipid hydroperoxides could be much more concentrated. In this regard, it is of note that approximately half of the total catalase is located in the periplasm of H. pylori cells (43).

In summary, our results showed that lipid hydroperoxides accumulate in ahpC mutant cells and that lipid hydroperoxides inhibit catalase activity via the formation of a catalytically incompetent compound II species. This observation indicates a novel physiological role of the AhpC system in protecting catalase, thereby demonstrating a connection unrecognized previously in the function of two major proteins in a pathogenic bacterium.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants 1-RO1-DK60061 (to R. J. M.) and GM62542 (to M. K. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Present address: Dept. of Microbiology, North Carolina State University, Raleigh NC 27695. Back

To whom correspondence should be addressed: Microbiology Dept., University of Georgia, 805 Biology Science Bldg., Athens, GA 30602. Tel.: 706-542-2323; Fax: 706-542-6874; E-mail: rmaier{at}uga.edu.

1 The abbreviations used are: AhpC, alkyl hydroperoxide reductase; t-BOOH, t-butyl hydroperoxide. Back



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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