• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of iaiPermissionsJournals.ASM.orgJournalIAI ArticleJournal InfoAuthorsReviewers
Infect Immun. Jan 2005; 73(1): 378–384.
PMCID: PMC538961

Contribution of the Helicobacter pylori Thiol Peroxidase Bacterioferritin Comigratory Protein to Oxidative Stress Resistance and Host Colonization


Peroxiredoxins, the enzymes that catalyze the reduction of hydrogen peroxide and organic hydroperoxides, are ubiquitous proteins that protect organisms from damage by reactive oxygen species. Helicobacter pylori contains three members of the peroxiredoxin family: AhpC (alkyl hydroperoxide reductase), Tpx (thiol-specific peroxidase), and bacterioferritin comigratory protein (BCP). In this study, we characterized H. pylori bcp mutant strains and wild-type BCP. Compared to the parent strain and the ahpC mutant strain, the bcp mutant showed moderate sensitivity to the superoxide-generating agent paraquat and to organic hydroperoxides. Upon exposure of 108 cells to air for 10 h, 106 wild-type cells survived but none of the 108 bcp mutant cells were recovered. Introduction of an intact bcp gene at an unrelated locus in the bcp strain restored the wild-type-like oxidative stress resistance phenotype. Purified BCP was shown to be a thiol peroxidase that depends on the reducing activity of thioredoxin and thioredoxin reductase. Among a series of peroxides tested, linoleic acid hydroperoxide was the preferred substrate of BCP. By examining the profiles of protein expression within H. pylori cells, we confirmed that AhpC is much more abundant than BCP. The overlapping functions and activities of BCP and AhpC probably explain why the bcp mutant displayed a relatively weak oxidative stress resistance phenotype. The bcp mutant strain could colonize mouse stomachs, although colonization by the wild-type strain was slightly better than that by the mutant strain at 1 week after host inoculation. However, at 3 weeks after inoculation, the colonization ability of the wild type was significantly greater than that of the bcp mutant; for example, H. pylori was recovered from 10 of 11 mouse stomachs inoculated with the wild-type strain but from only 4 of 12 mice that were inoculated with the bcp mutant strain. This indicates that H. pylori BCP plays a significant role in efficient host colonization.

Oxidative stress resistance is one of the key properties that enable pathogenic bacteria to survive the effects of the production of reactive oxygen by the host (21). Peroxiredoxins (Prx) are ubiquitous proteins that confer resistance to oxidative stress. They are enzymes lacking prosthetic groups that catalyze the reduction of hydrogen peroxide and organic hydroperoxides (26). Peroxiredoxins can be divided into two subgroups according to the number of conserved cysteines (Cys) within the proteins. Some peroxiredoxins contain only a single essential, N-terminal Cys residue per subunit (1-Cys Prx), and other peroxiredoxins contain an additional conserved Cys residue that links the two subunits via an intersubunit disulfide bond with the N-terminal Cys in the oxidized protein (2-Cys Prx).

Helicobacter pylori is a microaerophilic bacterium that causes peptic ulcers and is a risk factor for adenocarcinomas in humans (7). H. pylori contains three members of the Prx family of reductases, with the most important one being AhpC (alkyl hydroperoxide reductase). H. pylori AhpC is a 2-Cys Prx, and it uses reduced thioredoxin (Trx) as the electron donor to reduce hydrogen peroxide and organic hydroperoxides (3). It was also shown to have activity in reducing peroxynitrite (5). H. pylori AhpC plays a very important role in oxidative stress resistance and host colonization (16, 17). In addition to ahpC, two genes, tpx (HP0390) and bcp (HP0136), coding for two thiol peroxidases that belong to a 1-Cys Prx subgroup have been revealed by H. pylori genome sequence annotation (1, 22). Tpx was originally identified as an Escherichia coli homologue of scavengase p20, and a thioredoxin-linked peroxidase activity of Tpx protein was demonstrated (23, 28). An H. pylori tpx mutant strain is more sensitive to killing by peroxide and superoxide than the wild-type strain, and it has reduced ability to colonize mouse stomachs (6, 17). Bacterioferritin comigratory protein (BCP), originally identified in E. coli, was shown to be a new member of 1-Cys Prx. E. coli BCP shows a thioredoxin-dependent thiol peroxidase activity, with linoleic acid hydroperoxide (LOOH) being the preferred substrate over H2O2 (12). An E. coli bcp mutant shows the same level of hypersensitivity to H2O2 and organic peroxides as an ahpC mutant (12).

This study was initiated to characterize H. pylori BCP and to investigate the roles of this protein in oxidative stress resistance and in the colonization of the host stomach. Recently, Comtois et al. (6) constructed an H. pylori bcp mutant (in strain 26695) and observed a weak phenotype in vitro; thus, no further investigation was conducted. In the present study, we characterized in more detail the bcp mutants from different strains, particularly in comparison with an ahpC mutant strain. In addition, we purified H. pylori BCP and determined its peroxidase activity, which showed that BCP has a function overlapping that of AhpC. We report that the bcp mutant displays a relatively weak phenotype because of the presence of much more abundant AhpC within H. pylori cells. More importantly, with mouse colonization studies we demonstrated that H. pylori BCP contributes significantly to the bacterium's ability to colonize the host stomach, particularly in longer-term (3-week) colonization assays.



Unless otherwise stated, all biochemicals and reagents were from Sigma Chemicals.

H. pylori strains and growth conditions.

H. pylori strains SS1, ATCC 43504, and 26695 were used as the wild types. H. pylori was cultured on Brucella agar (Difco) plates supplemented with 10% defibrinated sheep blood or 5% fetal bovine serum (hereafter called BA plates). Chloramphenicol (50 μg/ml) or kanamycin (40 μg/ml) was added to the medium for culturing of mutants. Cultures of H. pylori were grown microaerobically at 37°C in a 5% CO2 incubator under continuously controlled levels of oxygen (2 or 4% partial pressure).

DNA techniques.

All DNA manipulations were performed as described previously (15). 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 using a Perkin-Elmer 2400 thermal cycler with Taq or Pfu DNA polymerase (Fisher). Oligonucleotide primers were synthesized by Integrated DNA Technologies, Coralville, Iowa.

Construction of H. pylori bcp mutant.

Primers bcpF (5′-AGCGTTATTGTTTGTGGCTTG-3′) and bcpR (5′-AACATGCTCCTTATTGGCGG-3′) were used to amplify by PCR a 910-bp fragment containing the H. pylori bcp gene (HP0136) with genomic DNA from strain ATCC 43504 as the template. There are two HindIII restriction sites (nucleotides 310 and 468 from the bcp start codon, 158 bp apart) within this PCR fragment (note that there is only one HindIII site in the sequence from strain 26695). The PCR fragment was directly cloned into pGEM-T vector according to the instructions of the manufacturer (Promega) to generate pGEM-bcp. The host strain used for cloning was E. coli DH5α. Subsequently, a chloramphenicol acetyltransferase cassette (CAT) was inserted within the bcp sequence of pGEM-bcp by replacing the 158-bp HindIII fragment. The recombinant plasmid was then introduced into H. pylori by natural transformation via allelic exchange, and chloramphenicol-resistant colonies were isolated by incubation under 4% partial O2 pressure conditions. The disruption of the gene in the genome of the mutant strain was confirmed by PCR showing an increase in the expected size of the PCR product.

Construction of H. pylori bcp complementation strain.

The kanamycin resistance cassette (Kan) was inserted behind the bcp gene in the plasmid pGEM-bcp, yielding pGEM-bcp-Kan. A fragment containing the bcp gene and Kan was then excised from pGEM-bcp-Kan and ligated into plasmid pEU39, yielding pEU-bcp-Kan. The plasmid pEU39 (from The Institute for Genomic Research) contains a 2.04-kb fragment of H. pylori genomic DNA covering the open reading frame HP0405 (nifS-like gene). Previously, investigators from our laboratory showed that disruption of HP0405 in H. pylori produces no obvious phenotype (17a). In plasmid pEU-bcp-Kan, HP0405 was disrupted into two pieces flanking bcp-Kan. When this plasmid was used to transform H. pylori SS1 bcp::CAT (selection is accomplished by chloramphenicol and kanamycin resistance), the intact bcp gene (with its promoter) and Kan were inserted into the genome at the HP0405 locus. This produced a merodiploid strain, SS1 bcp::CAT-bcp-Kan, which contains the original interrupted bcp and an intact copy of bcp at an unrelated site.

Paper disk assay for peroxide sensitivity.

Sensitivity to different oxidative agents was evaluated by disk assay. Sterile filter paper disks (7.5 mm in diameter) were applied to BA plates that had been streaked for confluent growth of H. pylori strains. Ten microliters of the indicated agents described in Table Table11 was applied to each disk. After the plates were incubated under 2% O2 conditions for 48 h, the clear zones surrounding the disks were measured. The data given represent the distances from the edges of the disks to the ends of the clear zones, where growth began.

Disk sensitivity assay resultsa

Air survival assay.

Survival of nongrowing H. pylori cells under atmospheric oxygen was assayed as follows. Wild-type or mutant H. pylori cells grown under 2% oxygen were suspended in phosphate-buffered saline (PBS) and incubated at 37°C under normal atmospheric conditions. Samples were removed at the times indicated in Fig. Fig.1,1, serially diluted, and spread onto BA plates. After 3 days of incubation in a 2% oxygen environment, the colony counts were recorded.

FIG. 1.
Survival of nongrowing H. pylori cells under atmospheric oxygen. H. pylori cells grown under 2% oxygen to late log phase were suspended in PBS and incubated at 37°C under normal atmospheric conditions. Samples were removed at the times indicated ...

Mouse colonization.

Mouse colonization assays were performed essentially as described earlier (17, 20, 24). Briefly, the wild-type SS1 or SS1 bcp::CAT mutant cells were harvested after 48 h of growth (37°C; 2% oxygen) on BA plates and suspended in PBS to an optical density at 600 nm (OD600) of 1.7. Headspace in the tube was sparged with Ar gas to minimize oxygen exposure. These suspensions were administered to C57BL/6J mice (1.5 × 108 CFU/mouse; inocula were kept constant for each experiment) via oral gavage. The inoculum dose was determined from reproducible standard curves of OD600 values versus viable cell numbers from counts on plates. After 1 or 3 weeks, the mice were sacrificed and the stomachs were removed, weighed, and homogenized in Ar-sparged PBS. Homogenate was plated onto BA plates supplemented with bacitracin (200 μg/ml) and nalidixic acid (10 μg/ml) and was incubated for 5 to 7 days before examination for the presence of H. pylori colonies.

Construction of plasmid for overproduction of H. pylori six-His-tagged BCP.

With the use of genomic DNA from strain ATCC 43504 as the template and primers BCP-F (5′-CGCGCGGCATATGGAAAAATTAGAAGTAGGG-3′) and BCP-R (5′-GCATAGGCTCGAGCTCCAAACTCTCTAAAAC-3′), a DNA fragment containing the complete bcp gene (~480 bp) was amplified by PCR. The PCR product was digested by NdeI and XhoI and cloned into pET-21a vector (Novagen), previously digested with the same restriction enzymes, to generate pET-BCP-6His. The recombinant plasmid was used to transform E. coli BL21 Origami (Novagen).

Overexpression and purification of H. pylori six-His-tagged BCP.

E. coli BL21 Origami cells harboring pET-BCP-6His were grown at 37°C to an OD600 of 0.5 in 500 ml of Luria-Bertani medium with ampicillin (100 μg/ml) and kanamycin (40 μg/ml). Expression of BCP was induced by addition of 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside) into the medium followed by further incubation for 3 h, and the cells were harvested by centrifugation (5,000 × g; 15 min; 4°C). All subsequent steps were performed at 4°C. Cells were washed with 200 ml of buffer A (20 mM Na2HPO4 [pH 7.4], 500 mM NaCl, and 5 mM imidazole) and resuspended in 5 ml of the same buffer. Cells were lysed by two passages through a cold French pressure cell at 18,000 lb/in2. Cell debris was removed by centrifugation at 20,000 × g. The supernatant was applied to a nickel-nitrilotriacetic acid affinity column (QIAGEN), and buffer A was used to wash the resin until the A280 reached the baseline. Proteins were washed with buffer B (buffer A with 30 mM imidazole) until the A280 reached the baseline and were finally eluted with buffer C (buffer A with 250 mM imidazole). Extracts of E. coli BL21 Origami containing the vector only did not result in retrievable proteins from this purification (Ni affinity) procedure. Fractions were analyzed by gel electrophoresis, and the BCP-positive fractions were pooled. Protein concentrations were determined with a bicinchoninic acid protein assay kit (Pierce, Rockford, Ill.).

Gel electrophoresis of cell extracts.

Plate-grown H. pylori cells were harvested and suspended in PBS. The cells were collected by centrifugation (5,000 × g for 10 min), resuspended in PBS, and broken by two passages through a French pressure cell. Crude extracts were then cleared of unbroken cells by centrifugation at 10,000 × g for 10 min. For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 5 μg of cell extract was placed into SDS buffer, boiled for 5 min, and applied to a denaturing 12.5% acrylamide gel.

Enzyme activity assays.

The peroxidase activity of purified BCP linked to NADPH oxidation via the thioredoxin-thioredoxin reductase system was determined by monitoring the decrease of absorbance at 340 nm. The reaction mixture (0.6 ml) contained 50 mM HEPES-NaOH (pH 7.0), 0.4 μM TrxR, 2 μM Trx1, 2 μM BCP, 0.2 mM NADPH, and various concentrations of either H2O2, tert-butyl hydroperoxide (tBOOH), or LOOH. The reaction was started by the addition of NADPH and was carried out at room temperature. As a control, BCP was omitted from the assay mixture. The purified H. pylori Trx1 and TrxR proteins were kindly provided by Leslie Poole. LOOH was generated by incubating 0.1 mM linoleic acid with 10 μg of soybean lipoxygenase/ml in 50 mM HEPES-NaOH (pH 7.0) at room temperature for 10 min. The concentration of LOOH was determined spectrophotometrically (epsilon234 = 25,000 M−1 cm−1).


Sensitivity of H. pylori bcp mutant to oxidative stress.

To investigate the physiological role of BCP in H. pylori, we created bcp mutants. The bcp gene was disrupted by insertion of CAT into the mouse-adapted strain SS1 as well as into the strains ATCC 43504 and 26695.

To characterize the oxidative stress resistance phenotype of the bcp mutant, we used disk inhibition assays to test the sensitivity of the mutant to a series of oxidative reagents in comparison to the sensitivities of the wild type and the ahpC mutant strain (Table (Table1).1). Inhibition zones around stress agent-saturated paper disks were measured. The bcp mutant showed moderate sensitivity to the superoxide-generating agent paraquat and to organic hydroperoxides but was not sensitive to H2O2. In comparison, the ahpC mutant was highly sensitive to all of these reagents. These results on sensitivity of the bcp mutant are basically in agreement with the data reported by Comtois et al. for H2O2 and cumene hydroperoxide (6); those authors did not test the sensitivity to tBOOH and paraquat.

Another in vitro phenotype of the bcp mutant that was assayed was the ability of nongrowing cells to survive periods of air exposure (Fig. (Fig.1).1). Upon exposure of approximately 108 cells to air for 10 h, 106 viable cells of the wild-type strain SS1 could be recovered. This is in contrast to no viable cell recovery for the bcp mutant. During the first 4 h of air exposure, the bcp mutant strain behaved like the wild-type strain. After the 4-h period, the viability of the bcp mutant strain dropped much more rapidly, so that the numbers of cells recovered at 6, 8, or 10 h were markedly lower than those of cells of the wild-type strain (Fig. (Fig.1A).1A). In comparison, the ahpC mutant was highly sensitive to air, with complete loss of viability after 6 h of exposure to air. We noticed variation in the phenotypes of the bcp mutants in different background strains. As shown in Fig. 1B and C, the bcp mutants in strains ATCC 43504 and 26695 showed weaker phenotypes than the mutant in the SS1 background. Nevertheless, significant sensitivity of the mutant in all backgrounds was observed after a longer time of exposure to air. The bcp mutant cells were completely killed at 10 or 8 h, but a significant number of wild-type cells survived that exposure. Comtois et al. (6) reported that the wild-type strain 26695 is completely killed at the same air exposure time (~8 h) as the bcp mutant strain, leading to a claim that BCP deficiency does not affect the oxygen sensitivity. To clarify this discrepancy, we also performed the air exposure experiment using their conditions, namely, suspending cells in the rich growth medium instead of PBS buffer. Again, we obtained results similar to those shown in Fig. Fig.1C.1C. We suspect that in their experiment the wild-type cells may have suffered oxidative damage before exposure to air. We note that our H. pylori cells were grown in a controlled low-O2 atmosphere (always maintained at 2% partial O2 pressure) and harvested in exponential phase (never in stationary phase) before testing for sensitivity to air exposure. Based on our results with different strains and different conditions, combined with results of disk sensitivity assays, we conclude that BCP in H. pylori has a physiological role in combating oxidative stress, although the role of BCP in doing so may not be as important as that of the AhpC enzyme.

To ensure that the observed sensitivity to oxidative stress was completely attributable to inactivation of bcp, we introduced a functional copy of the bcp gene back into the SS1 bcp mutant strain for complementation. The complemented strain (SS1 bcp::Cm-bcp+-Kan) contained a mutated bcp gene at the original locus and an intact bcp gene at an unrelated site (Materials and Methods). We determined the sensitivity of the complemented strain to oxidative reagents (Table (Table1)1) and the oxygen-dependent cell-killing effect of this strain (Fig. (Fig.1A).1A). This strain exhibited the same phenotype as the wild-type strain. This finding indicated that introduction of a functional bcp gene at another chromosomal locus restored the cell's ability to resist oxidative stress, meaning that the phenotype observed for the mutant was indeed due to lack of BCP.

Ability of H. pylori bcp mutant to colonize mouse stomachs.

Previously, investigators from our laboratory performed a series of mouse colonization experiments to examine the role of oxidative stress resistance factors in H. pylori virulence. The mutant strains tested included those with mutations in sodB, ahpC, tpx, and mdaB genes (17, 20, 24). To determine whether loss of BCP activity has a significant effect on H. pylori colonization in the host, the relative abilities of the wild-type and the bcp-deficient mutant strains to colonize the mouse stomach were evaluated (Fig. (Fig.2).2). Four groups of C57BL/6J mice (11 or 12 mice in each group) were inoculated with either the wild-type strain SS1 or the mutant strain SS1 bcp::CAT; the inoculants were incubated in low-O2 conditions (2% partial O2 pressure) for administration to the animals. H. pylori cells were recovered from the mouse stomachs either 1 or 3 weeks after oral administration. To avoid O2 exposure of the stomach isolates, the stomachs were homogenized in low-O2 conditions (by use of argon-sparged PBS in sealed tubes), and after plating of the cells from the stomach homogenate dilutions, the plates were immediately transported to the incubator containing 2% O2.

FIG. 2.
Results of mouse colonization assay of the H. pylori SS1 wild-type strain (SS1 WT) and its isogenic bcp::CAT mutant. The mice were inoculated with H. pylori two times (2 days apart) with a dose of 1.5 × 108 cells. Colonization of mouse stomachs ...

One week after inoculation, H. pylori was recovered from the stomachs of all 11 mice inoculated with the wild-type strain, with numbers ranging from 104 to 106 CFU/g of stomach. Of the 11 mice inoculated with the bcp mutant strain, 3 were not colonized by H. pylori (the bacterial counts were below the detection limit). According to Wilcoxon rank statistical analysis of the 1-week data, the colonization by the wild-type strain may have been better than that by the mutant strain but at a low confidence level (P = 0.1). Three weeks after inoculation, H. pylori was recovered from 10 of 11 mouse stomachs inoculated with the wild-type strain, with numbers ranging from 103 to 105 CFU/g of stomach. However, only 4 of 12 mice that were inoculated with the bcp mutant strain were found to harbor H. pylori in the 3-week colonization assay. In addition, the bacterial titers in these four H. pylori-positive stomachs were significantly lower than those in the stomachs colonized by the wild-type strain (see the legend to Fig. Fig.2).2). At 3 weeks, the statistical difference between the colonization abilities of the two strains was highly significant (P = 0.008). These results indicate that H. pylori BCP contributes to both the process of establishment and especially persistence within the host environment.

Thiol peroxidase activity of H. pylori BCP.

To examine the biochemical activity of BCP, the H. pylori bcp gene was cloned into pET21a, expressed in E. coli strain BL21 Origami, and purified to near homogeneity by using the His tag and nickel-nitrilotriacetic acid resin (Fig. (Fig.3,3, lane 2). The migration of purified six-His-tagged BCP on SDS-PAGE gel was in agreement with the protein's predicted molecular mass of 17 kDa plus 1 kDa for the six-His tag. The majority of the expressed protein present was soluble, although a small amount was contained in the membrane fraction (data not shown).

FIG. 3.
SDS-PAGE showing profiles of total proteins within H. pylori cells and purified six-His-tagged BCP. Five micrograms of cell extract from H. pylori wild-type strain SS1 (WT) and the isogenic ahpC or bcp mutant was loaded into each lane as indicated. Lane ...

A BCP homologue is present in many pathogenic bacteria, including E. coli, Haemophilus influenzae, H. pylori, and Mycobacterium tuberculosis (12). E. coli BCP has been characterized. It is a 1-Cys Prx with the Cys45 in the form of cysteine sulfenic acid (Cys-SOH) as a catalytic intermediate. E. coli BCP preferentially reduces LOOH rather than H2O2 or tBOOH, with the use of Trx as the immediate electron donor in the assays (12).

To investigate the peroxidase activity of H. pylori BCP, we measured its ability to reduce hydroperoxides with H. pylori Trx1 and TrxR provided as the reducing system. The proteins were mixed with NADPH along with different hydroperoxides, and the change in A340 (oxidation of NADPH) was monitored (Fig. (Fig.4).4). Peroxidase activity was expressed as absorbance units per minute. First, we tested whether the BCP alone was active in reducing peroxides by using NADPH. When the Trx1-TrxR proteins were omitted from the reaction mixture, no change in A340 was observed, indicating that the BCP activity of reducing peroxides is dependent on the thioredoxin system. In the absence of BCP (control), the Trx1-TrxR system alone had NADPH oxidation activity, as a steady decrease in A340 was observed. With all three proteins present, a higher rate of change in A340 was observed, demonstrating the activity of BCP. With 100 μM H2O2 or 100 μM tBOOH, the rate of NADPH oxidation increased from 0.03 absorbance U/min (for the control without BCP) to 0.08 absorbance U/min (for H2O2) or 0.10 absorbance U/min (for tBOOH). Therefore, the specific activities of BCP for 100 μM H2O2 and 100 μM tBOOH were 0.05 and 0.07 absorbance U/min, respectively (Fig. 4A and B).

FIG. 4.
Thiol peroxidase activity of H. pylori BCP. NADPH oxidation was monitored as the decrease in absorbance at 340 nm. The assay mixture in HEPES-NaOH (pH 7.0) buffer contained 0.2 mM NADPH, different concentrations of peroxides as indicated, 0.4 μM ...

As LOOH was shown to be a preferable substrate for E. coli BCP, we also sought to determine whether this is the case for H. pylori BCP. Due to an insolubility problem, the maximum concentration of LOOH that could be critically tested in this assay system was 20 μM. Thus, we compared the BCP activities by using 20 μM (each) H2O2, tBOOH, or LOOH. No significant activity of BCP was detected for 20 μM (each) H2O2 or tBOOH (i.e., the number of absorbance units per minute was not significantly higher than the control level) (Fig. 4C and D). However, in the presence of 20 μM LOOH, significant activity was observed; the rate of NADPH oxidation increased from 0.02 absorbance U/min (control without BCP) to 0.09 absorbance U/min (Fig. (Fig.4E).4E). This result suggested that BCP may be a lipid peroxide scavenger specifically for the structurally complex peroxide molecules, such as those with fatty acid side chains.

Functional relationship of AhpC, Tpx, and BCP.

AhpC, Tpx, and BCP are structurally and functionally related peroxiredoxins. In H. pylori, they are all thioredoxin-dependent thiol peroxidases. The reason for H. pylori's producing these proteins with apparently overlapping functions and what their relative contributions are to the bacterium's ability to resist oxidative stress are just beginning to be understood. Possibly these enzymes have different specificities toward different peroxides. In addition, these proteins may be expressed at different levels within the cell or at different stages of cell growth. The data presented here, together with the results from other studies, give us some initial insights toward understanding these questions.

H. pylori AhpC was shown to be capable of reducing different peroxides, including H2O2, tBOOH, and LOOH, with similar rate constants (3). Also, H. pylori Tpx was shown to be a scavengase for H2O2, with an activity similar to those of its homologues in other bacteria (23). All three peroxiredoxins have some ability to reduce H2O2. However, the main task of dissipating H2O2 in H. pylori is carried out by an abundant catalase (8, 9, 18). One proposed function of peroxiredoxins may involve the regulation of H2O2 signaling (25, 27). Our enzyme activity result for H. pylori BCP is in agreement with the observation for E. coli BCP that LOOH is the preferred substrate. Unsaturated fatty acids have been well documented to be a constituent of lipids in H. pylori (11). Nevertheless, growth of H. pylori displayed sensitivity to unsaturated free fatty acids due to their incorporation into phospholipids of the membrane, leading to membrane dysfunction (14). The growth inhibition was dependent on the degree of unsaturation, and one of the potent inhibitory fatty acids was linoleic acid (14). Thus, under the physiological (oxidative stress) condition, there may be a steady accumulation of damaging lipid hydroperoxides within H. pylori cells; this situation would require an abundant lipid peroxide reductase activity to remove the damaging organic hydroperoxides. In contrast, E. coli lacks the polyunsaturated fatty acids (4), and the major physiological substrate of E. coli AhpC was suggested to be H2O2 (19). However, results from our laboratory and others suggest that under physiological conditions the major function of H. pylori AhpC is to reduce organic hydroperoxides (3, 16; our unpublished results). Thus, H. pylori AhpC and BCP have apparently redundant functions in detoxifying lipid peroxides.

The E. coli bcp mutant displayed hypersensitivity to both H2O2 and organic peroxides, a phenotype like that of the E. coli ahpC mutant (12). In contrast, the H. pylori bcp mutant showed only moderate sensitivity to organic peroxides (and to paraquat) compared to the hypersensitivity of the ahpC mutant to all the oxidative agents tested (Table (Table11 and Fig. Fig.1).1). To explain this, we examined the profiles of protein expression in various strains by SDS-PAGE (Fig. (Fig.3).3). In the wild-type strain, the amount of AhpC protein accounts for more than 2% of the total separated cell proteins, based on the densitometric measurement of the protein bands. This is in agreement with the results of the proteome analysis by Jungblut et al. (13), which showed that AhpC (TsaA) is the third most abundant protein in H. pylori. In comparison, the expression level of BCP is very low, so that it is almost undetectable by SDS-PAGE. We reason that the H. pylori bcp mutant displayed a weak phenotype because of the presence of an abundant AhpC protein that also detoxifies peroxides. A double mutant strain with mutations in both ahpC and bcp would be expected to be highly susceptible to oxidative stress and in particular to lipid peroxide-mediated damage.

H. pylori infection induces an inflammatory response (gastritis) within the host, which leads to an increase in the level of toxic oxygen species in the gastric mucosa and the gastric juice (2, 10, 18). H. pylori is able to tolerate oxidative stress from the host immune response because it possesses an array of detoxification enzyme systems. Previous studies demonstrated an association of H. pylori antioxidant activities with host colonization proficiency (9, 17, 20, 24). Among the oxidative stress resistance gene mutants, sodB and ahpC mutants display the most severe phenotypes and are almost completely lacking ability to colonize the mouse stomach. The bcp mutant displays much lower sensitivity to oxidative stress agents in vitro than the ahpC mutant. Nevertheless, the mouse colonization result indicates that H. pylori BCP significantly contributes to the process of host colonization and in particular to persistence abilities. Elucidation of the regulation and expression patterns of AhpC, Tpx, and BCP in vivo is expected to provide more insights into the functional relationship among these peroxiredoxins in H. pylori and other pathogenic bacteria.


This work was supported by NIH grant 1-RO1-DK60061.

We thank Sue Maier for her expertise and assistance. We are particularly grateful to L. B. Poole of Wake Forest University School of Medicine, Winston-Salem, N.C., for providing the purified H. pylori Trx1 and TrxR proteins.


Editor: J. N. Weiser


1. Alm, R. A., L. S. Ling, D. T. Moir, B. L. King, E. D. Brown, P. C. Doig, D. R. Smith, B. Noonan, B. C. Guild, B. L. deJonge, G. Carmel, P. J. Tummino, A. Caruso, M. Uria-Nickelsen, D. M. Mills, C. Ives, R. Gibson, D. Merberg, S. D. Mills, Q. Jiang, D. E. Taylor, G. F. Vovis, and T. J. Trust. 1999. Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397:176-180. [PubMed]
2. Bagchi, D., G. Bhattachatya, and S. J. Stohs. 1996. Production of reactive oxygen species by gastric cells in association with Helicobacter pylori. Free Radic. Res. 24:439-450. [PubMed]
3. Baker, L. M., A. Raudonikiene, P. S. Hoffman, and L. B. Poole. 2001. Essential thioredoxin-dependent peroxiredoxin system from Helicobacter pylori: genetic and kinetic characterization. J. Bacteriol. 183:1961-1973. [PMC free article] [PubMed]
4. Bielski, B. H. J., R. L. Arudi, and M. W. Sutherland. 1983. A study of the reactivity of HO2/O2 with unsaturated fatty acids. J. Biol. Chem. 258:4759-4761. [PubMed]
5. Bryk, R., P. Griffin, and C. Nathan. 2000. Peroxynitrite reductase activity of bacterial peroxiredoxins. Nature 407:211-215. [PubMed]
6. Comtois, S. L., M. D. Gidley, and D. J. Kelly. 2003. Role of the thioredoxin system and the thiol-peroxidases Tpx and Bcp in mediating resistance to oxidative and nitrosative stress in Helicobacter pylori. Microbiology 149:121-129. [PubMed]
7. Dunn, B. E., H. Cohen, and M. J. Blaser. 1997. Helicobacter pylori. Clin. Microbiol. Rev. 10:720-741. [PMC free article] [PubMed]
8. Harris, A. G., F. E. Hinds, A. G. Beckhouse, T. Kolesniow, and S. L. Hazell. 2002. Resistance to hydrogen peroxide in Helicobacter pylori: role of catalase (KatA) and Fur, and functional analysis of a novel gene product designated ‘KatA-associated protein,’ KapA (HP0874). Microbiology 148:3813-3825. [PubMed]
9. Harris, A. G., J. E. Wilson, S. J. Danon, M. F. Dixon, K. Donegan, and S. L. Hazell. 2003. Catalase (KatA) and KatA-associated protein (KapA) are essential to persistent colonization in the Helicobacter pylori SS1 mouse model. Microbiology 149:665-672. [PubMed]
10. Hazell, S. L., A. G. Harris, and M. A. Trend. 2001. Evasion of the toxic effects of oxygen, p. 167-175. In H. L. T. Mobley, G. L. Mendz, and S. L. Hazell (ed.), Helicobacter pylori: physiology and genetics. ASM Press, Washington, D.C.
11. Hazell, S. L., and D. Y. Graham. 1990. Unsaturated fatty acids and viability of Helicobacter pylori. J. Clin. Microbiol. 28:1060-1061. [PMC free article] [PubMed]
12. Jeong, W., M.-K. Cha, and I.-H. Kim. 2000. Thioredoxin dependent hydroperoxidase activity of bacterioferritin comigratory protein (BCP) as a new member of the thiol-specific antioxidant protein (TSA)/alkyl hydroperoxide peroxidase C (AhpC) family. J. Biol. Chem. 275:2924-2930. [PubMed]
13. Jungblut, P. R., D. Bumann, G. Haas, U. Zimny-Arndt, P. Holland, S. Lamer, F. Siejak, A. Aebischer, and T. F. Meyer. 2000. Comparative proteome analysis of Helicobacter pylori. Mol. Microbiol. 36:710-725. [PubMed]
14. Khulusi, S., H. A. Ahmed, P. Patel, M. A. Mendall, and T. C. Northfield. 1995. The effects of unsaturated fatty acids on Helicobacter pylori in vitro. J. Med. Microbiol. 42:276-282. [PubMed]
15. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
16. Olczak, A. A., J. W. Olson, and R. J. Maier. 2002. Oxidative-stress resistance mutants of Helicobacter pylori. J. Bacteriol. 184:3186-3193. [PMC free article] [PubMed]
17. Olczak, A. A., R. W. Seyler, J. W. Olson, and R. J. Maier. 2003. Association of Helicobacter pylori antioxidant activities with host colonization proficiency. Infect. Immun. 71:580-583. [PMC free article] [PubMed]
17a. Olson, J. W., N. S. Mehta, and R. J. Maier. 2001. Requirement of nickel metabolism proteins HypA and HpyB for full activity of both hydrogenase and urease in Helicobacter pylori. Mol. Microbiol. 39:176-182. [PubMed]
18. Ramarao, N., S. D. Gray-Owen, and T. F. Meyer. 2000. Helicobacter pylori induces but survives the extracellular release of oxygen radicals from professional phagocytes using its catalase activity. Mol. Microbiol. 38:103-113. [PubMed]
19. Seaver, L. C., and J. A. Imlay. 2001. Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli. J. Bacteriol. 183:7173-7181. [PMC free article] [PubMed]
20. Seyler, R. W., Jr., J. W. Olson, and R. J. Maier. 2001. Superoxide dismutase-deficient mutants of Helicobacter pylori are hypersensitive to oxidative stress and defective in host colonization. Infect. Immun. 69:4034-4040. [PMC free article] [PubMed]
21. Storz, G., and M. Zheng. 2000. Oxidative stress, p. 47-60. In G. Storz and R. Hengge-Aronis (ed.), Bacterial stress responses. ASM Press, Washington, D.C.
22. Tomb, J. F., O. White, A. R. Kerlavage, R. A. Clayton, G. G. Sutton, R. D. Fleischmann, K. A. Ketchum, H. P. Klenk, S. Gill, B. A. Dougherty, K. Nelson, J. Quackenbush, L. Zhou, E. F. Kirkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H. G. Khalak, A. Glodek, K. McKenney, L. M. Fitzegerald, N. Lee, M. D. Adams, J. C. Venter, et al. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547. [PubMed]
23. Wan, X. Y., Y. Zhou, Z. Y. Yan, H. L. Wang, Y. D. Hou, and D. Y. Jin. 1997. Scavengase p20: a novel family of bacterial antioxidant enzymes. FEBS Lett. 407:32-36. [PubMed]
24. Wang, G., and R. J. Maier. 2004. An NADPH quinone reductase of Helicobacter pylori plays an important role in oxidative stress resistance and host colonization. Infect. Immun. 72:1391-1396. [PMC free article] [PubMed]
25. Woo, H. A., H. Z. Chae, S. C. Hwang, K. S. Yang, S. W. Kang, K. Kim, and S. G. Rhee. 2003. Reversing the inactivation of peroxiredoxins caused by cysteine sulfinic acid formation. Science 300:653-656. [PubMed]
26. Wood, Z. A., E. Schroder, J. Robin Harris, and L. B. Poole. 2003. Structure, mechanism and regulation of peroxiredoxins. Trends Biochem. Sci. 28:32-40. [PubMed]
27. Wood, Z. A., L. B. Poole, and P. A. Karplus. 2003. Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling. Science 300:650-653. [PubMed]
28. Zhou, Y., X. Y. Wan, H. L. Wang, Z. Y. Yan, Y. D. Hou, and D. Y. Jin. 1997. Bacterial scavengase p20 is structurally and functionally related to peroxiredoxins. Biochem. Biophys. Res. Commun. 233:848-852. [PubMed]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...