Department of Molecular Biology & Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK
Correspondence
David J. Kelly
d.kelly{at}sheffield.ac.uk
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ABSTRACT |
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Abbreviations: GSNO, S-nitrosoglutathione; SNP, sodium nitroprusside
Present address: Microscience Ltd, 545 Eskdale Road, Winnersh Triangle, Wokingham, Berkshire RG41 5TU, UK.
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INTRODUCTION |
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Oxidative and nitrosative stress resistance are key properties that enable pathogenic bacteria to survive the effects of the production of reactive oxygen and nitrogen species by the host (Storz & Zheng, 2000). H. pylori seems well equipped to deal with peroxide stress, as it contains an active catalase and a number of peroxidases, including a periplasmic cytochrome c peroxidase (Tomb et al., 1997
; Kelly, 1998
; Alm et al., 1999
). Interestingly, it possesses several enzymes of the peroxiredoxin family which may play key roles. The best studied of these enzymes is alkyl-hydroperoxide reductase (AhpC), which was proposed to be essential for viability (Baker et al., 2001
). However, ahpC mutants can be isolated if mutant selection is performed under low oxygen tensions (Olczak et al., 2002
). The AhpC enzyme probably has several roles in protecting against oxidative stress by reducing a variety of organic peroxides produced during oxidative damage and, by analogy with E. coli (Costa Seaver & Imlay, 2001
), may be a major contributor to hydrogen peroxide disposal at concentrations too low to be dealt with efficiently by catalase. AhpC may also be important in protecting H. pylori against the damaging effects of nitric oxide (NO). NO is a bactericidal agent produced in the host by macrophages and other cells, which can combine with superoxide to form peroxynitrite (ONOO-), an extremely toxic and reactive nitrogen species (Hughes, 1999
). H. pylori AhpC can reduce peroxynitrite to the relatively harmless nitrite (Bryk et al., 2000
), at least in vitro. At least two other peroxidases are encoded in the genome of H. pylori. One, encoded by HP390 in strain 26695, is a thiol-peroxidase that was originally identified by Wan et al. (1997)
as scavengase p20 and is divergently transcribed from the superoxide dismutase gene (sodB). The other enzyme is encoded by HP0136 in strain 26695 and is a homologue of the E. coli bacterioferritin co-migratory protein (Bcp), now known to be a thiol-peroxidase (Jeong et al., 2000
).
AhpC, Tpx and Bcp are all members of the peroxiredoxin family (Schröder & Ponting, 1998; Jeong et al., 2000
), which use reduced thioredoxin as an electron donor for the catalytic reduction of their respective substrates. H. pylori possesses two distinct thioredoxins, Trx1 and Trx2 (Windle et al., 2000
; Baker et al., 2001
), encoded by HP0824 and HP1458, respectively, in strain 26695. A thioredoxin reductase is encoded by trxB (HP0825 in strain 26695; Tomb et al., 1997
). The H. pylori Trx1 thioredoxin has been characterized biochemically (Windle et al., 2000
). Experiments with the purified proteins in vitro have shown that Trx1 (but not Trx2) acts as the electron donor to AhpC (Baker et al., 2001
). In view of the fact that AhpC plays a critical role in the H. pylori oxidative stress response, it would be predicted that Trx1 is also of central importance, but this and the possibility that Trx2 or another reductant can substitute in vivo has not been investigated. Indeed, the normal physiological role of Trx2 is unclear, as are the functions of the additional peroxidases known to be encoded in the genome of H. pylori. In this study, we have constructed mutants in the trxA1, trxA2, tpx and bcp genes in order to determine the role of the thioredoxins and thiol-peroxidase proteins of H. pylori in contributing to the resistance of this bacterium to the effects of oxidative and nitrosative stress. Phenotypic analyses of these mutants show that both Trx1 and Trx2 have roles in oxidative and nitrosative stress resistance. tpx mutants were found to be more sensitive to killing by peroxide and superoxide (but not oxygen or NO) compared to the wild-type parent strain, while bcp mutants had a similar but much weaker phenotype.
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METHODS |
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Inactivation of H. pylori genes.
A set of PCR primers were designed to amplify the entire coding regions of the trxA1, trxA2, tpx and bcp genes for insertional activation after cloning of the products into pGEM T-easy or pGEM-3-Zf (Promega). The primers used were TrxA1C-F (5'-GCTCGAATTCACGATCCGCTGTTATT-3'), TrxA1C-R (5'-CGAGGAATTCTAACACCGCCTCTAGT-3'), TrxA1K-F (5'-CTGAAGAAGAAGCTTGCGGA-3'), TrxA1K-R (5'-CATCTGGCGCAAGTGCTAAC-3'), TrxA2(a)-F (5'-ATCACTGCAGGCTAATGCGGCTAATG-3'), TrxA2(a)-R (5'-GCACCTGCAGCGCCCTTTATGATACC-3'), TrxA2(b)-F (5'-ACGCAGAGAAAATCGCTCAT-3'), TrxA2(b)-R (5'-GCGTCTTCAATCGGTTTTTG-3'), Bcp-F (5'-GCCGGACAGCTGACATTGAA-3'), Bcp-R (5'-AGCGATCTTAGGGTTGTAGG-3'), Tpx-F (5'-CTCCTTGTGGATCAGATAGCC-3') and Tpx-R (5'-GCCAGAGACTTTGAGCGATA-3'). The TrxA1C and TrxA2(a) primers introduced EcoRI or PstI restriction sites (shown in bold italics) for the cloning of trxA1 and trxA2, respectively, into pGEM-3-Zf(-). PCRs were performed with Taq polymerase using genomic DNA from strain 26695 as a template. pGEM-3Zf(-) was linearized with EcoRI or PstI and ligated with the products resulting from PCR with the above primers after digestion with the same restriction enzymes, generating plasmids pSLC1 and pSLC4 (Table 1). The TrxA2(b) primers amplified an internal fragment of trxA2 and were used for mutant verification. The remaining primers were used for cloning into pGEM T-easy. All the resulting plasmids were linearized at unique restriction sites within the H. pylori gene inserts, as detailed in Table 1
. Protruding 5' termini were in-filled using the Klenow fragment of the E. coli DNA polymerase I. For insertional inactivation, a chloramphenicol acetyltransferase (cat) cassette or an aminoglycoside phosphotransferase gene (aphAIII) derived from Campylobacter coli (Wang & Taylor, 1990
; Pittman et al., 2001
) were used. The 749 bp cat cassette excised from pUCAT by HincII digestion was used to inactivate trxA1 and trxA2. The cassette was purified using a Gel Extraction Kit (Qiagen) and blunt-end ligated into pSLC1 and pSLC4, respectively. Plasmids pSLC7 and pSLC10 were generated (see Table 1
). To construct a plasmid suitable for generating a H. pylori trxA1 trxA2 double mutant, the aphAIII gene was amplified from pUKAN with Pfu polymerase, using primers Kan-F (5'-ACTGAGATCTACTCTATGAAGCGCCATATT-3') and Kan-R (5'-CAATAGATCTTTTAGACATCATCTAAATCTAGG-3'). pSLC3 was linearized at the unique BstXI site within the trxA2 gene, in-filled using the Klenow fragment and blunt-end ligated with the aphAIII gene. The resulting plasmid was designated pSLC9. Plasmids pSLC11 and pSLC12 were derived from pSLC5 and pSLC6 by insertion of aphAIII into the bcp and tpx genes, respectively.
Transformation of H. pylori 26695 was carried out with pSLC7, pSLC10, pSLC11 and pSLC12 to generate single gene mutants in trxA1 (strain SLC100), trxA2 (strain SLC200), bcp (strain SLC400) and tpx (strain SLC500), respectively. Strain SLC100 was transformed with pSLC9 to generate a trxA1 trxA2 double mutant (strain SLC300). H. pylori 1061 was transformed with pSLC8 to construct a non-polar trxA1 mutant. Natural transformations were carried out as described by Ferrero et al. (1992). Genomic DNA was extracted from putative recombinants, and correct insertion of the resistance cassettes was evaluated by PCR using the above primers.
Effect of oxidative and NO stress on cell viability.
Wild-type and mutant H. pylori strains were grown on Columbia blood agar plates for 4872 h, harvested and resuspended in BHI/FCS to an OD600 value of 1·0. Viable counts showed that this corresponded to about 107109 c.f.u. ml-1. Aliquots (2 ml) of this cell suspension were distributed to a series of 20 ml tissue-culture flasks, to which either no additions were made (control) or 50 mM H2O2, 10 mM methyl viologen (paraquat; a superoxide generator), 10 mM sodium nitroprusside (SNP; an NO+ donor) or 5 mM S-nitrosoglutathione (GSNO; an NO releaser and NO+ donor) were added. The flasks were incubated at 37 °C under microaerobic conditions with orbital shaking at 120 r.p.m. One flask with no additions was also incubated under fully aerobic conditions (21 %, v/v, O2) at 37 °C with orbital shaking at 250r.p.m. to determine the effect of oxygen stress. Periodically, samples were taken from the flasks, serially diluted in BHI and then plated onto blood agar. Colonies were counted after 96120 h incubation.
Cumene hydroperoxide disc-diffusion assay.
H. pylori was grown on a blood agar plate for 4872 h, after which time the growth was scraped off and resuspended in 1 ml BHI broth. Aliquots (100 µl) of this suspension were spread out onto fresh blood agar plates and sterile 5 mm diameter Whatman filter paper discs were placed in the centre of the plates. Five microlitres of a 10 % (v/v) cumene hydroperoxide solution in DMSO was pipetted onto the discs and the plates were incubated for 4872 h at 37 °C. The diameter of the zone of growth inhibition was measured. No growth inhibition was observed when DMSO alone was placed on the filter discs. Six replicate assays were performed for each strain, and the data were subjected to Student's t-test to evaluate their statistical significance.
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RESULTS |
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Growth characteristics of the trx, tpx and bcp mutants: thioredoxin deficiency results in growth inhibition by molecular oxygen
Strains SLC100 (26695 trxA1 : : cat), SLC200 (26695 trxA2 : : cat), SLC300 (26695 trxA1 : : cat trxA2 : : aphAIII) and SLC600 (1061 trxA1 : : aphAIII) all grew poorly on blood agar plates incubated under standard microaerobic conditions (with 10 %, v/v, O2), forming smaller colonies than the respective 26695 or 1061 parent strains. The nature of the growth defects in the 26695 thioredoxin mutants was determined in BHI/FCS liquid batch cultures incubated in gas atmospheres containing either 10 or 2 % (v/v) O2 (Fig. 2). The wild-type grew at a similar rate in either gas atmosphere (doubling time of
3 h), but reached slightly higher final cell densities when cultured under 10 % (v/v) O2 (Fig. 2a
). Strain SLC100 (trxA1 : : cat) showed an extended lag period and a significantly reduced exponential growth rate (doubling time of
6 h) at 10 % (v/v) O2 (Fig. 2b
), but cultures eventually reached the same final cell density as the wild-type parent strain (compare Fig. 2a, b
). However, when cultured with 2 % (v/v) O2 in the gas atmosphere, strain SLC100 grew as well as the wild-type (Fig. 2a, b
), even reaching slightly higher final cell densities. At 10 % (v/v) O2, strain SLC200 (trxA2 : : cat) showed a severe reduction in both the rate and extent of growth compared to its isogenic parent, but grew as well or better than the parent strain at 2 % (v/v) O2 (Fig. 2a, c
). As expected, the SLC300 trxA1 trxA2 double mutant showed a severe growth defect at 10 % (v/v) O2 but not at 2 % (v/v) O2 (Fig. 2d
), similar to that of strain SLC200. The data clearly show that both thioredoxin 1 and 2 are important for the normal microaerobic growth of H. pylori, with a deficiency of either resulting in an increased sensitivity to molecular oxygen. The isolation of strain SLC300, however, indicates that neither thioredoxin is absolutely essential for viability.
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Differential roles of thioredoxins 1 and 2 in protection against oxidative stress
Fig. 3 shows the effects of exposure to air, hydrogen peroxide or superoxide stress on the viability of cell suspensions of the wild-type and trx mutants, in comparison to control cells incubated under standard microaerobic conditions at 10 % (v/v) O2. H. pylori wild-type cells taken from 23-day-old plate cultures and resuspended to densities of 107109 c.f.u. ml-1 showed a biphasic loss of viability after exposure to 21 % (v/v) O2 in air, 50 mM H2O2 or 10 mM paraquat. An initial gradual decline was followed by a phase in which the cells lost viability much more rapidly (Fig. 3b
d). In contrast, incubation under microaerobic conditions resulted in maintenance of the initial level of viability in each of the strains for the duration of the experiment (Fig. 3a
). Strain SLC100 (trxA1) was killed much more rapidly than its isogenic parent by each of the oxidative stress treatments imposed (Fig. 3b
d) and was particularly sensitive to hydrogen peroxide killing (Fig. 3c
). Strain SLC200 (trxA2) also showed an increase in sensitivity to killing by oxidative stress, but this was much less pronounced than with SLC100. As expected from these results, strain SLC300 (trxA1 trxA2) was slightly more sensitive than SLC100 to loss of viability upon exposure to oxygen, hydrogen peroxide or paraquat. The data show that a deficiency in TrxA1 results in greater sensitivity of H. pylori to killing by several types of oxidative stress compared to TrxA2 deficiency.
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DISCUSSION |
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The H. pylori thioredoxins are likely to interact with many cellular proteins, but analysis of the effects of the trxA1 mutation fit in well with a major in vivo role for TrxA1 as the electron donor to AhpC. Like mutants in the ahpC gene (Olczak et al., 2002), the trxA1 mutant was more oxygen sensitive than the parent strain; it grew less well in 10 % (v/v) O2 compared to 2 % (v/v) O2 and cell suspensions were killed more rapidly than the parent strain after exposure to 21 % (v/v) O2. It also showed greater sensitivity to hydrogen peroxide, cumene hydroperoxide and the superoxide generator paraquat. Our results also demonstrate a particularly important role for Trx1 in resistance to nitrosative stress. NO reacts rapidly in aqueous solution with superoxide to form the extremely toxic peroxynitrite (Hughes, 1999
). Studies with purified AhpC from H. pylori have demonstrated that it can catalyse the reduction of peroxynitrite to nitrite (Bryk et al., 2000
) and hence detoxify this compound. An increased sensitivity of the trxA1 mutant to NO releasers would thus be expected if a major source of electrons for the reduction of peroxynitrite by AhpC was Trx1.
The role of Trx2 in oxidative stress management in H. pylori is less clear than with Trx1. It has been shown that, in vitro, Trx2 does not act as an electron donor to AhpC (Baker et al., 2001). Nevertheless, the trxA2 mutant constructed in this study was more sensitive to both oxidative and nitrosative stress than the parent strain. One dramatic effect of the inactivation of trxA2 was a much greater inhibition of growth at 10 % (v/v) O2 compared to that seen with the trxA1 mutant. This was particularly apparent in BHI liquid batch cultures, whereas growth on blood agar plates was affected less severely, possibly due to a protective effect of haem in removing reactive oxygen species. Some of the effects of the trxA2 mutation were more apparent in a trxA1 background. The two thioredoxins may be able to substitute for some functions in vivo, but it is likely that TrxA2 is acting as an electron donor for another reductase, peroxidase or peroxiredoxin that contributes to the removal of reactive oxygen or nitrogen species, in addition to the Trx1/AhpC system. Obvious candidates would be Tpx or Bcp, although these enzymes seem not to be important for NO detoxification as judged from the phenotypes of the cognate mutants. Studies with the purified proteins will be needed to clarify this.
In many bacteria, the thioredoxin system acts as the electron donor for ribonucelotide reductase (RNR; Jordan & Reichard, 1998). H. pylori contains only a single RNR, a heterodimeric oxygen-dependent class I type enzyme encoded by the nrdA and nrdB genes (Tomb et al., 1997
; Alm et al., 1999
). Reliance on this type of RNR would prevent H. pylori from growing strictly anaerobically, as is the case with its close relative Campylobacter jejuni (Sellars et al., 2002
). The activity of this enzyme is therefore essential for DNA synthesis and thus for cell viability under normal microaerobic conditions. Our finding that it was possible to isolate a trxA1 trxA2 double mutant must imply that the H. pylori RNR can be supplied with electrons from a source other than Trx1 or Trx2, which is interesting in view of the fact that the bacterium appears to be deficient in other reduction systems that might substitute for Trx, particularly glutathione and the glutaredoxin system (Tomb et al., 1997
; Alm et al., 1999
; Baker et al., 2001
). Although the double mutant clearly had a very severe deficiency in oxidative stress resistance, compensatory up-regulation of a Trx-independent antioxidant system may be able to compensate to some extent, as has been reported in the case of ahpC mutants of H. pylori in which expression of the iron-binding NapA protein is increased (Olczak et al., 2002
).
The role of the thiol peroxidase Tpx in oxidative stress resistance has also been investigated in this study. Wan et al. (1997) first identified the Tpx protein in H. pylori and the partially purified enzyme was shown to protect glutamine synthetase from oxidative inactivation and to possess thioredoxin-linked peroxidase activity, when assayed using E. coli Trx. Tpx is an abundant protein in H. pylori as judged by two-dimensional gel analyses (Jungblut et al., 2000
) and the tpx gene is divergently transcribed from sodB, encoding superoxide dismutase (Tomb et al., 1997
; Wan et al., 1997
). The increased sensitivity of the tpx mutant to hydrogen peroxide, cumene hydroperoxide and the superoxide generator paraquat confirms a significant in vivo role for Tpx as an antioxidant protein in H. pylori. That the tpx mutant did not show an increased sensitivity to killing by nitrosative stress indicates a specific role for Tpx in detoxifying reactive oxygen species, contrasting with AhpC, which appears to be a more general peroxiredoxin.
H. pylori also contains another member of the peroxiredoxin family, the Bcp protein encoded by HP0136 in 26695. This is a homologue of the E. coli Bcp protein, which was shown by Jeong et al. (2000) to be a thioredoxin-dependent thiol peroxidase with a preference for certain organic peroxides such as linoleic acid hydroperoxide. Unlike an E. coli bcp null-mutant, which had a prolonged lag phase in liquid batch cultures and a pronounced hypersensitivity to hydrogen peroxide and organic peroxides (Jeong et al., 2000
), the H. pylori bcp mutant constructed in this study had only a weak phenotype. A slight perturbation in growth characteristics (slightly slower growth rate) compared to the wild-type was reproducibly noted, but the mutant was not significantly more hydrogen peroxide sensitive and only slightly more superoxide sensitive than the parent strain. Thus, Bcp is not a major contributor to general oxidative stress resistance in H. pylori, but instead could have a more specific role, for example, in the removal of fatty acid hydroperoxides produced during metabolism or by oxidative stress.
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ACKNOWLEDGEMENTS |
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Received 23 July 2002;
revised 8 October 2002;
accepted 9 October 2002.