Oral Microbiology Unit, Department of Oral and Dental Science, University of Bristol Dental School, Lower Maudlin Street, Bristol BS1 2LY, UK1
Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK2
Author for correspondence: Howard F. Jenkinson. Tel: +44 117 928 4358. Fax: +44 117 928 4313. e-mail: howard.jenkinson{at}bris.ac.uk
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ABSTRACT |
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Keywords: manganese transport, oral streptococci, peroxide, reactive oxygen species
Abbreviations: NBT, nitro-blue tetrazolium; ROS, reactive oxygen species; SOD, superoxide dismutase
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INTRODUCTION |
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It is suggested that anions do not directly degrade DNA or polyunsaturated lipids to a significant extent, but that
-mediated cellular damage arises primarily by the release of Fe from proteins or chelates (Touati, 2000
). This can facilitate Fenton-type reactions, in which the oxidation of metal ions, such as Fe2+ by hydrogen peroxide (H2O2), releases highly reactive hydroxyl radicals (HO·). Many mitis-group streptococci produce relatively large amounts of H2O2 during aerobic growth by the action of oxidase enzymes, e.g. NADH oxidase (Nox) and pyruvate oxidase (SpxB), the latter of which is up-regulated in response to O2 (Auzat et al., 1999
; Pericone et al., 2000
). The expression of sodA is also enhanced under oxidative stress in some streptococci (Gibson & Caparon, 1996
; Yesilkaya et al., 2000
). However, relatively little is known about the function or regulation of streptococcal peroxidase enzymes, which inactivate H2O2 directly. In silico analysis of the Streptococcus pyogenes genome revealed genes encoding glutathione peroxidase (GpoA) and alkyl hydroperoxide reductase (AhpC) that were found to be involved in protection against
and organic peroxides (King et al., 2000
). In mitis-group streptococci (Whiley & Beighton, 1998
), including Streptococcus gordonii, Streptococcus parasanguis and Streptococcus pneumoniae, orthologous genes encoding proteins with about 50% amino acid sequence identities to Escherichia coli thiol peroxidase (Tpx) have been identified (Fenno et al., 1995
; Dintilhac & Claverys, 1997
; Kolenbrander et al., 1998
). Purified Tpx from S. pneumoniae catalyses the degradation of H2O2 and protects glutamine synthetase from H2O2-mediated inactivation (Wan et al., 1997
).
Manganese (Mn2+) plays a major role in oxidative stress tolerance in a number of different bacteria. For example, SOD activity in Streptococcus suis is strongly influenced by Mn2+ concentration in the growth medium (Niven et al., 1999 ). Also in Lactobacillus plantarum and Neisseria gonorrhoeae, which do not apparently produce MnSOD, Mn2+ can directly protect cells against ROS (Archibald & Fridovich, 1981
; Tseng et al., 2001
). In S. gordonii, which colonizes the oral cavity and nasopharynx, and can cause endocarditis (Douglas et al., 1993
), Mn2+ uptake under low Mn2+ (<0·5 µM) is mediated by an ATP-binding cassette (ABC)-type transporter, composed of ScaC (ATP-binding protein), ScaB (hydrophobic membrane protein) and ScaA (solute-binding lipoprotein) (Kolenbrander et al., 1998
). Expression of the Sca permease is regulated in response to Mn2+ by a metallorepressor protein ScaR (Jakubovics et al., 2000
). In this study we demonstrate that the Sca (Mn2+) permease is essential for oxidative stress tolerance in S. gordonii and that Mn2+ regulates expression of superoxide dismutase and thiol peroxidase activities.
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METHODS |
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The tpx gene was inactivated by allelic exchange with the erythromycin resistance determinant ermAM. PCR amplification with primers tpxf2/tpxr2 (5'-CGTCCGATGAAGACCGTTTC-3', 5'-CGCTGTAACCATCAATGCGG-3') of S. gordonii DL1 DNA template generated a 1190 bp fragment comprising the entire tpx gene (492 bp) and flanking sequences that was cloned into pGEM-T. A DNA fragment (1052 bp) containing the ermAM gene (GenBank accession no. AB057644) was PCR-amplified from plasmid pVA838 (Macrina et al., 1983 ) using primers ermf/ermr (5'-CCATATCATAAAAATCGATACAGC-3', 5'-CCTTATCGATACAAATTCCCCG-3') that contained ClaI restriction sites. The PCR product was digested with ClaI and ligated into a unique ClaI site within the cloned tpx gene, thus generating plasmid pGEM-tpx.ermAM (Table 1
). The insert DNA was excised with SalI, gel-purified and transformed into S. gordonii DL1 (to generate strain UB1313 tpx), and into S. gordonii UB1083 sodA (to generate double mutant UB1314 sodA tpx) (Table 1
). Confirmation of predicted insertions was obtained by appropriate PCR-amplification and sequencing of products, or by DNA blot hybridization.
Determination of SOD activity.
For visualization of SOD activity on non-denaturing polyacrylamide gels, S. gordonii cells were cultured to stationary phase in BHY medium and harvested by centrifugation at 5000 g for 10 min. Cells were washed once in 50 mM potassium phosphate buffer, pH 7·8, and suspended in 0·05 ml spheroplasting buffer (26%, w/v, raffinose, 10 mM MgCl2, 20 mM Tris/HCl, pH 6·8) (Demuth et al., 1996 ). Mutanolysin (25 U, Sigma) and PMSF (0·5 mM final concentration) were added and samples were incubated at 37 °C for 15 min. Cells were then broken by adding an equal volume of 0·10 mm glass beads (Sigma) and vortexing the suspensions vigorously for 2 min. Cell debris was pelleted by centrifugation at 12000 g for 2 min, and the cell-free supernatant was retained. Protein concentrations were determined by a modified Bradford assay (Bio-Rad), using bovine immunoglobulin as standard. Non-denaturing PAGE of proteins was performed by the method of Davies (1964)
. Gels were stained with Coomassie brilliant blue, or SOD activity was detected as described elsewhere (Beauchamp & Fridovich, 1971
). Briefly, gels were incubated at ambient temperature in 2·45 mM nitro-blue tetrazolium (NBT) solution for 20 min. This solution was replaced with riboflavin buffer (36 mM potassium phosphate, pH 7·8, containing 28 mM tetramethylethylenediamine and 28 µM riboflavin) for 15 min. Gels were then transferred into distilled water and exposed to light from a 15 W fluorescent lamp in a foil-lined box for 515 min. Gel images were digitized using an Astra 1220U scanner and intensities of bands were quantified with Kodak digital science 1D image analysis software. SOD activities in S. gordonii cell extracts were quantified using the McCordFridovich assay (McCord & Fridovich, 1969
). Early exponential-phase cells (OD600
0·3) were harvested by centrifugation (5000 g for 10 min) and washed in PE buffer (26·5 mM KH2PO4, 0·33 M K2HPO4, 0·1 mM EDTA, pH 7·8). Bacteria were then suspended in spheroplasting buffer and cell-free extracts prepared as described above. Samples were stored on ice and SOD activities were determined from triplicate readings within 2 h of extract preparation. Specific SOD activities per mg protein were calculated as means from between five and seven independent cultures, where 1 U was defined as the amount of SOD required to reduce the initial rate of change of absorbance at 550 nm (
A550) by 50% (McCord & Fridovich, 1969
).
Determination of growth and kill rates.
To assess the effects of atmospheric conditions on the growth of S. gordonii, cultures were incubated at 37 °C anaerobically, aerobically (static incubation) or under vigorous aeration. Susceptibilities to paraquat and to H2O2 were determined as follows. Exponential-phase cells (OD6000·3) were harvested by centrifugation at 5000 g for 10 min and suspended at a density of 1x109 cells ml-1 in 1% (w/v) Bacto-peptone (Difco) at 37 °C. Paraquat (final concentration 10 mM) or H2O2 (final concentration 25 mM) was added and the suspensions were incubated at 37 °C for 1 h. Samples were removed at time zero and at intervals over 60 min, serially diluted, plated onto BHYN agar and numbers of c.f.u. were determined following 36 h incubation at 37 °C. S. gordonii strains formed only short chains of cells (usually 24 cells per chain) and so sonication, often employed to break chains of cells, was not applied.
Northern analysis.
RNA was extracted from streptococcal cells as previously described (Jakubovics et al., 2000 ), separated by electrophoresis through 0·8% (w/v) agarose gels containing 3% (v/v) formaldehyde and transferred to Hybond-N+ membranes (Pharmacia). A probe comprising the internal coding region of sodA (418 bp) was generated by PCR amplification as described above. The primer pair tpxf1/tpxr1 (5'-CATCTAGAAGTAGGCGACACAGC-3', 5'-GCTATTGCCGGATCCTAGTCAGG-3') was employed to amplify an internal fragment (431 bp) of tpx (GenBank accession no. L11577). PCR products were labelled with 32P using Prime-a-Gene (Promega) and purified on NICK columns (Pharmacia). Hybridizations were performed for 16 h at 68 °C in Church & Gilbert (1984)
medium. Blots were washed twice in 2x SSC, 0·1% (w/v) SDS for 5 min at 20 °C and twice for 20 min in the same medium at 68 °C. Membranes were exposed to Fuji HR-E 30 X-ray film for 15 days and the intensities of the bands were quantified using Kodak digital science 1D image analysis software.
Statistical analyses.
All statistical analyses of data were performed by Students t-test using Microsoft Excel software. Values of P<0·05 were considered to be statistically significant.
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RESULTS |
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Relative roles of SOD and Tpx in detoxification of ROS
To determine more precisely the requirements for SOD and Tpx in protection against ROS, we generated an isogenic double mutant, UB1314 sodA tpx, as described in Methods. The effects of O2 on growth, and of paraquat and H2O2 on survival of wild-type or mutant strains were then tested. Wild-type cells entered stationary phase in batch culture at lower density when aerated as opposed to when grown anaerobically (Fig. 3). Inactivation of sodA or tpx, or of both genes in UB1314, did not affect growth of cells under anaerobic conditions (data not shown). However, the growth rate of UB1083 sodA (doubling time td=76·4±6·5 min) in aerated culture was significantly (P<0·01) less than that of the wild-type (td=44·8±3·3 min) (Fig. 3
). Although the growth rate of UB1313 tpx was not significantly different from that of DL1, the growth rate (td=81·3±9·5 min) and growth yield of double mutant UB1314 were severely attenuated (Fig. 3
). These data suggest that SOD is more important than Tpx for growth of S. gordonii in the presence of O2.
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SOD activity depends upon Mn2+ availability
To investigate the effect of Mn2+ on SOD in S. gordonii, enzyme activities in cell-free extracts from wild-type or mutant cells were measured (McCord & Fridovich, 1969 ). SOD activities in wild-type DL1 or UB1313 tpx cells grown statically in BHY medium were similar (Table 2
) and, as expected, SOD was undetectable in cell extracts of UB1083 sodA (Table 2
). SOD activity was significantly reduced (P<0·01) in a Sca permease-deficient mutant, PK3041 scaC (Table 2
) that is defective in uptake of Mn2+ (Kolenbrander et al., 1998
). Addition of 10 µM Mn2+ to BHY medium improved the growth yields of all strains, and there were concomitant increases in SOD activities for DL1, UB1313 and PK3041 (Table 2
). These data suggest that BHY medium does not satisfy fully the Mn2+ requirement for optimal growth of S. gordonii DL1.
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DISCUSSION |
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In streptococci, which do not produce catalase, protection against H2O2 may be afforded by peroxidase enzymes. In S. gordonii, the tpx gene, located immediately downstream of the sca Mn2+-permease operon (Kolenbrander et al., 1998 ), encodes a protein with significant sequence identity to E. coli Tpx. It appears that SOD and Tpx may form part of a co-ordinated response to oxidative stress, since the expression of both genes was up-regulated >10-fold under vigorous aeration of cultures. Tpx expression is also up-regulated by O2 in S. parasanguis (Spatafora et al., 2002
). A major role for S. gordonii Tpx appears to be in protection against H2O2. In aerated cultures the tpx mutant (and tpx sodA mutant) entered stationary phase at a lower cell density than the wild-type. It has been shown that inhibition of S. gordonii growth under these conditions is partly the result of accumulation of H2O2 (Barnard & Stinson, 1996
).
Our data suggest that a complex Mn2+-dependent regulatory system operates in the control of the oxidative stress response in S. gordonii. Under low Mn2+ there is reduced expression of sodA and of tpx, but the enzymes are sufficiently active for normal growth, even under conditions of mild oxidative stress. The mechanism by which Mn2+ influences transcription of these genes is unknown, but it is unlikely to involve the metallorepressor protein ScaR (Jakubovics et al., 2000 ) since transcription of sodA and tpx in scaR mutants is still Mn2+-responsive (data not shown). It is possible that decreased sodA expression under low Mn2+ may benefit cells by co-ordinating SOD production with the amount of Mn2+ available for enzyme activation. Alternatively, SOD may act as an intracellular store for Mn, in which case up-regulation of sodA expression under high Mn2+ might regulate the concentration of free intracellular Mn2+ cations. In contrast, there is no evidence that Tpx binds Mn2+ and peroxidase activity is dependent upon oxidation and reduction of one or more active site cysteine residues (Zhou et al., 1997
).
In other Gram-positive bacteria more details are known about the regulatory networks that co-ordinate Mn2+-sensing and ROS-sensing pathways. In B. subtilis, several peroxide stress genes are repressed by PerR, a metalloprotein that is activated by Mn2+ or Fe2+ cations and is susceptible to H2O2-inactivation, specifically in the Fe form (Bsat et al., 1998 ; Herbig & Helmann, 2001
). Staphylococcus aureus PerR controls the expression of at least eight genes (Horsburgh et al., 2001
). In S. pyogenes, PerR is essential for induced resistance to peroxide (King et al., 2000
), although it does not apparently regulate expression of ahpC, encoding alkyl hydroperoxide reductase. It is possible therefore that a PerR orthologue may be involved in co-ordinating gene regulation in response to metal ions and ROS in S. gordonii. We are currently investigating this hypothesis with the aim of identifying PerR-regulated genes in S. gordonii and their respective regulatory promoter sequences.
For S. gordonii cells subjected to aerobic stress (in aerated culture) the Mn2+ requirement must be substantially increased to provide for the large amounts of SOD enzyme being produced. Thus if Mn2+ homeostasis is disrupted, then aerobic growth will be compromised. It is clearly evident that in low Mn2+ (<0·1 µM) environments, such as are reported to be present in serum and peripheral tissues (Krachler et al., 1999 ), the Sca permease is required to provide for Mn2+ homeostasis. In the absence of the permease there is no other mechanism in S. gordonii that will provide sufficient Mn2+ for growth of the cells under aerobic or other oxidative stress conditions. These observations would also suitably account for the defects in virulence of Mn2+ permease mutants of Streptococcus mutans, S. parasanguis and S. pneumoniae (Burnette-Curley et al., 1995
; Berry & Paton, 1996
; Kitten et al., 2000
; Tseng et al., 2002
). Identifying the critical genes and proteins involved in oxidative stress tolerance in these organisms may provide new targets for anti-streptococcal therapies.
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ACKNOWLEDGEMENTS |
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Received 22 May 2002;
accepted 9 July 2002.