Department of Biology, Middlebury College, Middlebury, VT 05753, USA1
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405, USA2
Author for correspondence: Grace Spatafora. Tel: +1 802 443 5431. Fax: +1 802 443 2072. e-mail: spatafor{at}middlebury.edu
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ABSTRACT |
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Keywords: peroxidase, oral streptococci, oxidative stress
Abbreviations: GS, glutamine synthetase; MCO, metal-ion-catalysed oxidation; SOD, superoxide dismutase; TpxEc, periplasmic thiol peroxidase of Escherichia coli; TpxSp, putative streptococcal thiol peroxidase
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INTRODUCTION |
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FimA belongs to the family of LraI proteins that are known to play a dual role in adhesion and metal ion transport (Dintilhac & Claverys, 1997 ; Dintilhac et al., 1997
; Fenno et al., 1995
; Janulczyk et al., 1999
; Kolenbrander et al., 1998
; Spellerberg et al., 1999
). In S. parasanguis FW213, the fimA gene is preceded by an ATP-binding protein sequence (fimC) and a transmembrane transporter sequence (fimB) (Fenno et al., 1995
). This organization constitutes an ABC-transporter system on the S. parasanguis chromosome, a system which is highly conserved in a variety of other microbial pathogens. For instance, the Streptococcus gordonii scaA and Streptococcus pneumoniae psaA genes, both located within ABC-transport systems, encode homologous lipoproteins that function in Mn2+ transport (Dintilhac et al., 1997
; Kolenbrander et al., 1998
). An LraI protein described in S. pyogenes belongs to a family of ABC transporters having specificity for zinc and iron (Janulczyk et al., 1999
). Moreover, metal-ion-uptake experiments support a role for FimA in S. mutans iron transport, and expression studies support the iron-mediated regulation of the fimA gene product in this oral pathogen (Spatafora et al., 2001
).
The possible transport of iron via the S. parasanguis FimA lipoprotein would ultimately give rise to toxic hydroxyl radicals in the presence of molecular oxygen by means of Fenton and HaberWeiss reactions. Specifically, the incomplete reduction of oxygen in bacterial cultures grown under aerobic conditions generates oxygen radicals that can react with bacterial nucleic acids, lipids and proteins, thereby promoting their degradation (Stadtman & Oliver, 1991 ). To prevent the lethal effects of such metal-ion-catalysed oxidation (MCO), bacterial cells have evolved protective mechanisms to neutralize the formation of toxic oxygen radicals. For instance, small molecule antioxidants, such as catalases and peroxidases, have been reported to play protective roles in the enteric bacteria (Cha et al., 1995
, 1996
; Kim et al., 1996
), in Pseudomonas sp. (Hassett et al., 1996
) and in Bacteroides sp. (Rocha et al., 1996
). In contrast, streptococci lack cytochromes and catalase and some streptococcal species produce relatively low levels of superoxide dismutase (SOD) (Zitzelsberger et al., 1984
) despite their ability to be aerotolerant. This suggests the existence of other enzymes that are important for aerobic streptococcal growth.
An ORF (ORF3) located immediately downstream of fimA on the S. parasanguis chromosome (Fenno et al., 1995 ) encodes a protein of 218 aa (20 kDa). Reports in the literature reveal 47% aa identity between the ORF3 sequence and a periplasmic thiol peroxidase in Escherichia coli (TpxEc) (Cha et al., 1995
, 1996
). Similar ORFs that encode gene products of approximately 20 kDa are located downstream of other streptococcal metal-ion-transporter operons, including Streptococcus sanguis SsaB (Ganeshkumar et al., 1993
), S. gordonii ScaA (Kolenbrander et al., 1998
) and S. pneumoniae PsaA (Sampson et al., 1994
). Cysteine residues at amino acid position 81 in TpxEc, and which are present in other Gram-negative thiol peroxidases, form putative disulfide bridges with a second cysteine residue that is perfectly conserved at position 94. Examination of the S. parasanguis ORF3 amino acid sequence (accession no. P31307) confirms the presence of a cysteine residue at position 94, which is also present in S. sanguis and S. gordonii Tpx homologues (Cha et al., 1995
). Site-directed mutagenesis of the disulfide bond in TpxEc abolishes thiol peroxidase activity (Chae et al., 1997
). This suggests that the cysteine residues located at positions 61 and 94 in the S. parasanguis ORF3 sequence may constitute a similar functional group. The present study was undertaken to determine if ORF3 in S. parasanguis functions as a thiol-dependent antioxidant, as reported for the E. coli orthologue.
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METHODS |
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Nucleic acid isolation.
Plasmid DNA was isolated from E. coli using Qiagen mini-prep spin columns in accordance with the recommendations of the supplier. Total RNA was isolated from S. parasanguis as described previously (Fenno et al., 1995 ).
Transformation of competent E. coli.
Competent E. coli tpx cells were incubated on ice in 10 mM CaCl2 and transformed with plasmid pVT828 carrying S. parasanguis ORF3, according to established protocols (Sambrook et al., 1989 ). Transformants were selected on L-agar supplemented with ampicillin (100 µg ml-1) and were confirmed by restriction enzyme mapping of plasmid DNA, isolated as described above.
Northern blotting.
Total RNA was resolved on 0·8% formaldehyde/agarose gels (in 20 mM MOPS) which were run overnight at 40 V. The RNAs were transferred onto nylon membranes (Schleicher and Schuell) according to established protocols (Sambrook et al., 1989 ) and cross-linked in a FisherBrand FB-UVXL-1000 cross-linker.
32P-dATP (New England Nuclear) was used to radiolabel a 280 bp BclI gene probe, which is internal to the S. parasanguis ORF3 sequence, by nick-translation (Gibco). Primers SMrRNA-F (5'-GCAGGCGGTCAGGAAAGT-3') and SMrRNA-R (5'-GAGATTAGCTTGCCGTCA-3') were used to PCR amplify a 0·7 kb 16S rDNA probe that served as an internal control in these experiments. The blots were hybridized at 60 °C in a solution containing 1% BSA, 7% SDS, 1 mM EDTA and 0·25 M Na3PO4.12H2O (pH 7·0) for 16 h and then washed in a solution containing 7% SDS, 1 mM EDTA and 0·25M Na3PO4.12H2O (pH 7·0). They were then exposed overnight to X-ray film (Kodak XAR) at -80 °C with an intensifying screen.
Preparation of bacterial cell lysates.
Bacterial cells (35 g wet wt) were harvested by centrifugation at 6000 g for 5 min at 4 °C. The pellets were washed three times in ice-cold breaking buffer (0·1 M KH2PO4, pH 7·2) and stored as dry pellets overnight at -80 °C. The pellets were thawed at room temperature and resuspended in 1015 ml of cold breaking buffer containing 200 mg DNase and RNase. One-third volume zirconium beads (0·1 mm) was added to the cell suspension, which was then incubated on ice for 15 min. The cells were mechanically disrupted in a Braun homogenizer for a total of 6 min, with intermittent CO2 cooling. Breakage was monitored by light microscopy on a Zeiss phase-contrast microscope. The resulting cell lysates were incubated on ice for 5 min to allow the zirconium beads to settle. The supernatant was then transferred to 30 ml Corex tubes and centrifuged at 20000 g for 20 min. The pellet was discarded and the supernatant transferred to a fresh tube for further clarification by centrifuging for an additional 20 min at 20000 g. Proteins from cell lysates were concentrated using Centricon concentrators (Amicon) and protein determinations were performed with a BCA protein assay kit (Pierce), according to the manufacturers recommendations, using BSA as a standard.
SDS-PAGE and Western blot analysis.
Crude protein extracts derived from E. coli JC7623, a JC7623 tpx mutant, or S. parasanguis FW213 were loaded onto 515% SDS-PAGE gradient gels (100 µg lane-1) and subsequently transferred onto nitrocellulose membranes in a Hoeffer transfer apparatus. Immunoblots were performed as described by Towbin et al. (1979) , using a polyclonal antiserum directed against the 20 kDa ORF3 protein from S. parasanguis (Fenno et al., 1995
) or against the 20 kDa thiol peroxidase (Tpx) from E. coli (Cha et al., 1995
). A peroxidase-conjugated goatanti-rabbit anti-IgG antibody (Sigma) was subsequently applied to the membrane; reacting proteins were visualized using enhanced chemiluminescence according to the recommendations of the supplier (Amersham).
Glutamine synthetase (GS) protection assays.
An assay mixture (23 ml) containing 150 mM L-glutamine (Sigma), 40 mM NH2OH, 50 mM 3,3'-dimethylglutarate, 400 µM ADP, 300 µM KCl and 20 mM KAsO4 was prepared. The solution was adjusted to pH 7·6 with triethanolamine and HCl, after which 1 ml 0·01M MnCl2 was added. The mixture was then dispensed into 1·2 ml portions and warmed to 37 °C.
Separate reaction tubes containing 0·2 M imidazole/HCl (pH 7·0), 0·2 M GS, 1 mg of E. coli or S. parasanguis protein lysate and 40 µl of an inactivation solution containing 50 µM FeCl3 and 100 mM DTT were prepared. The final volume in all of the tubes was brought to 100 µl with sterile dH2O and the solutions were incubated at 37 °C for 15 min. Following inactivation, the reaction and assay mixtures were combined, incubated for 15 min and then transferred to borosilicate glass tubes containing 2 ml stop solution (0·12 M trichloroacetic acid and 0·12 M FeCl3). A colour change was monitored spectrophotometrically at A540 to reveal the production of -glutamyl hydroxamate. The active GS control was exposed to the assay mixture, but not to the inactivation mixture, whereas the inactive GS control was exposed to the inactivation mixture in the absence of cell lysate.
H2O2 filter disk assay.
Filter disks saturated with 0·5 M H2O2 were placed onto THA plates that had been seeded with 105 c.f.u. ml-1 of the S. parasanguis fimA mutant (VT930), the fimA tpx double mutant (VT929) or the FW213 wild-type progenitor. After overnight growth in a GasPak anaerobic jar (510% CO2), zones of inhibition surrounding each filter disk were measured to assess bacterial sensitivity to H2O2.
Co-precipitation studies.
S. parasanguis FW213 cell lysates derived from cultures grown in THB to mid-exponential phase (OD540 0·5) were prepared as described above and immunoprecipitated with polyclonal rabbit anti-FimA or anti-TpxSp antisera diluted 1:500 in PBS. Immunoprecipitate reaction mixtures were incubated in 2 ml microcentrifuge tubes at 4 °C for 3 h, with gentle agitation. The reactions were then washed repeatedly in PBS and the immunoprecipitates were pelleted by centrifugation for 1 min at 10000 g. The precipitated proteins were resolved on 515% non-denaturing gradient gels and transferred onto nitrocellulose membranes for subsequent immunoblotting with anti-FimA or anti-TpxSp antisera, as described above.
Statistical analysis.
Students t-tests were applied to the analysis of GS and H2O2 disk assays using an SPSS statistical analysis software package. For E. coli and S. parasanguis protection assays, two independent experiments were performed in duplicate. For each of four independent H2O2 disk assays, the results of five replicates were averaged prior to analysis. Values are reported as the means plus or minus the standard deviations. In all cases, a 95% confidence interval was assumed for statistical significance.
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RESULTS |
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Western blots of E. coli and S. parasanguis protein extracts were reacted with a rabbit polyclonal antiserum directed against the 20 kDa ORF3 protein from S. parasanguis (TpxSp) or against TpxEc (Fig. 1). Immunoblots reacted with the former confirmed the presence of a 20 kDa protein in S. parasanguis cytosolic extracts and revealed that the protein was efficiently expressed in the E. coli tpx- mutant that had been transformed with the S. parasanguis ORF3 gene contained on pVT828. However, the TpxSp antiserum did not react with the 20 kDa TpxEc protein in E. coli JC7623 tpx+ or in the JC7623 tpx mutant. Minor bands in these lanes were the result of cross-reactivity of the secondary antiserum with proteins in the E. coli cell extract.
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TpxSp has antioxidant activity in E. coli
To guard against reactive oxygen species, micro-organisms have evolved a variety of antioxidant defence mechanisms that include enzymes that reduce or eliminate free radicals, such as superoxides and peroxides. Thiol-specific antioxidants protect enzymes, including GS, from oxidative inactivation by decomposing H2O2 and preventing the formation of reactive oxygen species during MCO of thiol compounds. Given the similarities between TpxEc and TpxSp we decided to test their thiol-dependent antioxidant activities in a GS protection assay, as described by Stadtman & Oliver (1991) . In this system, GS is oxidized and inactivated by a hydroxyl radical that is formed as ferrous iron catalyses the thiol-dependent generation of H2O2. Importantly, protection in the E. coli tpx mutant was significantly less than that in the JC7623 wild-type progenitor (P<0·05) (Fig. 2
) and transformation of the E. coli tpx mutant with the S. parasanguis tpx gene complemented the peroxidase-deficient host, restoring GS protection to near wild-type levels. We presume endogenous GS is responsible for the greater levels of protective activity in E. coli cell lysates relative to the active GS control.
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DISCUSSION |
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Peroxidases are widely distributed among plants, animals and micro-organisms, where they perform essential roles in metabolism. The studies presented here demonstrate that TpxSp is necessary for the aerobic metabolism of S. parasanguis. Our hypothesis that TpxSp activity functions to nullify the toxic effects of peroxide radicals in S. parasanguis is supported by in trans complementation of an E. coli tpx mutant transformed with the S. parasanguis tpx gene (Fig. 2). Specifically, the 20 kDa tpxSp gene product protected GS activity from metal-ion catalysed protein oxidation in the E. coli mutant at levels similar to those of the wild-type. Moreover, protection of GS was significantly compromised in S. parasanguis cell extracts prepared from a fimA tpx double mutant (VT929) relative to a mutant that harbours a single mutation in the fimA gene (VT930) (Fig. 3
). In addition, greater zones of clearing were noted for the double mutant in H2O2 disk assays (Table 2
). These observations lend further support to a role for TpxSp in protecting S. parasanguis from oxidative stress.
The remarkable sequence identity that is shared between TpxSp and other bacterial thiol peroxidases includes a pair of cysteine residues, one of which is perfectly conserved at position 94 and comprises part of the functional group for thiol peroxidase activity (Cha et al., 1996 ). The specific mechanism(s) by which TpxSp protects S. parasanguis from the MCO of the protein may therefore be similar to those described for other thiol-specific antioxidants (Chae et al., 1997
). For instance, the enzyme may work to catalyse a reaction between a thiol donor (reducing agent) and the peroxide radical. Alternatively, the thiol donor may reduce the disulfide bond between adjacent cysteine residues to form two thiol groups. The dithiol may then reduce peroxides to form non-toxic by-products. Preliminary studies conducted in our laboratory support the former mechanism for antioxidation by TpxSp (data not shown). Specifically, upon substituting DTT in GS protection assays with Tris(2-carboxyethyl)phosphine, a compound which reduces disulfide bonds to dithiol groups (Pierce), protection of GS activity was nearly abolished in E. coli tpx mutants transformed with the S. parasanguis tpx gene. This suggests that the active protein, in its oxidized state, catalyses the rapid detoxification of peroxides by other reducing agents.
Reports in the literature reveal 47% identity between the protein sequences of TpxSp and TpxEc (Cha et al., 1996 ). In the present study, however, Western blot analysis of S. parasanguis whole-cell lysates revealed no detectable reactivity with the TpxEc antiserum and there was no reactivity between the TpxSp antiserum and cell lysates derived from E. coli JC7623 (Fig. 1
). These data suggest that the TpxSp protein is not recognized by the TpxEc antiserum and vice versa, owing to different antigenic epitopes on the two strains or epitope masking during electrophoresis.
The results of Northern hybridization experiments (Fig. 4) revealed a 0·5 kb tpx-specific transcript that is expressed in S. parasanguis cultures grown aerobically, but which is not expressed when grown anaerobically. At first, this was surprising given that the H2O2-sensitivity data support a tpx-specific protective response in S. parasanguis cells grown anaerobically. In fact, these findings are consistent with the results of Northern hybridization experiments conducted in our laboratory with S. mutans RNA isolated from cultures grown in the presence of H2O2 under conditions of strict anaerobiosis. These studies revealed that expression of a S. mutans peroxidase is induced by H2O2 in the absence of oxygen (data not shown). Thus, we propose that H2O2 can induce expression of the S. parasanguis tpxSp gene under similar growth conditions. These experiments also revealed transcription of tpxSp as part of a 3·3 kb polycistronic mRNA which is derived from the upstream fimA promoter under aerobic conditions (Fenno et al., 1995
), indicating that Fe2+-sensing may be linked to the oxidative stress response in S. parasanguis. This is consistent with a role for Mn2+ in the readthrough transcription of the psaD thiol peroxidase in S. pneumoniae (Novak et al., 1998
). In contrast, there is no evidence to support regulation of the S. gordonii thiol peroxidase by Mn2+ since Northern blots do not reveal co-transcription of the tpx gene and the upstream permease operon (Jakubovics et al., 2000
).
Repeated attempts to generate a knockout mutation in the S. parasanguis tpx gene were unsuccessful when the allelic exchange was selected under aerobic or anaerobic conditions. S. parasanguis VT929, a mutant which harbours mutations in both fimA and tpx, is viable under these growth conditions, however, so we hypothesized a possible interaction between the fimA and tpxSp gene products. In fact, the results of co-precipitation studies did not support a direct interaction between these two proteins (Fig. 5). An alternative hypothesis was derived from the results of metal-ion-uptake assays conducted in our laboratory, which implicate the 36 kDa S. parasanguis fimA gene product in iron transport (unpublished data). Specifically, we propose that the FimA metal-ion transporter contributes to oxidative stress by generating hydroxyl radicals capable of causing irreparable damage to the cell via the Fenton reaction. A fimA mutation would therefore alleviate the MCO of bacterial nucleic acids, lipids and proteins, and so protect S. parasanguis from the effects of toxic oxygen radicals. Such protection imparted by a fimA mutation is consistent with the results of GS and H2O2 disk assays, which indicate that VT930 is less sensitive to oxidative stress than the FW213 wild-type progenitor (Fig. 3
and Table 2
). Thus, S. parasanguis tpx mutants may be non-viable when grown aerobically owing to the oxidative stress that is derived from FimA-mediated iron uptake coupled with the absence of a functional peroxidase. The inability to isolate such a mutant under conditions of strict anaerobiosis, however, suggests that tpx inactivation may also be lethal for reasons unrelated to aerobic conditions and oxidative stress.
In summary, we report that the 20 kDa tpx gene product in S. parasanguis functions as a protective antioxidant and that the results of enzymic analyses are consistent with a specific thiol-peroxidase-like activity for this protein. Since a direct assay for thiol-dependent peroxidase activity is unavailable, site-directed mutagenesis experiments are planned to confirm the putative involvement of the cysteine residues in S. parasanguis antioxidation. That peroxidases appear to be present in a variety of microbial pathogens supports their role in providing protection from the peroxide-mediated oxidative stress imposed by infectious processes. Studies aimed at investigating the relationships between metal-ion availability, metal-ion binding and transport by fimA, and S. parasanguis peroxidase activity will elucidate the regulatory mechanism(s) that are likely to govern this series of complex interactions between host and pathogen.
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ACKNOWLEDGEMENTS |
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Received 7 August 2001;
revised 28 October 2001;
accepted 2 November 2001.