Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405, USA1
Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, MD, USA2
Author for correspondence: Keith P. Mintz. Tel: +1 802 656 4271. Fax: +1 802 656 8749. e-mail: kmintz{at}zoo.uvm.edu
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ABSTRACT |
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Keywords: adhesion, oxidative stress, periodontal disease
a The GenBank accession number for the msrA sequence reported in this paper is AY026361.
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INTRODUCTION |
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MsrA is a ubiquitous enzyme that is found in a variety of animal tissues and organisms (Brot et al., 1981 ; Kuschel et al., 1999
; Moskovitz et al., 1996a
). The presence of MsrA in a variety of tissues and organisms suggests that this protein has an important function in providing cells with a defence against oxidative stress. A role of MsrA in protecting cells against oxidative damage is supported by the decrease in viability of null mutants during oxidative stress (Dhandayuthapani et al., 2001
; Hassouni et al., 1999
; Moskovitz et al., 1995
, 1998
, 2001
; Vriesema et al., 2000
). Furthermore, the overexpression of MsrA in Escherichia coli, yeast and stable transfected human T cells provides higher resistance to hydrogen peroxide treatment (Moskovitz et al., 1995
, 1998
). In addition to a role in protection to oxidative damage, MsrA has been proposed to be a virulence factor for numerous micro-organisms. Inactivation of msrA reduces bacterial adhesion of Streptococcus pneumoniae to human cells and reduces type I fimbriae-mediated haemagglutination of red blood cells by enteropathogenic E. coli (Wizemann et al., 1996
). This enzyme has also been shown to be a virulence determinant in Erwinia chrysanthemi, Mycoplasma genitalium and Staphylococcus aureus (Dhandayuthapani et al., 2001
; Hassouni et al., 1999
; Singh et al., 2001
).
The oral pathogen Actinobacillus actinomycetemcomitans is implicated as the causative agent of localized juvenile periodontal disease as well as various forms of adult periodontitis and other non-oral infections (Asikainen & Alaluusua, 1993 ; Kaplan et al., 1989
; Slots et al., 1986
; Zambon, 1985
). Periodontal diseases are chronic inflammatory diseases that are mediated in part by virulence factors that are capable of direct tissue damage (Fives-Taylor et al., 1999
). In addition, the host also mediates much of the pathogenic effects of the bacteria through a multitude of immune mediators including neutralization of the bacteria by oxidative mechanisms (Zadeh et al., 1999
). Therefore, it was of interest to determine if msrA is present in the A. actinomycetemcomitans genome and if this enzyme contributes to the virulence of this pathogen. In the present study, the gene encoding MsrA of A. actinomycetemcomitans was identified and isolated. An isogenic mutant was developed by allele replacement mutagenesis and the functions associated with MsrA activity in other organisms were investigated.
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METHODS |
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Allele replacement mutagenesis.
A unique StuI restriction site within msrA was engineered by inverse PCR (Ochman et al., 1989 ) of the T/A cloning vector containing the 1·8 kb DNA fragment. The primers used will result in a loss of 500 bp of the coding region of msrA. The spectinomycin gene from plasmid pDL269 (LeBlanc et al., 1991
) was isolated and ligated with the inverse PCR product restricted with StuI (Mintz & Fives-Taylor, 2000
). The ligation mixture was transformed into E. coli JM109 cells by electroporation and plated on LB agar plates containing 50 µg spectinomycin ml-1. Spcr colonies were selected and the plasmids isolated using a rapid plasmid purification scheme (Berghammer & Auer, 1993
). The construct was confirmed by restriction mapping and PCR. The disrupted gene was released from the plasmid by digestion with EcoRI and ligated with the mobilizable plasmid pVT1460 restricted with EcoRI (Mintz et al., 2002
). Electrocompetent DH5
(
pir) cells were transformed with the ligation mixture and transformants were selected on LB agar containing 50 µg Spc ml-1. Plasmids were isolated and the construct was confirmed by PCR. Plasmid containing the disrupted gene was purified using Qiagen spin columns and transformed by electroporation into E. coli SM10(
pir) cells for conjugation. Mobilization of the plasmid containing the disrupted gene from E. coli SM10(
pir) to A. actinomycetemcomitans strain VT1169 (Rifr/Nalr) was accomplished by conjugation as described previously (Mintz & Fives-Taylor, 2000
). Bacteria were plated on TSBYE agar containing Nal, Rif and Spc (50, 100 and 50 µg ml-1, respectively) and incubated as described above for 34 days. Spectinomycin-resistant colonies were replica-plated on TSBYE plates containing 100 µg Kan ml-1. Bacteria that were Spcr, Kans were analysed by colony PCR to confirm the presence of mutant msrA. The allelic replacement of the genomic copy of msrA was confirmed by Southern blot analysis.
Plasmid construction for restoration of MsrA activity in A. actinomycetemcomitans.
A derivative of the A. actinomycetemcomitans replicating plasmid pPK1 (LeBlanc et al., 1993 ), pVT1503 was used to restore MsrA activity in the msrA mutant. The spectinomycin adenyltransferase (aad9) was deleted by incubation with KpnI and BamHI and then separated by agarose gel electrophoresis. The aminoglycoside phosphotransferase gene (kan) derived from pUC-4K (Pharmacia Biotech) previously subcloned into the SmaI site of pBluescript II SK (Stratagene), was isolated following restriction with KpnI and BamHI. The 1·1 kb DNA fragment containing kan was ligated with the pPKI plasmid backbone, transformed into E. coli JM109 cells and grown on LB agar plates containing 50 µg kanamycin ml-1. Transformants were confirmed by restriction analysis.
The msrA and flanking regions derived from amplification of chromosomal DNA from A. actinomycetemcomitans SUNY 465 (described above) was released from the T/A cloning vector by incubation with EcoRI and purified by gel electrophoresis. The purified product was ligated with pVT1503 restricted with EcoRI, transformed into E. coli JM109 cells and selected on Kan plates. The purified plasmid was transferred into A. actinomycetemcomitans by electroporation as described previously (Sreenivasan et al., 1991 ).
Southern analysis.
msrA strains were grown in TSBYE containing 50 µg Spc ml-1 and the DNA isolated using Puregene DNA extraction kit (Gentra Systems). Chromosomal DNA was restricted with EcoRI and the fragments separated on a 0·7% agarose gel in TAE buffer. The DNA fragments were transferred to Hybond nylon membranes (Amersham Life Sciences) and the membranes were treated following the method of Sambrook et al. (1989) . The membranes were hybridized with DNA probes conjugated with horseradish peroxidase using the conditions suggested by the manufacturer (Amersham Life Sciences). Hybridizing fragments were visualized using the ECL detection system (Amersham Life Sciences) and exposure to photographic film (XAR-5, Eastman Kodak).
Determination of MsrA activity.
Overnight bacterial cultures (50 ml) were harvested by centrifugation and the bacteria resuspended in 400 µl PBS containing 2 mM PMSF and 1 mM EDTA, pH 8·0. The bacteria were disrupted by the addition of 70 mg glass beads (150212 µm, Sigma) and shaking in a Fast Prep FP120 (Savant Instruments) at setting 6 with 4x30 s pulses at 4 °C. Whole bacteria and membrane fragments were collected by centrifugation for 20 min at 20000 g at 4 °C. The supernatants were removed, snap-frozen in methanol/dry ice and stored at -80 °C. Protein concentrations were determined by the BCA Protein Assay Kit following the manufacturers instructions (Pierce) with BSA as the standard. The reduction of protein-bound methionine sulfoxide by MsrA was assayed using dabsyl-Met(O) as described previously (Moskovitz et al., 1999 , 2000
). Briefly, 200 µM dabsyl-Met(O) was incubated with cell extract or recombinant pure protein in the presence of 20 mM DTT and 20 mM Tris/HCl, pH 7·4. Following incubation at 37 °C for 30 min, formation of dabsyl-Met was analysed by product separation on a reverse phase C-18 column using HPLC system, as previously described (Moskovitz et al., 1999
).
To differentiate between the presence of the enzyme in the cytoplasmic and periplasmic space, spheroplasts were generated by incubation of bacteria with 30000 U lysozyme in the presence of 20% sucrose, 1 mM EDTA, pH 8·0, for 5 min on ice (Feilmeier et al., 2000 ). The resulting spheroplasts were recovered by centrifugation for 2 min at 12000 g. The supernatant containing the periplasmic proteins was made to 1 mM PMSF and stored frozen at -80 °C before assaying for MsrA activity. The pellet was resuspended in 1 ml water containing 1 mM EDTA, pH 8·0 and 1 mM PMSF, mixed thoroughly and incubated at room temperature for 5 min. The cytoplasmic proteins were recovered in the supernatant obtained by centrifugation at 138000 g for 1 h following the disruption of the spheroplasts with water. The cytoplasmic proteins were removed and stored at -80 °C before assaying for MsrA activity.
Disk inhibition assay.
Bacterial cells were grown to exponential or stationary phase in TSBYE media and equal numbers of bacteria (108) were diluted in TSBYE agar at 42 °C and poured into plates. A 6 mm filter disk was placed in the centre of the agar plate and a 15 µl aliquot of hydrogen peroxide (1001000 mM) was applied to the disk. The plates were incubated overnight as described above.
Adhesion of A. actinomycetemcomitans to epithelial cells and intramacrophage survival.
The adherence of A. actinomycetemcomitans to the KB oral carcinoma human epithelial cell line was performed as previously described using an ELISA format (Mintz & Fives-Taylor, 1994 ). RAW 264.7 murine macrophages derived from BALB/c mice (ATCC TIB-71) were used for intramacrophage survival studies. The cells were grown under 5% CO2 in Dulbeccos Modified Eagles Medium (DMEM) supplemented with 10% foetal bovine serum containing 4 mM L-glutamine, 1 mM pyruvate, 0·15% sodium bicarbonate and 0·45% glucose at 37 °C. All cells were used between passages 3 and 20 of the ATCC stock.
Intramacrophage survival was assessed using the gentamicin-protection assay as described by Guy et al. (2000) . Cells were seeded into wells of sterile 24-well tissue culture plates and incubated for 24 h as described above. Macrophages (5x105) were infected at a m.o.i. of
50:1 with bacteria grown to mid-exponential phase. The plates were centrifuged at
170 g for 5 min to synchronize infection and incubated for 20 min to allow internalization. The infected monolayers were washed twice with PBS at 37 °C followed by addition of warmed DMEM (1 ml per well) containing 100 µg gentamicin ml-1 to kill extracellular bacteria. After 2 h, the growth medium was replaced with DMEM containing 10 µg gentamicin ml-1 to minimize the killing of intracellular bacteria. At the desired time points, the appropriate wells were washed twice with pre-warmed PBS and the monolayers were lysed by incubation in 1% Triton X-100 in PBS (0·5 ml per well) for 10 min. Following vigorous mechanical disruption, the lysate was diluted in PBS and plated onto TSBYE agar plates. The plates were incubated for 2 days as described above and the colonies were counted.
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RESULTS |
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A BLAST search of the amino acid sequence deduced from the ORF revealed homology with the MsrA protein of multiple bacterial species. Alignment of the protein sequence (Fig. 1) indicated that the A. actinomycetemcomitans protein had the highest sequence homology with MsrA of Helicobacter pylori (66% identical amino acids). The protein sequence was also homologous to the MsrA of Str. pneumoniae (58% identical amino acids) except the sequence alignment started 46 aa from the amino terminus of the MsrA of A. actinomycetemcomitans. Homology was also noted within the protein encoded by pilB of N. gonorrhoeae (54% identical amino acids). The A. actinomycetemcomitans sequence contains the putative active site of MsrA, GCFWG, (corresponding to aa 5458 in Fig. 1
) as determined for the Saccharomyces cerevisiae homologue (Moskovitz et al., 2000
).
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Cellular fractionation studies indicated that the MsrA enzyme activity was strictly associated with the soluble fraction [1389 ± 230 pmol min-1 (mg protein)-1]. No activity was associated with the membrane fraction. However, assay of the cytoplasmic and periplasmic space fractions showed that the MsrA activity was equally distributed between the two compartments (7043 and 6715 U, respectively), indicating that the enzyme may be transported across the inner membrane.
Inactivation of the gene encoding MsrA activity
The gene encoding MsrA was inactivated by insertion of an antibiotic cassette encoding resistance to spectinomycin into an engineered restriction site that resulted in the loss of 500 bp of the coding region. This construct was transferred to A. actinomycetemcomitans by conjugation and transconjugants were selected for spectinomycin resistance. Transconjugants were analysed for the disruption of msrA by colony PCR and the allele replacement of the genomic copy of msrA was confirmed by Southern blot analysis (Fig. 2
). A 6 kb EcoRI fragment hybridized with the msrA probe in the lane corresponding to the wild-type DNA, whereas a larger fragment (
6·6 kb) hybridized with the probe in all four putative mutant strains. An identical blot probed with spc hybridized exclusively with the fragment corresponding to the disrupted msrA in the blot in Fig. 2(b)
. Signal was absent in the lane corresponding to the wild-type DNA. No signal was generated in any of the lanes when the plasmid without insert was used as the probe (data not shown). These data indicate that the disrupted gene replaced the wild-type gene in the A. actinomycetemcomitans genome in a site-directed and specific manner. One of these strains was further characterized.
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Mononuclear phagocytes, including macrophages and monocytes, are present in diseased periodontal tissues and participate in host defence processes. Therefore, to determine if this gene product is involved in the survival of the bacterium in a more biologically relevant system, both strains were tested for survival inside macrophages. Uptake of approximately equal numbers of bacteria was synchronized by centrifugation and incubation at 37 °C for 20 min before the addition of antibiotic to kill external bacteria. The survival kinetics of the wild-type and mutant strains are depicted in Fig. 3. These data clearly demonstrate that A. actinomycetemcomitans is rapidly killed following internalization by macrophages. In addition, the data also suggest that the survival kinetics of the wild-type and the msrA null mutant strains are similar. These data indicate that inactivation of MsrA does not affect the intracellular survival of A. actinomycetemcomitans in macrophages.
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DISCUSSION |
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The protein from H. pylori is suggested to be released into the extracellular space by multiple mechanisms including specific secretion pathways, autolysis and membrane vesicle formation (Cao et al., 1998 ). The results in this study suggest that MsrA of A. actinomycetemcomitans is transported across the cytoplasmic membrane and is most likely located in the periplasmic space or loosely associated with the inner membrane. A. actinomycetemcomitans also secretes membranous vesicles (Lai et al., 1981
; Meyer & Fives-Taylor, 1994
; Nowotny et al., 1982
) and therefore may release the enzyme in a manner analogous to H. pylori.
The protective role of MsrA to oxidative damage has been demonstrated in a number of bacterial systems (Dhandayuthapani et al., 2001 ; Hassouni et al., 1999
; Moskovitz et al., 1995
; Vriesema et al., 2000
). However, the data presented in this study indicate that MsrA of A. actinomycetemcomitans is not the predominant mechanism that protects the organism against oxidative damage. A. actinomycetemcomitans has been shown to contain a single gene for catalase, but may contain other undefined mechanisms to inactivate hydrogen peroxide (Thomson et al., 1999
). In addition, a Cu2+-Zn2+ superoxide dismutase homologue has also been identified (Fletcher et al., 2001
). Therefore, it is possible that these systems mask the antioxidant defence mechanism provided by MsrA. Interestingly, the A. actinomycetemcomitans strain overexpressing MsrA activity at levels 810 times that of wild-type levels did not show any difference in growth inhibition compared with the parent strain in disk inhibition assays (data not shown).
Downstream of msrA is an ORF encoding an MsrB homologue, which exhibits a high specificity for reduction of the R forms of free and protein-bound methionine sulfoxide (Moskovitz et al., 2002 ). MsrA exhibits a high specificity for the reduction of the S form of the substrates. The substrate used in the detection of MsrA enzymic activity contains both R and S stereoisomer forms (J. Moskovitz, unpublished). Therefore, the complete absence of enzymic activity in the mutant strain suggests that disruption of msrA also results in the loss of MsrB activity. This suggests that the disruption of msrA in this study has a polar effect on msrB transcription. msrA and msrB have been determined to be transcribed as part of a polycistronic message in Sta. aureus (Singh et al., 2001
).
MsrB shares sequence homology with PilB of N. gonorrhoeae, a transcriptional regulator. Therefore, a regulatory function for MsrB is plausible and may explain the results of the complementation studies. Wild-type levels of enzyme activity were achieved following transformation of the mutant strain with a low-copy-number plasmid containing msrA. This result by itself would suggest that msrB transcription was not affected by the disruption of msrA. However, transformation of the parent strain with the same plasmid increased enzymic activity up to 10 times that found in the same strain when transformed with the plasmid backbone. This dramatic difference suggests that a gene product(s) that is present in the parent strain, that results in increased enzymic activity when msrA is presented in trans is inactive in the mutant strain. Taken together, the results suggest that inactivation of msrA results in a polar mutation that disrupts msrB transcription and that msrA and msrB are transcribed as a polycistronic mRNA. In addition, the data also suggest that MsrB regulates transcription of msrA in A. actinomycetemcomitans. A msrB mutant is being constructed to investigate this hypothesis.
The inactivation of msrA in other organisms has led to changes in the surface ligands and adherence of multiple bacterial species. Genetic inactivation of msrA in Str. pneumoniae leads to a dramatic reduction in bacterial adherence (Wizemann et al., 1996 ). In enteropathogenic E. coli, loss of MsrA activity decreased type I fimbriae-mediated haemagglutination and restoration of the enzymic activity by introduction of plasmid containing msrA restored the haemagglutination activity back to wild-type levels. In contrast to a loss of surface ligands in msrA mutants, a hyperpiliation and hyperadherent phenotype is observed in N. gonorrhoeae msrA mutants (Taha et al., 1988
; Wizemann et al., 1996
). MsrA of N. gonorrhoeae is encoded by pilB, which is part of the pilApilB locus that is involved in transcriptional regulation of the expression of the pilin subunit, pilE (Taha et al., 1988
, 1992
). In our study with A. actinomycetemcomitans, inactivation of msrA, which results in abolishing the enzymic activity, does not affect the binding of the bacterium to epithelial cells.
Fresh isolates of A. actinomycetemcomitans from the oral cavity grow in a heavily fimbriated, rough phenotype (Inouye et al., 1990 ; Preus et al., 1988
; Rosan et al., 1988
; Scannapieco et al., 1987
). Following continuous in vitro culturing, many of the isolates lose some of the fimbriae and display a smooth phenotype. The isolate used in this study displays some fimbriae, displays a smooth phenotype and adheres to multiple surfaces (Meyer & Fives-Taylor, 1994
; Mintz & Fives-Taylor, 1994
, 1999
). The nature or number of the adhesins in A. actinomycetemcomitans is still ill-defined. Therefore, the lack of effect due to the disruption of msrA suggests that MsrA activity is either not involved in 1) the maintenance of the biological activity of the adhesin(s); 2) the regulation at the level of transcription of the adhesin or 3) that redundant adhesin molecules are present at the cell surface and inactivation of a single adhesin does not diminish the binding activity or the reduction in activity is below the sensitivity of the assay used in this study.
Methionine sulfoxide reductase has been described for a variety of organisms and common characteristics have been established. Before this study, this gene or gene product had not been characterized from an oral pathogen. In this study, a series of experiments have been attempted to demonstrate the protective role of MsrA as an antioxidant. However, the data suggests that this enzyme may only serve a minor role in the protection of the organism against oxidative damage. A wealth of evidence also exists that demonstrates the role of MsrA as a virulence determinant for pathogenesis in terms of adherence and motility of microorganisms (Hassouni et al., 1999 ; Wizemann et al., 1996
). In A. actinomycetemcomitans SUNY 465, the data indicate that MsrA is not involved in the stabilization or regulation of adhesin(s) mediating the interaction of the bacterium with human epithelial cells. Although in vitro models may provide insight into potential protein function, the role of MsrA in vivo awaits the development of suitable animal model systems for periodontal disease.
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ACKNOWLEDGEMENTS |
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Received 10 April 2002;
revised 15 July 2002;
accepted 1 August 2002.
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