Peptide methionine sulfoxide reductase (MsrA) is not a major virulence determinant for the oral pathogen Actinobacillus actinomycetemcomitansa

Keith P. Mintz1, Jackob Moskovitz2, Hui Wu1 and Paula M. Fives-Taylor1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Actinobacillus actinomycetemcomitans is an oral pathogen that is a causative agent for periodontal disease as well as other non-oral infections. The chronic inflammation associated with periodontal diseases suggests that the bacterium must be able to neutralize oxygen intermediates to survive in the host tissues. Methionine sulfoxide reductase (MsrA) is an enzyme that has been demonstrated to have a role in protection against oxidative damage and has also been identified to be required for the proper expression or maintenance of functional adhesins on the surface of several pathogenic bacteria. The A. actinomycetemcomitans homologue of msrA has been isolated and a chromosomal insertion mutant constructed by allele replacement mutagenesis. Inactivation of the gene led to a complete loss of enzymic activity toward a synthetic substrate. However, the isogenic mutant was not more sensitive to oxidative stress or less adherent to epithelial cells as compared with the parent strain. These data suggest that this strain of A. actinomycetemcomitans has redundant systems that compensate for the MsrA activities ascribed for other organisms.

Keywords: adhesion, oxidative stress, periodontal disease

a The GenBank accession number for the msrA sequence reported in this paper is AY026361.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Exposure to reactive oxygen intermediates such as superoxide anions, hydrogen peroxide and hydroxyl radicals, which are by-products of aerobic metabolism, can damage proteins, nucleic acids and cell membranes (Storz & Imlay, 1999 ). These intermediates can oxidize methionine residues in proteins to methionine sulfoxide, which may result in the loss of the biological function of the protein (reviewed by Brot & Weissbach, 1991 ). In vitro, the function can be restored by incubation of the oxidized protein with the enzyme methionine sulfoxide reductase, MsrA, in a thioredoxin-dependent reaction (Moskovitz et al., 2000 ).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and plasmids.
The A. actinomycetemcomitans strains developed in this study are derived from the clinical strain SUNY 465. All A. actinomycetemcomitans strains were grown statically in Trypticase soy broth supplemented with 0·6% yeast extract (TSBYE) in a humidified 10% CO2 atmosphere at 37 °C. All E. coli strains: JM109, DH5{alpha}({lambda}pir) and SM10({lambda}pir) were grown in Luria–Bertani (LB) broth at 37 °C with aeration. The non-replicating (in A. actinomycetemcomitans) mobilizable plasmid pVT1461 was constructed as described by Mintz et al. (2002) . The strains and plasmids used in this study are listed in Table 1.


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Table 1. Bacterial strains and plasmids

 
Cloning and sequence of the A. actinomycetemcomitans msrA.
Conserved amino acid sequences of MsrA from Str. pneumoniae and Neisseria gonorrhoeae were used as a probe to detect the presence of a homologous protein sequence deduced from the available A. actinomycetemcomitans strain HK1651 DNA sequence at the University of Oklahoma. Based on this homology, oligonucleotide primers were synthesized (Operon Technologies) to amplify the A. actinomycetemcomitans msrA including flanking regions from the chromosome of strain SUNY 465. A DNA fragment was generated in a PCR reaction using Taq polymerase (Gibco BRL) using the sense primer corresponding to a sequence 356 bp 5' to the start codon of msrA (5'-CACACGACTGTCCGAAC-3') and the antisense sequence 432 bp 3' from the stop codon of the gene (5'-CGTAGACAAGACCGCAACA-3') of strain HK1651 in a Perkin-Elmer PCR machine. The PCR products were separated by agarose gel electrophoresis and a 1860 bp fragment was purified using QIAquick gel extraction kit following the manufacturer’s directions (Qiagen) and ligated with the T/A cloning vector, pGEM T-Easy (Promega). The ligation mixture was transformed into JM109 E. coli cells and plated on LB agar plates containing 50 µg ampicillin ml-1, IPTG and X-Gal (Sambrook et al., 1989 ). Plasmids with disrupted ß-gal were purified, restricted with EcoRI to release the insert and characterized by agarose gel electrophoresis. An individual plasmid containing the correct size insert was used for sequencing. Both DNA strands of the insert were sequenced using the dideoxy terminator cycle sequencing kit (Applied Biosystems) and analysed using an Applied Biosystems automated DNA sequencer. DNA sequencing was performed at the Vermont Cancer Center DNA Analysis Facility. The complete msrA sequence can be obtained from GenBank (accession no. AY026361).

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{alpha}({lambda}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({lambda}pir) cells for conjugation. Mobilization of the plasmid containing the disrupted gene from E. coli SM10({lambda}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 3–4 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 (150–212 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 manufacturer’s 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 (100–1000 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 Dulbecco’s Modified Eagle’s 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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isolation of A. actinomycetemcomitans msrA
The gene encoding MsrA was identified in A. actinomycetemcomitans using a conserved sequence of the protein to probe the available genomic sequence of A. actinomycetemcomitans. The entire ORF and adjoining sequences were amplified by PCR and sequenced. The ORFs flanking msrA were found to encode a homologue of carbonic anhydrase (upstream) and cytochrome c-type biogenesis protein which has some homology to MsrB (downstream) of MsrA. The msrA DNA sequence determined from the strain used in this study (SUNY 465) was identical to the prototype strain HK1651 sequenced at the University of Oklahoma.

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 54–58 in Fig. 1) as determined for the Saccharomyces cerevisiae homologue (Moskovitz et al., 2000 ).



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Fig. 1. Sequence alignment of MsrA from A. actinomycetemcomitans. The alignment was generated using CLUSTAL W (http://www.clustalw.genome.ad.jp/) and Boxshade 3.21 (http://www.ch.embnet.org/software/BOX_form.html). The putative active site of MsrA is shown in bold. ACTAC, A. actinomycetemcomitans; HELPY, H. pylori (Cao et al., 1998 ; Tomb et al., 1997 ); STRPN, Str. pneumoniae (Wizemann et al., 1996 ); NEIGO, N. gonorrhoeae (Taha et al., 1988 ; Wizemann et al., 1996 ).

 
Cellular localization of MsrA
The amino terminal sequence of MsrA of A. actinomycetemcomitans displays features that are characteristic of prokaryotic signal sequences (von Heijne, 1986 ). A run of charged amino acids after the start methionine is followed by a central hydrophobic region and then a more polar COOH-terminal domain adjacent to the mature protein. Following this polar amino acid region, a putative signal peptidase cleavage site (the 17th amino acid, ala, from the start methionine) is predicted (von Heijne, 1986 ).

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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Fig. 2. Southern blot analysis of chromosomal DNA. Chromosomal DNA was isolated from wild-type and msrA strains and incubated with EcoRI. Products were separated by agarose gel electrophoresis and transferred to nylon membranes. Duplicate membranes were probed with labelled msrA (a) or spectinomycin adenyltransferase (aad9) (b). +, wild-type DNA; -, mutant DNA. Arrows correspond to migration of selected DNA markers.

 
Characterization of A. actinomycetemcomitans MsrA activity
MsrA activity was determined using dabsyl-Met(O) as the substrate, as described previously (Moskovitz et al., 2000 ). The MsrA activity of A. actinomycetemcomitans was found to be comparable to the activity found for other bacterial species (Table 2). A complete absence of MsrA activity was observed for the msrA mutant strain, verifying that the targeted gene is linked with MsrA activity. The activity was restored to wild-type levels when a replicating plasmid containing the complete msrA sequence was introduced into the mutant strain by electroporation (see Table 2). Transformation of the mutant or wild-type strain with the plasmid vector did not significantly alter the enzymic activity. Overexpression of MsrA activity was observed when the plasmid containing msrA was transformed into the wild-type strain.


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Table 2. Measurement of MsrA enzyme activity

 
Oxidation of methionine residues by biologically reactive oxygen intermediates, such as superoxides, hydrogen peroxide and hydroxyl radicals may lead to the loss of biological function (Brot & Weissbach, 1991 ). The role of msrA in oxidative stress in A. actinomycetemcomitans was investigated by several approaches. The sensitivity of A. actinomycetemcomitans to hydrogen peroxide was investigated using disk inhibition assays. Concentrations of hydrogen peroxide were varied from 100 mM to 1 M and the zone of growth inhibition was similar for the mutant as compared with the parent strain (Table 3) at all concentrations. Similar results were obtained when stationary phase bacteria were used in the assay. No difference in the growth inhibition was observed with 1% to 3·8% paraquat (methyl viologen), an uncoupler of oxidative phosphorylation, which leads to superoxide production (data not shown).


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Table 3. Effect of H2O2 treatment on A. actinomycetemcomitans growth

 
Growth conditions were varied to determine if environmental factors regulated the expression of msrA. No difference in the area of growth inhibition between the wild-type and mutant strains was observed when the bacteria were grown and plated under anaerobic conditions, at an acidic pH (6·5 versus 7·2) or in the presence of cysteine, which can substitute for yeast extract in the growth media (Sreenivasan et al., 1993 ) (data not shown). To exclude the possibility that the growth media may be masking the difference between the two strains, equal numbers of bacteria were washed with PBS and resuspended in PBS containing 1 mM hydrogen peroxide. Aliquots were taken at timed intervals and plated to determine bacterial viability. Again, no difference in viability over time was observed (data not shown).

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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Fig. 3. Intramacrophage survival of A. actinomycetemcomitans msrA+ and msrA- strains. Equal numbers of wild-type ({diamondsuit}) and mutant ({blacksquare}) bacteria were incubated with mouse macrophages. Bacterial survival was determined using a gentamicin protection assay. At the indicated times, cells were disrupted with detergent and the bacteria were plated. The values are the means of two experiments, each time point was performed in triplicate.

 
MsrA activity has been demonstrated to be important in maintaining bacterial surface structures involved in adhesion (Wizemann et al., 1996 ). Therefore, we investigated the adhesion properties of the msrA null mutant to human epithelial cells. Equal numbers of bacteria (wild-type and mutant) were incubated with the epithelial cells and bacterial binding was detected by purified anti-A. actinomycetemcomitans immunoglobulins. No difference in the binding of the mutant compared with the parent strain to epithelial cells was detected (0·6±0·07 vs 0·6±0·03 relative absorbance units, respectively). These data suggest that MsrA is not involved in maintaining the adhesin(s) required for binding to epithelial cells in this strain of A. actinomycetemcomitans.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study we have identified and isolated the gene encoding MsrA activity in A. actinomycetemcomitans strain SUNY 465. MsrA is a ubiquitous protein that has been found in both eukaryotic and prokaryotic organisms (Cao et al., 1998 ; Dhandayuthapani et al., 2001 ; Hassouni et al., 1999 ; Moskovitz et al., 1996b , 1998 ; Rahman et al., 1992 ; Vriesema et al., 2000 ; Wizemann et al., 1996 ). The amino acid sequence identity ranges between 33% and 88% among members of this family (Lowther et al., 2000 ). The A. actinomycetemcomitans MsrA has the greatest amino acid homology with the MsrA of H. pylori. Based on the protein sequence, both of these sequences appear to contain a cleavable signal sequence based on signal sequence algorithms (http://psort.nibb.ac.jp/form.html). None of the other bacterial MsrA proteins investigated contained these sequences. Recently, a fragment of DNA encoding the signal sequence and a portion of the A. actinomycetemcomitans MsrA was identified using alkaline phosphatase fusions of secreted proteins (Ward et al., 2001 ). In fact, the gene encoding the H. pylori MsrA was isolated using antisera raised against antigens found in broth culture supernatants (Cao et al., 1998 ). More recently, two forms of the N. gonorrhoeae homology of MsrA (PilB) have been shown to be synthesized (Skaar et al., 2002 ). One form of the polypeptide contains a signal sequence and is secreted from the bacterial cytoplasm to the outer membrane. The other form of the protein lacks a signal sequence and is cytoplasmic. Taken together, these examples and the data presented in this study suggest that MsrA of A. actinomycetemcomitans is transported across the cytoplasmic membrane in a sec-dependent pathway.

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 8–10 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.


   ACKNOWLEDGEMENTS
 
We would like to thank Mingyuan Shao for cloning msrA, Akamol Suvarnapunya for his assistance in the macrophage survival studies and Bruce Roe at the University of Oklahoma’s Advanced Center for Genome Technology for providing the A. actinomycetemcomitans HK1651 nucleotide sequence. This work was supported by Public Health Service grant RO1-DE09760 and in part by RO1-DE13824.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 10 April 2002; revised 15 July 2002; accepted 1 August 2002.



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