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Departments of Pharmacology,1 Oral Infectious Diseases and Immunology, Graduate School of Dental Science, Kyushu University,4 Special Patient Oral Care Unit, Kyushu University Dental Hospital, Fukuoka 812-8582,2 Division of Microbiology and Oral Infection, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588,3 Department of Microbiology, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan5
Received 29 April 2002/ Returned for modification 14 August 2002/ Accepted 18 November 2002
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ABSTRACT |
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INTRODUCTION |
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Porphyromonas gingivalis is a gram-negative obligate anaerobe belonging to the division Cytophagales (23). This bacterium is one of the organisms that is most strongly associated with chronic adult periodontitis and expresses numerous potential virulence factors, such as fimbriae, hemagglutinins, lipopolysaccharides, and various proteases that are capable of hydrolyzing collagen, immunoglobulins, iron-binding proteins, and complement factors (21, 27). P. gingivalis, by definition, cannot grow in aerobic conditions but exhibits a high degree of aerotolerance. This aerotolerance enables the organism to survive in periodontal pockets that are occasionally exposed to aerobic conditions.
P. gingivalis posseses SOD that is essential for tolerance to atmospheric oxygen, as revealed by the finding that P. gingivalis sod mutant shows a rapid viability loss by exposure to atmospheric oxygen (34), although it exhibits a marked resistance to peroxide stress. It has been demonstrated that the Dps (DNA-binding protein from starved cells) protein in E. coli plays an important role in the protection of cells from peroxide stress (1, 2). This protein is produced primarily in the stationary-phase cells, and its expression is regulated by 
38, 70, and OxyR. Structurally, the Dps protein forms a ferritin-like spherical oligomeric structure. In addition, the Dps monomer displays essentially the same protein fold (four-helix bundle) as the ferritin monomer (17). It is of special importance that E. coli Dps exhibits DNA- and iron-binding activities by which the cells probably gain the resistance to oxidative stresses. Recent studies have demonstrated that a diverse group of Dps homologues are found in various prokaryotes, including Synechococcus sp., Bacillus subtilis, Listeria innocua, Streptococcus mutans, and Bacteroides fragilis (5, 6, 36, 38, 49), and are related to the ferritin-bacterioferritin-rubrerythrin superfamily (3, 17).
In the present study, we provide the first evidence indicating the presence of a Dps homologue in P. gingivalis. We have also constructed the Dps-deficient mutants to analyze the function of this protein in the organism. The results clearly indicate that the Dps protein is implicated in the protection of the organism from peroxide stress, thereby contributing to its survival in periodontal pockets and host cells.
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MATERIALS AND METHODS |
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Purification of P. gingivalis ferritin-like protein and determination of its N-terminal amino acid sequence.
P. gingivalis ATCC 33277 was grown in enriched BHI broth for 48 h. The cells were harvested by centrifugation at 15,000 x g for 20 min at 4°C. The pellet was suspended in 30 ml of buffer A (10 mM Tris-HCl buffer [pH 7.5]), and N-
-p-tosyl-L-lysine chloromethyl ketone (Sigma Chemical Co., St. Louis, Mo.) and leupeptin (Peptide Institute, Osaka, Japan) were added to final concentrations of 0.1 and 1 mM, respectively. The cells were broken by sonication (25 W; 30 pulses/min; 1-s pulse length) in a Branson sonicator with 1-min intervals for 10 min on ice. The sonicate was shaken for 15 min at 37°C and centrifuged to remove the unbroken cells. The supernatant was saved, and CsCl was added to a final concentration of 40% (wt/vol), followed by centrifugation at 80,000 x g for 24 h at room temperature. The solution was carefully separated into fractions, and each fraction was dialyzed overnight against buffer A by changing the buffer at 6-h intervals. The fractions were then examined by electron microscopy. The fraction containing ferritin-like particles was concentrated by ultrafiltration with a Microcon YM-10 (Millipore Corp., Bedford, Mass.). For further purification, the concentrated ferritin-like particles were subjected to gel filtration on a Sephadex FPLC column (FPLC column TM200; Pharmacia, Uppsala, Sweden). Proteins were eluted with buffer A at a flow rate of 0.5 ml/min. Protein elution was monitored by measuring the absorbance at 280 nm. Each fraction was examined by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the presence of ferritin-like particles.
To determine the amino-terminal amino acid sequence of this ferritin-like protein, the protein on the polyacrylamide gel was electrophretically transferred onto polyvinylidene difluoride membrane and stained with Coomassie brilliant blue R-250. The stained protein band was cut out and analyzed in an automatic protein sequencer (Applied Biosystems model 476A; Perkin-Elmer Cetus, Norwalk, Conn.).
Molecular cloning of P. gingivalis dps and construction of recombinant plasmids and bacterial strains.
Sau3AI plasmid library (pUC19) of P. gingivalis ATCC 33277 chromosomal DNA was introduced to E. coli TA4112 (oxyR) after amplification of the library in DH5 and a recombinant plasmid clone (pKD386) that conferred resistance to t-butyl hydroperoxide on the oxyR strain was obtained. The BstXI site within dps of pKD386 was eliminated with T4 DNA polymerase to yield pKD387. A tetQ DNA block of pKD375 (40) was inserted into the BstXI site of pKD386 to yield pKD388. The dps gene DNA was PCR amplified from pKD386 with primers (an upper primer [CCATATGAAAAAGATTCTTGAAGTAACGGG] and a lower primer [GGGATCCTTACTTGGCAGCGTAGGCAGACA]) and introduced to pET11a, resulting in pKD390.
For construction of the lacZ reporter suicide-integration plasmid, a 3.1-kb lacZ region DNA was PCR amplified from pRS414 DNA (41) with PCR primers (an upper primer [CGGAATTCCCGGGGATCCCGTCGTT] and a lower primer [AAGATCTTATTTTTGACACCAGACCAACTGG]) and ligated to the linear pCR2.1 DNA by using the TA cloning method, resulting in pKD391. E. coli DH5 harboring pKD391 showed ß-galactosidase activity, indicating that the subcloned lacZ gene was functional. A 3.1-kb EcoRI fragment of pKD391 containing the lacZ gene was then inserted to the EcoRI site of pKD355, resulting in pKD392. EcoRI and BamHI sites other than the EcoRI-SmaI-BamHI multiple cloning sites were eliminated in pKD392 by partial restriction digestion and Klenow filling, giving rise to pKD393.
For construction of a dps'-'lacZ fusion plasmid, a DNA fragment comprising the 5'-terminal region of dps and its upstream region was PCR amplified from the chromosomal DNA of P. gingivalis ATCC 33277 with primers (an upper primer [CGAATTCCTCTAGAGGATCTTCTTC] and a lower primer [GGGATCCAAACCCGTTACTTCAAGA]). The upper primer can hybridize to the chromosomal DNA 1.6-kb upstream of the start codon of dps and generate an EcoRI site at one end of the PCR product, whereas the lower primer can hybridize to the chromosomal DNA within the dps gene and generate a BamHI site at the other end of the PCR product. The amplified DNA fragment was cloned into pCR2.1, sequenced, excised by double digestion with EcoRI and BamHI, and ligated to the EcoRI-BamHI region of pKD393. The resulting plasmid pKD394 produced the dps'-'lacZ fusion protein with the N-terminal 10 amino acids of Dps.
P. gingivalis oxyR preliminary sequence data was obtained from The Institute for Genomic Research website (http://www.tigr.org). The P. gingivalis oxyR gene region (927 bp) was PCR amplified from the chromosomal DNA of ATCC 33277 with PCR primers (an upper primer [CCATATGAATATACAGCAGCTCGAA] and a lower primer [CGGATCCTCAAGCCAAATGCTGCCCTGT]), cloned into pCR2.1, sequenced, excised with EcoRI, and ligated to the EcoRI site of pUC19, resulting in pKD396. The BspEI site within the oxyR gene of pKD396 was converted to BglII by using a BglII linker DNA and the tetQ DNA block was inserted into the BglII site, resulting in pKD397. ATCC 33277 and KDP146 (dps+ dps'-'lacZ) were transformed to tetracycline resistance by electrotransformation with the PstI-linearized pKD397 DNA to yield KDP143 (oxyR::Tcr) and KDP148 (oxyR::Tcr dps+ dps'-'lacZ), respectively.
For construction of P. gingivalis dps mutants, ATCC 33277 and KDP139 were transformed to tetracycline resistance with linearized pKD388 (dps::Tcr) DNA to yield KDP141 (dps::Tcr) and KDP142 (ftn::Emr dps::Tcr), respectively.
Purification of Dps from a dps-overexpressing E. coli. E. coli BL21(DE3) harboring pKD390 was grown to an optical density at 540 nm (OD540) of 0.5 in 300 ml of L broth containing 50 µg of ampicillin/ml. IPTG (isopropyl-ß-D-thiogalactopyranoside) was added to the culture at 1 mM, followed by incubation for 2 h to overproduce the recombinant Dps protein. The cells were collected by centrifugation at 9,000 x g for 20 min, resuspended in 10 ml of 20 mM phosphate buffer (pH 7.0), and disrupted by sonic oscillation (1 min each, 10 times). After centrifugation at 10,000 x g for 15 min, the supernatant was saved, to which ammonium sulfate was added at 85% saturation, and the precipitate was dissolved in 3 ml of phosphate buffer. After centrifugation to remove insoluble materials, the supernatant was dialyzed against the same phosphate buffer with a seamless cellulose tube (UC36-32-100; Sankohjunyaku Co., Tokyo, Japan) for 24 h and applied to a column (2.0 by 25 cm) of DEAE-Sepharose (Pharmacia) equilibrated with the same buffer. Proteins were eluted with 180 ml of a 0 to 1,000 mM linear NaCl gradient in the same buffer. The recombinant Dps protein was usually eluted at 400 mM NaCl. After concentration of the protein with a Microcon YM-10, the protein sample was applied to Mono-Q column (Pharmacia) and eluted with 180 ml of a 0 to 1,000 mM linear NaCl gradient in the same buffer. The Dps protein fraction that was eluted at 400 mM NaCl was then subjected to gel filtration on Superdex 200 column (Pharmacia). The system was equilibrated with the same buffer, proteins were eluted at a flow rate of 0.3 ml/min and 2-ml fractions were collected. The fractions constituting the prominent protein peak were pooled and concentrated with a Microcon YM-10. The iron-loaded Dps was obtained by incubating 50 µg of the recombinant Dps with 1 mM ferrous ammonium sulfate in 20 mM morpholinepropanesulfonic acid-NaOH (pH 7.0) at 4°C for 1 h (45). To remove excess iron, the mixture was extensively dialyzed against the same morpholinepropanesulfonic acid buffer by using the seamless cellulose tube (UC36-32-100). The iron-loaded Dps was concentrated by using the Microcon YM-10.
DNA-binding activity. Recombinant P. gingivalis Dps (10 µg) treated with or without ferrous ammonium sulfate was mixed with 500 ng of linear DNA (1-kb DNA Ladder; Promega, Madison, Wis.) and kept at 4°C for 1 h. The mixture was subjected to agarose gel electrophoresis (1%; Tris-acetate buffer).
Iron starvation. To determine the ability to grow under iron starvation, cells of test strains were first grown in the presence of hemin and then deprived of the iron source. The initial inoculum that had been prepared by growing the strains in hemin-containing enriched BHI medium overnight was diluted 10-fold with hemin-free enriched BHI medium and incubated. Every 24 h, the OD540 of the cultures was measured, and a 10-fold dilution of the cultures with hemin-free enriched BHI medium was repeated.
Agar diffusion assay. P. gingivalis cells were anaerobically grown in enriched BHI medium and spread on enriched TS plates, and a sterile disk containing 4 µl of 6% hydrogen peroxide, 15% hydrogen peroxide, 1 mg of mitomycin C/ml, or 0.5 mg of metronidazole/ml was placed at the center of each plate and incubated anaerobically at 37°C for 4 days.
Sensitivity of P. gingivalis to hydrogen peroxide in aerobic conditions. P. gingivalis cells grown in enriched BHI medium for 48 h were diluted twofold with fresh enriched BHI medium with or without 1.0 mM hydrogen peroxide and incubated aerobically at 37°C with shaking (125 cpm). To determine the number of survivals, cultures were withdrawn at intervals and plated on enriched TS plates after adequate dilution. The plates were anaerobically incubated for 7 days at 37°C
Cell culture and infection of HUVEC with P. gingivalis. Human ambilical vein endothelial cells (HUVEC) obtained from Cell Applications, Inc. (San Diego, Calif.) was maintained in MCDB151 medium (Sigma) containing 15% fetal calf serum, acidic fibroblast growth factor (Sigma), heparin (Sigma), and kanamycin sulfate. For infection, cells were seeded at 5 x 105 cells per well in six-well tissue culture dishes with 2 ml of the same medium. P. gingivalis cells were grown in enriched BHI medium to a mid-logarithmic phase, washed with phosphate-buffered saline (PBS), and suspended into kanamycin sulfate- and fetal bovine serum-free MCDB151 medium. The bacterial suspension was then added to the HUVEC monolayer at a multiplicity of infection of 10,000. After incubation at 37°C for the indicated period, the infected cells were collected, washed with the same medium containing 300 µg of gentamicin and 400 µg of metronidazole/ml, suspended in the antibiotic-containing medium, and incubated at 37°C for 1 h to kill extracellular bacterial cells. The infected HUVEC were collected, washed with PBS, and suspended in MCDB151 medium. After freezing and thawing of the HUVEC suspension, enriched BHI medium was added to the suspension. The mixture was vortexed for 15 s and serially diluted, and each dilution was plated on enriched TS agar and then incubated anaerobically at 37°C for 5 days to determine the survival of the intracellular bacterial cells. All assays were conducted independently at least five times. The results are expressed as the means ± the standard deviations (SD) of multiple experiments.
Confocal laser scanning microscopy. P. gingivalis cells of a fresh overnight culture were precipitated by centrifugation at 6,000 rpm for 10 min, washed, and suspended in PBS. The cell suspension was supplemented with 2,7-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein (BCECF; 10 mM stock solution; Molecular Probes, Inc., Eugene, Oreg.) at a final concentration of 5 µM and incubated anaerobically at 37°C for 30 min. After centrifugation at 6,000 rpm for 5 min, the precipitated bacterial cells were suspended in MCDB151 medium without antibiotics. HUVEC that had been grown on a glass coverslip in a six-well tissue culture plate were washed twice with PBS and infected with the BCECF-labeled P. gingivalis cells at a multiplicity of infection of 10,000. After a 10-min incubation at 37°C, the infected HUVEC were washed twice with PBS and fixed in 10% formaldehyde in PBS at room temperature for 15 min. The fixed cells were washed twice with PBS and treated with PBS containing 50 mM NH4Cl and 0.3% Tween 20 for 10 min at room temperature. After being washed with PBS twice, the HUVEC were subjected to confocal laser scanning microscopy (DMIRB/E; Leica Microsystems, Wetzlar, Germany). From confocal laser scanning images, incorporated bacterial cells were counted in a depth of 1.0 µm at the central section of a single HUVEC. Data were expressed as the means of more than 50 determinations ± the SD.
Other methods. Iron staining with Ferene S and heme staining with 3,3',5,5'-tetramethylbenzidine (TMBZ) were performed as described previously (37). Electrotransformation of P. gingivalis cells was done as previously described (35). SDS-PAGE was performed under reducing conditions on 15% gels essentially as described previously (25). The gels were stained with 0.1% Coomassie blue R-250. For immunoblot analysis, proteins on SDS-PAGE gels were electrophretically transferred to nitrocellulose membranes as previously described (46). The membranes were immunostained with a 5,000-fold dilution of an anti-P. gingivalis Dps antiserum. The antiserum was prepared from a rabbit immunized with P. gingivalis Dps purified from the E. coli strain overproducing P. gingivalis Dps. The reacting proteins were detected by using the ECL Western blotting system (Amasham).
DNA sequencing was carried out by using a dideoxy sequencing kit (Auto Read Sequencing kit; Pharmacia) with plasmid templates and an automated DNA sequencer (ALF DNA Sequencer; Pharmacia). The sequence data were analyzed with the GeneWorks software program (IntelliGenetics, Mountain View, Calif.).
Colony hybridization and Southern blot hybridization were performed by standard methods (39, 47).
Statistical analysis. The Student t test was used to compare differences in CFU numbers among bacterial strains by using StatView J4.5 software (Abacus Concepts, Inc., Berkeley, Calif.).
Nucleotide sequence accession number. The nucleotide sequence reported in the present study was deposited in the DDBJ/EMBL/GenBank database under accession no. AB025779.
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RESULTS |
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Molecular cloning of a P. gingivalis gene conferring peroxide resistance on the E. coli oxyR mutant. Apart from the biochemical characterization of the ferritin-like protein, we tried to isolate P. gingivalis genes responsible for peroxide resistance. P. gingivalis chromosomal DNA library was introduced into the E. coli oxyR mutant and t-butyl hydroperoxide-resistant transformants were isolated. All of the transformants harbored recombinant plasmids containing the same chromosomal DNA region, revealed by restriction enzyme analysis (data not shown). One of the recombinant plasmids (pKD386) contained a 2.6-kb chromosomal DNA fragment. Subcloning analysis located the peroxide-resistant gene on a 1.1-kb DNA region within the 2.6-kb fragment. Unexpectedly, one open reading frame in the 1.1-kb region encoded a protein with 159 amino acid residues, the N-terminal sequence of which was the same as that of the ferritin-like particle protein. As shown in Fig. 1, the protein had a marked resemblance of the amino acid sequence to Dps proteins from other bacterial species. Therefore, this protein was referred to as P. gingivalis Dps.
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P. gingivalis Dps purified from the recombinant E. coli cells was mixed with ferrous ammonium sulfate, electrophoresed through nondenaturing gels, and stained with Ferene S and TMBZ. Ferene S but not TMBZ stained P. gingivalis Dps, suggesting that P. gingivalis Dps is able to bind nonheme iron (data not shown). The DNA-binding activity of Dps was assayed by using linear plasmid DNA as described previously (1). The linear DNA mixed with the iron-loaded Dps did not migrate into an agarose gel, indicating the DNA-binding activity of Dps (Fig. 2).
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(ii) Sensitivity to hydrogen peroxide, mitomycin C, and metronidazole. To determine the sensitivity of the dps mutants to hydrogen peroxide, mitomycin C, and metronidazole, we used agar diffusion assays under anaerobic conditions (Fig. 5). The dps and ftn dps mutants were more sensitive to hydrogen peroxide than were the wild-type and ftn strains (Fig. 5A). There was no difference in sensitivity to mitomycin C and metronidazole among these strains (Fig. 5B and C). These results indicated that Dps contributed to protection against peroxide in P. gingivalis cells.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of Dps homologue genes of various microorganisms is upregulated by exposure to oxidative stress (1, 2, 6, 22, 38). In E. coli and B. fragilis, the stress response of their dps genes is regulated by OxyR (2, 38). In the present study, we constructed the lacZ reporter suicide-integration plasmid for analysis of gene expression of P. gingivalis. Using this lacZ reporter system, we found that the P. gingivalis dps gene was constitutively expressed but was upregulated by exposure to atmospheric oxygen and that this induction was totally dependent on OxyR. There was an approximately 10-fold increase in B. fragilis dps expression upon exposure to atmospheric oxygen (38), whereas P. gingivalis dps expression increased by only 25%, indicating that the degree of constitutive expression of P. gingivalis dps was much greater than that of B. fragilis dps. B. fragilis possesses catalase, whereas P. gingivalis does not possess catalase (31, 38). The high degree of constitutive expression of Dps in P. gingivalis might compensate for the absence of catalase in this organism.
Analysis of the P. gingivalis dps mutant revealed that P. gingivalis Dps was responsible for protection against peroxides, especially against hydrogen peroxide. Previous studies suggest that the ability of Dps to protect cells from oxidative damage may be derived from DNA condensation and masking with the Dps protein and from sequestration of iron ions that might otherwise generate detrimental free radicals (17, 24, 29, 48, 49). If DNA condensation and mechanical masking with Dps contributes to the protection of chromosomal DNA from peroxides, Dps-deficient mutants might show sensitivity to other DNA-damaging agents as well. However, the P. gingivalis dps mutant had no sensitivity to mitomycin C or metronidazole that can damage DNA. Nondenaturing PAGE profiles showed that P. gingivalis Dps contained nonheme iron. However, P. gingivalis Dps may not contribute to the iron storage of this organism, as revealed by the iron deprivation experiment. In addition, the ftn dps double mutant showed the same sensitivity to hydrogen peroxide as did the dps single mutant. These results suggest that, in P. gingivalis Dps, the sequestration of iron ions might not be plausible to explain the ability to protect cells from peroxides. We found in a previous study (34) that P. gingivalis sod mutant shows a rapid viability loss upon exposure to atmospheric oxygen. In contrast, we found in the present study that the dps mutant showed no viability loss upon exposure to atmospheric oxygen for 6 h. On the other hand, the dps mutant showed a viability loss by the treatment of hydrogen peroxide in aerobic conditions and that this viability loss was not enhanced by the addition of the ftn mutation. These results strongly suggest that P. gingivalis Dps is responsible for protection against specific ROS such as hydrogen peroxide. In this context, Dunkan and Touati (14) have shown that the E. coli dps mutant as well as a catalase-null mutant shows increased sensitivity to both hypochlorous acid and a catalase-null mutant. Hassett et al. (20) reported hat overproduction of Pseudomonas aeruginosa Dps provides protection against hydrogen peroxide but increases sensitivity to cumen hydroperoxide in P. aeruginosa oxyR mutant. Synechococcus sp. DpsA has catalase activity (36). In addition, rubrerythrin belonging to the ferritin-bacterioferritin-rubrerythrin superfamily, which is in turn related to the Dps family, has been found to have NADH peroxidase activity (9). P. gingivalis Dps might have peroxide-reducing activity, although we have not yet found this activity. To elucidate the mechanism of the peroxide resistance conferred by P. gingivalis Dps, the molecular and catalytic properties of the Dps should be explored.
Recently, a number of epidemiological studies have revealed a positive correlation between periodontal disease and coronary heart disease (4, 30). Patients with periodontal disease are more likely to experience transient bacteremias produced by flossing, mastication, and toothbrushing, which can occasionally let periodontal bacteria localize to endothelial cells (10). In fact, P. gingivalis has been immunolocalized in the shoulders of atherosclerotic plaques (7), and P. gingivalis DNA has been found in endarterectomy samples of patients with carotid stenosis by PCR analysis with primers specific to P. gingivalis DNA (19). The microorganism can invade various host cells, including epithelial cells and endothelial cells (11, 13, 26, 28). Survival of P. gingivalis cells in these host cells is important for the development of infection. The present study has provided the finding that Dps contributed to the intracellular survival of P. gingivalis cells. As far as we know, the present study is the first description of the significance of Dps in the survival of microorganisms in host cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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, ATCC 33277 (wild type); 
, KDP139 (ftn); •, KDP141 (dps); 
, KDP142 (ftn dps).