Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Asai, Y. Articles by Ogawa, T. Search for Related Content PubMed PubMed Citation Articles by Asai, Y. Articles by Ogawa, T.

 Previous Article  |  Next Article 

Infection and Immunity, April 2005, p. 2157-2163, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.2157-2163.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Lipopolysaccharide Preparation Extracted from Porphyromonas gingivalis Lipoprotein-Deficient Mutant Shows a Marked Decrease in Toll-Like Receptor 2-Mediated Signaling

Yasuyuki Asai,¹ Masahito Hashimoto,¹ Hansel M. Fletcher,² Kensuke Miyake,³ Shizuo Akira,⁴ and Tomohiko Ogawa^1*

Department of Oral Microbiology, Asahi University School of Dentistry, Gifu,¹ Division of Infectious Genetics, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo,³ Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan,⁴ Division of Microbiology and Molecular Genetics, School of Medicine, Loma Linda University, Loma Linda, California²

Received 8 October 2004/ Returned for modification 11 November 2004/ Accepted 8 December 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
We recently demonstrated that a new PG1828-encoded lipoprotein (PG1828LP) was able to be separated from a Porphyromonas gingivalis lipopolysaccharide (LPS) preparation, and we found that it exhibited strong cell activation, similar to that of Escherichia coli LPS, through a Toll-like receptor 2 (TLR2)-dependent pathway. In order to determine the virulence of PG1828LP toward cell activation, we generated a PG1828-deficient mutant of P. gingivalis strain 381 by allelic exchange mutagenesis using an ermF-ermAM antibiotic resistance cassette. A highly purified preparation of LPS from a PG1828-deficient mutant (PG1828-LPS) showed nearly the same ladder-like patterns in silver-stained gels as a preparation of LPS from a wild-type strain (WT-LPS), as well as Limulus amoebocyte lysate activities that were similar to those of the WT-LPS preparation. However, the ability of the PG1828-LPS preparation to activate NF-B in TLR2-expressing cells was markedly attenuated. Cytokine production by human gingival fibroblasts was also decreased in response to the PG1828-LPS preparation in comparison with the WT-LPS preparation, and the activity was comparable to the stimulation of highly purified lipid A of P. gingivalis by TLR4. Further, lethal toxicity was rarely observed following intraperitoneal injection of the PG1828-deficient mutant into mice compared to that with the wild-type strain, while the PG1828-LPS preparation showed no lethal toxicity. Taken together, these results clearly indicate that PG1828LP plays an essential role in inflammatory responses and may be a major virulence factor of P. gingivalis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Porphyromonas gingivalis has been implicated as a major etiological agent in the development and progression of chronic periodontitis, which is a destructive inflammatory disease of the supporting tissues of the teeth (38). This bacterium is a gram-negative, obligate anaerobic, oral black-pigmented rod that possesses a large number of potential virulence factors, such as fimbriae, hemagglutinin, lipopolysaccharide (LPS), and various proteases (15).

Among these virulence factors, LPS is well known as a major component of the outer membranes of gram-negative bacteria, and it exhibits powerful immunostimulatory and inflammatory activities (32). However, P. gingivalis LPS has a lower level of endotoxic potency than other types of enterobacterial LPSs (21, 27), while it and its active center, lipid A, have been shown to have other properties, such as an ability to activate cells from LPS-hyporesponsive C3H/HeJ mice as well as those from LPS-responsive C3H/HeN mice (18, 42).

Toll-like receptor 4 (TLR4) and its accessory protein MD-2 are known to function as signaling receptors for various LPSs (44), and C3H/HeJ mice have been demonstrated to be hyporesponders, due to a natural point mutation of TLR4 (30). Further, TLR2 has been shown to be an essential signal-transducing molecule for P. gingivalis LPS preparations (4, 13), although P. gingivalis LPS is thought to be associated with quite different lipid A phosphorylation and acylation patterns (25). More recently, Darveau et al. (6) indicated that P. gingivalis LPS activates cells through both TLR2 and TLR4, because it possesses multiple lipid A species. In contrast, it was also reported that P. gingivalis LPS exerted antagonistic effects toward TLR4-dependent cell activation by Escherichia coli LPS (5, 45).

We previously demonstrated that highly purified lipid A from P. gingivalis and its synthetic counterpart activated cells via a TLR4/MD-2-dependent pathway but not via TLR2, which was in contrast to the cell activation activities of a P. gingivalis LPS preparation, which were shown to occur via TLR2 (28). In addition, we recently showed that a PG1828-encoded lipoprotein (PG1828LP) was able to be separated from a P. gingivalis LPS preparation by using a detergent-modified phenol-water extraction method, and we found that it exhibited TLR2-dependent cell activation and possessed strong cell-activating capacities in comparison with E. coli LPS (11). In the present study, we generated a PG1828-deficient mutant of P. gingivalis and showed that its LPS preparation significantly reduced cell activation via TLR2.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, growth conditions, and plasmid. P. gingivalis strain 381 was grown anaerobically at 37°C in brain heart infusion (BHI) broth (Difco, Detroit, Mich.) containing 0.5% yeast extract (Difco), 5 µg of hemin per ml, and 1 µg of vitamin K₃ per ml. E. coli strain DH5 was used for cloning and was grown on Luria-Bertani agar base (Difco) or in LB broth (Difco). Plasmid pVA2198, which carries the ermF-ermAM gene and confers erythromycin resistance, was also used (9). For selection or maintenance of the plasmid-containing strains, antibiotics (1 µg of clindamycin per ml for P. gingivalis and 100 µg of ampicillin per ml for E. coli) were added to the media.

DNA sequencing. A DNA fragment from PG1825 to PG1829 of P. gingivalis 381 was obtained by PCR, using synthesized primers designed according to the DNA sequence of P. gingivalis W83 (24). The sequence of the product was determined with the ABI PRISM 3100-Avant automated DNA sequencer (Applied Biosystems, Foster City, Calif.), using the BigDye Terminator version 3.1 cycle sequencing kit.

Construction of the PG1828-deficient mutant. To generate a mutant deficient in the PG1828-encoded lipoprotein, plasmid DNA constructs were made by replacing the PG1828 gene with an ermF-ermAM cassette (Fig. 1A). The BamHI/EcoRI fragment of pVA2198 containing the ermF-ermAM gene was ligated with BamHI/EcoRI-digested pBluescript II SK(+) (Stratagene, La Jolla, Calif.). The upstream flanking regions, including the entire PG1825, PG1826, and PG1827 genes, and the downstream flanking regions, including the entire PG1829 gene, were amplified from P. gingivalis 381 chromosomal DNA by using the following primers: 1825-dw (5'-CGCCGGTCTAGACTAAGGATTGATTAAAC-3' [the XbaI site is underlined]), 1827-dw (5'-TTTATTACTAGTGTGTGTGCTGAGCAGGC-3' [the SpeI site is underlined]), 1829-dw (5'-GAGTGTGATATCCGCATGGCTATTCCTTT-3' [the EcoRV site is underlined]), and 1829-up (5'-AAGTATCTCGAGCTGCTCGCCTTTTGATT-3' [the XhoI site is underlined]). Fragments digested with enzymes were cloned into pBluescript II SK(+) carrying the ermF-ermAM gene and transformed into E. coli DH5 by standard techniques (34). The BssHII fragment of the resulting construct containing the ermF-ermAM gene (Fig. 1A) was transformed into P. gingivalis 381 by electroporation as previously described, with some modifications (22, 33). Briefly, P. gingivalis cells were anaerobically grown at 37°C in 50 ml of BHI broth containing 0.5% yeast extract (Difco), 5 µg of hemin per ml, and 1 µg of vitamin K₃ per ml and then were washed with cold distilled water twice and resuspended in 200 µl of 1 mM MgCl₂ containing 10% glycerol. Linearized DNA samples (10 µg) were added to an 80-µl cell suspension and electroporated in a 0.1-cm-diameter cuvette at 200 , 1.8 kV, and 25 µF (Gene Pulser II system; Bio-Rad, Hercules, Calif.). The cells were then immediately mixed with 10 ml of prewarmed BHI broth containing 0.5% yeast extract, 5 µg of hemin per ml, and 1 µg of vitamin K₃ per ml and incubated anaerobically at 37°C for 16 h, after which they were plated onto BHI agar plates containing 0.5% yeast extract, 5 µg of hemin per ml, 1 µg of vitamin K₃ per ml, and 1 µg of clindamycin per ml. The plates were incubated for 5 to 7 days anaerobically at 37°C to develop colonies.

View larger version (23K): [in this window] [in a new window]

FIG. 1. Inactivation of PG1828 in P. gingivalis. (A) Construction of a PG1828-deficient mutant of P. gingivalis strain 381 by allelic exchange mutagenesis with an ermF-ermAM antibiotic resistance cassette (as described in Materials and Methods). (B) Predicted maps of the genomes of the wild-type and the PG1828-deficient mutant. Arrowheads indicate the numbers and positions of oligonucleotide primers for PCR analysis (as described in Materials and Methods). (C) PCR analysis of WT and PG1828. Numbers above the lanes indicate the primer pairs (B) used for the PCR analysis. Lane M, DNA marker; left margin, molecular sizes.

PCR analysis. Chromosomal DNA samples from the wild-type strain and PG1828-deficient mutant were amplified by using primers 1 (5'-CGATGAGCTTGCGGTCAATA-3'), 2 (5'-CCATGTACGTGAGCGGATAT-3'), 3 (5'-GAAGCTGTCAGTAGTATACC-3'), 4 (5'-GTTGCTTTTGCAGCT-3'), and 5 (5'-CCATGACGAAGAGTTCGAGA-3'), as shown in Fig. 1B, with the primer pairs indicated in Fig. 1C.

DNA probes and Southern blot analysis. An ermF-ermAM probe was prepared as a 2.1-kb fragment and amplified from pVA2198 by using the primers erm-up (5'-CCGCTATTGCTTTTTTGCTC-3') and erm-dw (identical to primer 3). The DNA probe was labeled with alkaline phosphatase (ALP) by using the AlkPhos direct labeling module (Amersham Biosciences, Piscataway, N.J). BamHI-digested chromosomal DNA samples from the wild-type strain and the PG1828-deficient mutant were electrophoresed on a 1% agarose gel and then transferred to a HyBond-N+ nylon membrane (Amersham Biosciences) by using an alkaline blotting protocol. The membrane was prehybridized at 60°C for 15 min in a hybridization buffer and then hybridized with an ALP-labeled probe at 60°C for 2 h. At the end of the procedure, the membrane was washed under highly stringent conditions, and then a chemiluminescence detection assay was carried out with the CDP-Star detection reagent (Amersham Biosciences). The membrane was exposed to X-ray film for 30 min, and signals were developed for each strain.

RT-PCR analysis. Total RNA samples were isolated from the wild-type strain and PG1828-deficient mutant by using TRIzol reagent (Gibco BRL, Gaithersburg, Md.) according to the manufacturer's instructions and then were treated with RNase-free DNase (TaKaRa, Shiga, Japan) based on a method described previously (7). Reverse transcription (RT) was conducted with avian myeloblastosis virus reverse transcriptase (TaKaRa) with random hexamers, and the cDNA was subjected to PCR to amplify the PG1828 gene by using the primers 1828-up (identical to primer 4) and 1828-dw (5'-TTGAGCGGCTTGTTCAG-3'). For a negative control, a non-RT sample was also amplified by PCR.

LPS preparation and bacterial component. Extraction of LPS by a phenol-water method was performed as described previously (11). The preparation was repurified by a detergent-modified phenol-water extraction method, as described by Manthey and Vogel (20), and visualized by Tris-glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 15% polyacrylamide gel and periodic acid-silver (Ag) staining (43). P. gingivalis lipid A was isolated by aqueous acetic acid hydrolysis of an LPS preparation from the wild-type strain, followed by successive separation by thin-layer chromatography, as previously described (28). E. coli O55:B5 LPS was obtained from Sigma Chemical Co. (St. Louis, Mo.).

LAL assay. Various doses of the repurified LPS preparations from the wild-type strain (WT-LPS) and PG1828-deficient mutant (PG1828-LPS) were mixed separately with Limulus amoebocyte lysate (LAL) reagent and then incubated at 37°C for 30 min, after which the activities were determined with a quantitative chromogenic assay (Seikagaku Kogyo, Tokyo, Japan).

Visualization of PG1828LP. The LPS preparation was subfractionated by hydrophobic interaction chromatography according to our previously reported method (11), with a slight modification. Briefly, the LPS preparation was subjected to chromatography on an octyl-Sepharose 4FF column (Amersham Biosciences) and eluted with 0.1 M of ammonium acetate with a linear gradient of 1-propanol (15 to 60%). PG1828LP in the LPS preparation was separated by Tris-glycine SDS-PAGE with a 15% polyacrylamide gel and visualized by Coomassie brilliant blue (CBB) staining with Simply Blue SafeStain (Invitrogen Corp., Carlsbad, Calif.).

Luciferase assay. Interleukin-3 (IL-3)-dependent murine Ba/F3 pro-B cells stably expressing p55IgLuc and an NF-B/DNA binding activity-dependent luciferase reporter construct (Ba/B), murine TLR2 and a p55IgLuc reporter construct (Ba/mTLR2), and murine TLR4/MD-2 and a p55IgLuc reporter construct (Ba/mTLR4/mMD-2), were used to detect NF-B-dependent luciferase activity, as described previously (12, 28). Briefly, the cells were inoculated onto 96-well plates at 10⁵ cells/100 µl of RPMI 1640 supplemented with 10% fetal bovine serum and were stimulated separately with the indicated doses of the WT-LPS or PG1828-LPS preparations. After 4 h at 37°C, 100 µl of Bright-Glo luciferase assay reagent (Promega, Madison, Wis.) was added to each well, and luminescence was quantified with a luminometer (Turner Designs luminometer model TD-20/20; Promega). E. coli LPS was used as a positive control for TLR4/MD-2 ligand.

Human embryonic kidney 293 cells were transiently cotransfected with 1 µg of a human TLR2 expression vector or its empty vector, 50 ng of pNF-B Luc reporter plasmid (Stratagene), and 50 ng of pRL-TK (Promega) by using Lipofectamine 2000 (Invitrogen Corp.). Twenty-four hours after transfection, the cells were stimulated with the indicated doses of the WT-LPS or PG1828-LPS preparations for 12 h, after which luciferase activity was measured by using a dual-luciferase reporter assay system (Promega).

Mice. BALB/c mice (male, 8 weeks old) were obtained from Japan SLC, Inc., Shizuoka, Japan. The animals received humane care in accordance with our institutional guidelines and the legal requirements of Japan.

Cytokine assay. Human gingival fibroblasts were prepared from clinically normal gingival tissues according to a method similar to that described previously (11). After written informed consent under a protocol approved by the Institutional Review Board of Asahi University was received, tissue samples were collected. The cells were cultured in -MEM (Sigma Chemical Co.) containing 10% fetal bovine serum, 50 µg of gentamicin per ml, and 50 ng of amphotericin B per ml at 37°C in a 5% (vol/vol) CO₂ atmosphere and then were used for the assays at the 5th and 13th passages. The cells were stimulated with the indicated doses of the test specimens for 24 h at 37°C. Following incubation, the culture supernatants were collected and analyzed for secreted IL-8 by using an enzyme-linked immunosorbent assay (ELISA) kit (GT, Minneapolis, Minn.). In some experiments, mice were intraperitoneally injected with 100 µg of WT-LPS or PG1828-LPS preparation along with 18 mg of D-galactosamine (D-GalN). Serum specimens from the two groups (each containing five mice) were obtained 1 h after the injection of LPS preparations. These specimens were analyzed for tumor necrosis factor alpha (TNF-) and IL-6 concentrations by using an ELISA kit (eBioscience, San Diego, Calif.).

Virulence model. It was previously demonstrated that P. gingivalis LPS exhibited weak lethal toxicity (40). To sensitize mice to the lethal effects of LPS preparations, we used D-GalN (10). Briefly, a model for lethal toxicity in mice was established with an intraperitoneal injection of the indicated doses of the LPS preparations along with 18 mg of D-GalN diluted in 500 µl of saline. For some of the experiments, mice were injected peritoneally with the indicated doses of living P. gingivalis cells. Survival was monitored for up to 7 days, and the 50% lethal dose (LD₅₀) was calculated as previously described (16).

Analysis of enzymatic activity. The amounts of cell-associated enzymes in the wild-type strain and the PG1828-deficient mutant were determined by a method similar to that described previously (3). Briefly, the cells were centrifuged and suspended in phosphate-buffered saline for enzyme analysis. An API ZYM chromogenic assay system (Bio Mérieux, Marcy-l'Etoile, France) was used for estimation of enzyme levels, and the intensity of the color reaction was graded semiquantitatively in comparison to a standard API ZYM color reaction chart. The results were determined from three independent experiments.

Statistical analysis. IL-8 production induced by the various doses of PG1828-LPS was assessed with a one-way analysis of variance, using the Bonferroni or Dunn method, and the results are presented as the mean ± standard error of the mean. Serum cytokine concentrations in different experimental groups were analyzed for statistical significance by using Welch's t test.

Nucleotide sequence accession number. The DNA sequence obtained in this study has been entered into the DDBJ database under accession number AB189170.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of PG1828-deficient mutant of P. gingivalis. To evaluate the biological properties of PG1828LP isolated from the P. gingivalis LPS preparation, we constructed a PG1828-deficient mutant of P. gingivalis strain 381. The method used for this inactivation is shown in Fig. 1A. Erythromycin resistance was used as the selective marker for homologous recombination between the knockout cassette and chromosomal DNA. The PG1828 knockout cassette was formed by replacing PG1828 with the erythromycin resistance genes, ermF-ermAM. Following the electrotransformation of P. gingivalis 381 cells, multiple clindamycin-resistant colonies appeared and a representative colony was selected. The resulting transformants with erythromycin resistance were verified by PCR to have the desired ermF-ermAM gene insertion (Fig. 1B and C). Southern blot hybridization with an ermF-ermAM gene probe exhibited a single band for the BamHI-digested genomic DNA of the PG1828-deficient mutant (Fig. 2A), and the mutant expressed no PG1828 mRNA in an RT-PCR analysis (Fig. 2B). Further, the DNA sequences of PG1825, PG1826, PG1827, ermF-ermAM, and PG1829 of the mutant were checked with P. gingivalis 381 genomic DNA (accession no. AB189170) and that of the ermF-ermAM gene (accession no. AF219231).

View larger version (41K): [in this window] [in a new window]

FIG. 2. (A) Detection of ermF-ermAM by Southern blot analyses of BamHI-digested chromosomal DNA samples from the wild-type and PG1828-deficient mutant strains. BamHI-digested chromosomal DNA was electrophoresed on a 1% agarose gel (lane M, DNA marker; lane 1, WT; lane 2, PG1828) and transferred to a nylon membrane. The membrane was hybridized with an ALP-labeled ermF-ermAM probe, and a chemiluminescence detection assay was carried out (lane 3, WT; lane 4, PG1828). Left margin, molecular sizes. (B) RT-PCR analysis of PG1828 mRNA expression in P. gingivalis. The presence (+) or absence (–) of RT in each RT-PCR mixture is indicated above the lanes.

Characterization of LPS preparation from the PG1828-deficient mutant. We extracted an LPS preparation from the PG1828-deficient mutant and evaluated its characteristics. The PG1828-LPS preparation showed nearly the same ladder-like pattern as the WT-LPS preparation on Ag-stained gels (Fig. 3A). Further, the clotting activities of the PG1828-LPS preparation in an LAL assay were the same as those of the WT-LPS preparation (Fig. 3B). These results indicated that the PG1828-LPS preparation was equivalent to the LPS molecule of the WT-LPS preparation. We previously showed that PG1828LP could be visualized as a 16-kDa band by SDS-PAGE with CBB staining (11); therefore, the absence of PG1828LP in the PG1828-LPS preparation was confirmed by protein visualization. These preparations were subfractionated by using hydrophobic interaction chromatography to enrich PG1828LP as described previously (11). PG1828LP was then concentrated into specific fractions, and a major CBB-positive band was found at 16 kDa in the WT-LPS preparation; however, no CBB-positive 16-kDa band was found in the PG1828-LPS preparation (Fig. 3C).

View larger version (39K): [in this window] [in a new window]

FIG. 3. Characterization of the PG1828-LPS preparation. (A) Tris-glycine SDS-PAGE was performed with a 15% polyacrylamide gel, and proteins were visualized by Ag staining. Lanes: 1, WT-LPS; 2, PG1828-LPS. (B) The indicated doses of the LPS preparations were mixed with LAL reagent, and LAL activity was determined with a quantitative chromogenic assay. Representative results from three independent experiments are shown. (C) The LPS preparation was subjected to chromatography on an octyl-Sepharose 4FF column and eluted with 0.1 M ammonium acetate with a linear gradient of 1-propanol (15 to 60%). The components in the specific fraction were separated by Tris-glycine SDS-PAGE with a 15% polyacrylamide gel, and PG1828LP was visualized by CBB staining. Lanes: MP, protein marker; 1, WT-LPS preparation; 2, PG1828-LPS preparation. Left margin, molecular sizes.

Immunostimulatory activities of the LPS preparation from the PG1828-deficient mutant. We examined TLR-dependent cell activation by the LPS preparations (Fig. 4). The WT-LPS preparation from P. gingivalis 381 exhibited a much higher murine TLR2-dependent NF-B activation than the PG1828-LPS preparation from the PG1828LP-deficient mutant, whereas only marginal murine TLR4/MD-2-dependent NF-B activation compared to that with E. coli LPS was observed in both LPS preparations. The same results were obtained for the human TLR2-expressing cell line HEK293/hTLR2.

View larger version (22K): [in this window] [in a new window]

FIG. 4. TLR-dependent activation of the PG1828-LPS preparation. (A) Ba/B, Ba/mTLR2, and Ba/mTLR4/mMD-2 cells were stimulated with the indicated doses of WT-LPS or PG1828-LPS preparations for 4 h. E. coli LPS was used as a positive control for TLR4/MD-2 ligand. (B) HEK293/hTLR2 and HEK293/Empty were stimulated with the indicated doses of the WT-LPS or PG1828-LPS preparations for 12 h. NF-B activation was measured with a luciferase assay. Results are shown as relative luciferase activity, which was determined as the ratio of stimulated to nonstimulated activity.

To clarify the cell-activating capacities, we next examined the IL-8-producing activity of the PG1828-LPS preparation by using human gingival fibroblasts, which constitutively express TLR2 and TLR4 (39). PG1828-LPS showed a lower level of IL-8 production than the WT-LPS preparation, which was nearly the same as that of highly purified P. gingivalis lipid A (Fig. 5). These results demonstrated that PG1828LP is a major cell-activating component in the P. gingivalis LPS preparation.

View larger version (13K): [in this window] [in a new window]

FIG. 5. Cytokine production by human gingival fibroblasts stimulated with the PG1828-LPS preparation. The cells were stimulated with the indicated doses of the WT-LPS preparation, the PG1828-LPS preparation, or P. gingivalis lipid A for 24 h. IL-8 production was analyzed by ELISA. Data are shown as the means ± standard error of the means from three independent experiments. P. gingivalis lipid A was used for TLR4/MD-2 ligand in the P. gingivalis LPS preparation, and the data were described previously (11). *, significantly different from the mean value of each specimen dose against the PG1828-LPS preparation (P < 0.01).

Virulence of the PG1828-deficient mutant in vivo. To investigate whether PG1828LP is associated with virulence of the bacterium, murine lethal shock was examined. The PG1828-deficient mutant of P. gingivalis 381 exhibited a lower level of toxicity than the wild-type strain (Table 1). In addition, an intraperitoneal injection of the WT-LPS preparation resulted in lethal toxicity against D-GalN-sensitized mice, with an LD₅₀ of 37.8 µg/mouse (Table 2). On the other hand, the PG1828-LPS preparation induced no lethal activities at up to 100 µg/mouse. Further, the mice injected with the PG1828-LPS preparation showed a significant decrease in TNF- and IL-6 concentrations in serum compared to those injected with the WT-LPS preparation (Fig. 6).

View this table: [in this window] [in a new window]

TABLE 1. Lethal toxicity of P. gingivalis cells in BALB/c mice

View this table: [in this window] [in a new window]

TABLE 2. Lethal toxicity of P. gingivalis LPS preparations in D-GalN-sensitized BALB/c mice

View larger version (13K): [in this window] [in a new window]

FIG. 6. Cytokine concentrations in sera of mice treated with the PG1828-LPS preparation. Mice were intraperitoneally injected with 100 µg of WT-LPS or PG1828-LPS preparations along with 18 mg of D-GalN, and then serum specimens were collected 1 h after injection of the LPS preparations. TNF- and IL-6 concentrations were analyzed by ELISA. Error bars indicate standard errors of the means. *, significantly different from the mean value for the WT-LPS preparation (P < 0.01).

It was previously demonstrated that various proteinases contribute to the virulence of P. gingivalis organisms (17, 26, 31). Both wild-type and PG1828-deficient mutant strains exhibit significant levels of alkaline phosphatase, trypsin, acid phosphatase, and N-acetyl-ß-glucosaminidase, as well as weak butyrate esterase (C4), leucine arylamidase, cysteine arylamidase, and phosphoamidase activities (data not shown). The enzymatic characterizations of these organisms in the present study were consistent with those in a previous report (37). Together, these results show that the reduced lethal activity of the present PG1828-deficient mutant was not associated with its enzymatic properties.

Top Abstract Introduction Materials and Methods Results Discussion References

 
TLR2 is a main signal-transducing receptor that confers sensitivity to a variety of microbial pathogens, such as peptidoglycan, lipoarabinomannan, and bacterial lipoproteins, as well as others (41). Among those pathogens, lipoprotein has been reported to exhibit many biological activities associated with LPS (23, 46). Further, commercially available LPS preparations have been shown to slightly induce signals via TLR2 and TLR4, although the activities, caused by lipoproteins in those preparations, were shown to be removable by phenol reextraction (14, 19).

In the present study, we used a genetic approach to further examine the participation of lipoprotein in a P. gingivalis LPS preparation. The PG1828-deficient mutant failed to express PG1828 mRNA (Fig. 2), resulting in the disappearance of the 16-kDa CBB-positive stained band in the PG1828-LPS preparation assay results (Fig. 3). These findings indicate that the desired lipoprotein, PG1828LP, was successfully deleted from the deficient mutant. Further, the PG1828-LPS and WT-LPS preparations each showed a ladder-like pattern in Ag-stained gels, indicating the presence of a glycoconjugate with repeating units, which is a characteristic of LPS molecules, and there were no differences in LAL clotting activity between the two preparations (Fig. 3). On the other hand, the PG1828-LPS preparation exhibited a marked reduction of activation of TLR2-expressing cells compared to the WT-LPS preparation, while it slightly activated human gingival fibroblasts in a manner comparable to the stimulation of highly purified P. gingivalis lipid A by TLR4/MD-2 (Fig. 4 and 5). These results indicate that the cell activation induced by the PG1828-LPS preparation may have been be due to P. gingivalis LPS molecules.

Several bacterial lipoproteins have been identified and are thought to play important roles in bacterial pathogenesis. It was previously shown that a 47-kDa lipoprotein (GNA47) of Neisseria meningitidis was associated with peptidoglycan metabolism, cell separation, and cell membrane architecture and that it also caused bacteremia and mortality in infant rats (1). In addition, the spirochetal lipoprotein of OspC-deficient Borrelia burgdorferi was demonstrated to be unable to invade tick salivary glands, which is a critical step in transmission from the arthropod vector to mammalian host (29). Recently, a murein lipoprotein-deficient mutant of Salmonella enterica serovar Typhimurium was demonstrated to be nonvirulent in a murine lethal model (36). In the present study, the PG1828-deficient mutant produced a significantly reduced mortality rate in mice in comparison to the wild-type strain (Table 1), and an intraperitoneal injection of the PG1828-LPS preparation was not toxic in D-GalN-sensitized mice, even at the highest dose tested (100 µg/mouse) (Table 2). In another study, the lipoprotein obtained from E. coli cells was found to induce lethal shock against D-GalN-sensitized mice, and a heat-killed preparation from a lipoprotein-deficient E. coli mutant was less efficient than that from wild-type E. coli (46).

It was reported that various proteases were secreted by P. gingivalis organisms (17, 26, 31). We found here that both the wild-type and the PG1828-deficient mutant exhibited trypsin-like protease activity (data not shown). The intrinsic protease may digest PG1828LP released from the organisms. The protease, however, seems not to inhibit the virulence of PG1828LP (Table 1). Since mature PG1828LP contains two lysine at the 6th and 42nd amino acid residues from N-terminally lipidated cysteine, tryptic digestion of the lipoprotein gives three peptide fragments, i.e., residues 1 to 6, 7 to 42, and 43 to 52. Our previous study showed that the TLR2-mediated immunostimulating activity of isolated PG1828LP was reduced only about 10-fold by trypsin digestion and that the lipopeptide fragment from residue 1 to 6 still retained the activity (11). It has also been reported that proteinase K or trypsin digestion fails to inhibit the activities of lipoproteins from other bacterial species (8, 35). Further, synthetic lipopeptides with short peptides, such as Pam₃CSK₄, show strong TLR2-mediated activities (2). Thus, released PG1828LP, which exhibits immunostimulating activity in vivo and is possibly accessible to intrinsic proteases, acts as a virulence factor of P. gingivalis, even if it is digested.

In conclusion, we showed that PG1828LP, a PG1828-encoded lipoprotein from P. gingivalis, is a principal component for cell activation and virulence of the organism. The reduced activity of the PG1828-deficient mutant and its LPS preparation in proinflammatory cytokine production and lethal toxicity suggests that PG1828LP is a virulence factor associated with periodontal diseases.

 
This work was supported in part by a Grant-in-Aid for Scientific Research in Priority Areas (no. 16017299) of the Ministry of Education, Culture, Sports, Science and Technology, Japan.

We thank Chieko Kanamori for technical assistance and Mark Benton for critical reading of the manuscript.

 
* Corresponding author. Mailing address: Department of Oral Microbiology, Asahi University School of Dentistry, 1851-1 Hozumi, Mizuho, Gifu 501-0296, Japan. Phone: 81-58-329-1421. Fax: 81-58-329-1421. E-mail: tomo527{at}dent.asahi-u.ac.jp.

Editor: W. A. Petri, Jr.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Adu-Bobie, J., P. Lupetti, B. Brunelli, D. Granoff, N. Norais, G. Ferrari, G. Grandi, R. Rappuoli, and M. Pizza. 2004. GNA33 of Neisseria meningitidis is a lipoprotein required for cell separation, membrane architecture, and virulence. Infect. Immun. 72:1914-1919.[Abstract/Free Full Text]
2
2. Aliprantis, A. O., R. B. Yang, M. R. Mark, S. Suggett, B. Devaux, J. D. Radolf, G. R. Klimpel, P. Godowski, and A. Zychlinsky. 1999. Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2. Science 285:736-739.[Abstract/Free Full Text]
3
3. Asai, Y., T. Jinno, and T. Ogawa. 2003. Oral treponemes and their outer membrane extracts activate human gingival epithelial cells through Toll-like receptor 2. Infect. Immun. 71:717-725.[Abstract/Free Full Text]
4
4. Bainbridge, B. W., and R. P. Darveau. 2001. Porphyromonas gingivalis lipopolysaccharide: an unusual pattern recognition receptor ligand for the innate host defense system. Acta Odontol. Scand. 59:131-138.[CrossRef][Medline]
5
5. Coats, S. R., R. A. Reife, B. W. Bainbridge, T. T. Pham, and R. P. Darveau. 2003. Porphyromonas gingivalis lipopolysaccharide antagonizes Escherichia coli lipopolysaccharide at toll-like receptor 4 in human endothelial cells. Infect. Immun. 71:6799-6807.[Abstract/Free Full Text]
6
6. Darveau, R. P., T. T. Pham, K. Lemley, R. A. Reife, B. W. Bainbridge, S. R. Coats, W. N. Howald, S. S. Way, and A. M. Hajjar. 2004. Porphyromonas gingivalis lipopolysaccharide contains multiple lipid A species that functionally interact with both Toll-like receptors 2 and 4. Infect. Immun. 72:5041-5051.[Abstract/Free Full Text]
7
7. Dilworth, D. D., and J. R. McCarrey. 1992. Single-step elimination of contaminating DNA prior to reverse transcriptase PCR. PCR Methods Appl. 1:279-282.[Medline]
8
8. Dong, L., K. Shibata, Y. Sawa, A. Hasebe, Y. Yamaoka, S. Yoshida, and T. Watanabe. 1999. Transcriptional activation of mRNA of intercellular adhesion molecule 1 and induction of its cell surface expression in normal human gingival fibroblasts by Mycoplasma salivarium and Mycoplasma fermentans. Infect. Immun. 67:3061-3065.[Abstract/Free Full Text]
9
9. Fletcher, H. M., H. A. Schenkein, R. M. Morgan, K. A. Bailey, C. R. Berry, and F. L. Macrina. 1995. Virulence of a Porphyromonas gingivalis W83 mutant defective in the prtH gene. Infect. Immun. 63:1521-1528.[Abstract]
10
10. Galanos, C., M. A. Freudenberg, and W. Reutter. 1979. Galactosamine-induced sensitization to the lethal effects of endotoxin. Proc. Natl. Acad. Sci. USA 76:5939-5943.[Abstract/Free Full Text]
11
11. Hashimoto, M., Y. Asai, and T. Ogawa. 2004. Separation and structural analysis of lipoprotein in a lipopolysaccharide preparation from Porphyromonas gingivalis. Int. Immunol. 16:1431-1437.[Abstract/Free Full Text]
12
12. Hashimoto, M., Y. Asai, and T. Ogawa. 2003. Treponemal phospholipids inhibit innate immune responses induced by pathogen-associated molecular patterns. J. Biol. Chem. 278:44205-44213.[Abstract/Free Full Text]
13
13. Hirschfeld, M., J. J. Weis, V. Toshchakov, C. A. Salkowski, M. J. Cody, D. C. Ward, N. Qureshi, S. M. Michalek, and S. N. Vogel. 2001. Signaling by Toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect. Immun. 69:1477-1482.[Abstract/Free Full Text]
14
14. Hirschfeld, M., Y. Ma, J. H. Weis, S. N. Vogel, and J. J. Weis. 2000. Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine Toll-like receptor 2. J. Immunol. 165:618-622.[Abstract/Free Full Text]
15
15. Holt, S. C., L. Kesavalu, S. Walker, and C. A. Genco. 1999. Virulence factors of Porphyromonas gingivalis. Periodontol. 2000 20:168-238.[CrossRef]
16
16. Kärber, G. 1931. Beitrag zur kollektiven behandlung pharmakologischer reihenversuche. Arch. Exp. Pathol. Pharmakol. 162:480-483.[CrossRef]
17
17. Kesavalu, L., S. C. Holt, and J. L. Ebersole. 1996. Trypsin-like protease activity of Porphyromonas gingivalis as a potential virulence factor in a murine lesion model. Microb. Pathog. 20:1-10.[CrossRef][Medline]
18
18. Kirikae, T., T. Nitta, F. Kirikae, Y. Suda, S. Kusumoto, N. Qureshi, and M. Nakano. 1999. Lipopolysaccharides (LPS) of oral black-pigmented bacteria induce tumor necrosis factor production by LPS-refractory C3H/HeJ macrophages in a way different from that of Salmonella LPS. Infect. Immun. 67:1736-1742.[Abstract/Free Full Text]
19
19. Lee, H. K., J. Lee, and P. S. Tobias. 2002. Two lipoproteins extracted from Escherichia coli K-12 LCD25 lipopolysaccharide are the major components responsible for Toll-like receptor 2-mediated signaling. J. Immunol. 168:4012-4017.[Abstract/Free Full Text]
20
20. Manthey, C. L., and S. N. Vogel. 1994. Elimination of trace endotoxin protein from rough chemotype LPS. J. Endotoxin Res. 1:84-91.
21
21. Nair, B. C., W. R. Mayberry, R. Dziak, P. B. Chen, M. J. Levine, and E. Hausmann. 1983. Biological effects of a purified lipopolysaccharide from Bacteroides gingivalis. J. Periodont. Res. 18:40-49.[CrossRef][Medline]
22
22. Nakayama, K., T. Kadowaki, K. Okamoto, and K. Yamamoto. 1995. Construction and characterization of arginine-specific cysteine proteinase (Arg-gingipain)-deficient mutants of Porphyromonas gingivalis. Evidence for significant contribution of Arg-gingipain to virulence. J. Biol. Chem. 270:23619-23626.[Abstract/Free Full Text]
23
23. Neilsen, P. O., G. A. Zimmerman, and T. M. McIntyre. 2001. Escherichia coli Braun lipoprotein induces a lipopolysaccharide-like endotoxic response from primary human endothelial cells. J. Immunol. 167:5231-5239.[Abstract/Free Full Text]
24
24. Nelson, K. E., R. D. Fleischmann, R. T. DeBoy, I. T. Paulsen, D. E. Fouts, J. A. Eisen, S. C. Daugherty, R. J. Dodson, A. S. Durkin, M. Gwinn, D. H. Haft, J. F. Kolonay, W. C. Nelson, T. Mason, L. Tallon, J. Gray, D. Granger, H. Tettelin, H. Dong, J. L. Galvin, M. J. Duncan, F. E. Dewhirst, and C. M. Fraser. 2003. Complete genome sequence of the oral pathogenic bacterium Porphyromonas gingivalis strain W83. J. Bacteriol. 185:5591-5601.[Abstract/Free Full Text]
25
25. Netea, M. G., M. van Deuren, B. J. Kullberg, J. M. Cavaillon, and J. W. van der Meer. 2002. Does the shape of lipid A determine the interaction of LPS with Toll-like receptors? Trends Immunol. 23:135-139.[CrossRef][Medline]
26
26. O'Brien-Simpson, N. M., P. D. Veith, S. G. Dashper, and E. C. Reynolds. 2003. Porphyromonas gingivalis gingipains: the molecular teeth of a microbial vampire. Curr. Protein Pept. Sci. 4:409-426.[CrossRef][Medline]
27
27. Ogawa, T. 1994. Immunobiological properties of chemically defined lipid A from lipopolysaccharide of Porphyromonas (Bacteroides) gingivalis. Eur. J. Biochem. 19:737-742.
28
28. Ogawa, T., Y. Asai, M. Hashimoto, O. Takeuchi, T. Kurita, Y. Yoshikai, K. Miyake, and S. Akira. 2002. Cell activation by Porphyromonas gingivalis lipid A molecule through Toll-like receptor 4- and myeloid differentiation factor 88-dependent signaling pathway. Int. Immunol. 14:1325-1332.[Abstract/Free Full Text]
29
29. Pal, U., X. Yang, M. Chen, L. K. Bockenstedt, J. F. Anderson, R. A. Flavell, M. V. Norgard, and E. Fikrig. 2004. OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands. J. Clin. Investig. 113:220-230.[CrossRef][Medline]
30
30. Poltorak, A., X. He, I. Smirnova, M. Y. Liu, C. van Huffel, X. Du, D. Birdwell, E. Alejos, M. Silva, C. Galanos, M. Freudenberg, P. Ricciardi-Castagnoli, B. Layton, and B. Beutler. 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085-2088.[Abstract/Free Full Text]
31
31. Potempa, J., A. Banbula, and J. Travis. 2000. Role of bacterial proteinases in matrix destruction and modulation of host responses. Periodontol. 2000 24:153-192.[CrossRef]
32
32. Rietschel, E. T., H. Brade, O. Holst, L. Brade, S. Muller-Loennies, U. Mamat, U. Zahringer, F. Beckmann, U. Seydel, K. Brandenburg, A. J. Ulmer, T. Mattern, H. Heine, J. Schletter, H. Loppnow, U. Schonbeck, H. D. Flad, S. Hauschildt, U. F. Schade, F. Di Padova, S. Kusumoto, and R. R. Schumann. 1996. Bacterial endotoxin: chemical constitution, biological recognition, host response, and immunological detoxification. Curr. Top. Microbiol. Immunol. 216:39-81.[Medline]
33
33. Ross, B. C., L. Czajkowski, K. L. Vandenberg, S. Camuglia, J. Woods, C. Agius, R. Paolini, E. Reynolds, and I. G. Barr. 2004. Characterization of two outer membrane protein antigens of Porphyromonas gingivalis that are protective in a murine lesion model. Oral Microbiol. Immunol. 19:6-15.[CrossRef][Medline]
34
34. Sambrook, J., E. F. Fritsch, and T. Maniaatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
35
35. Scragg, I. G., D. Kwiatkowski, V. Vidal, A. Reason, T. Paxton, M. Panico, A. Dell, and H. Morris. 2000. Structural characterization of the inflammatory moiety of a variable major lipoprotein of Borrelia recurrentis. J. Biol. Chem. 275:937-941.[Abstract/Free Full Text]
36
36. Sha, J., A. A. Fadl, G. R. Klimpel, D. W. Niesel, V. L. Popov, and A. K. Chopra. 2004. The two murein lipoproteins of Salmonella enterica serovar Typhimurium contribute to the virulence of the organism. Infect. Immun. 72:3987-4003.[Abstract/Free Full Text]
37
37. Slots, J. 1981. Enzymatic characterization of some oral and nonoral gram-negative bacteria with the API ZYM system. J. Clin. Microbiol. 14:288-294.[Abstract/Free Full Text]
38
38. Socransky, S. S., A. D. Haffajee, M. A. Cugini, C. Smith, and R. L. Kent Jr. 1998. Microbial complexes in subgingival plaque. J. Clin. Periodontol. 25:134-144.[CrossRef][Medline]
39
39. Tabeta, K., K. Yamazaki, S. Akashi, K. Miyake, H. Kumada, T. Umemoto, and H. Yoshie. 2000. Toll-like receptors confer responsiveness to lipopolysaccharide from Porphyromonas gingivalis in human gingival fibroblasts. Infect. Immun. 68:3731-3735.[Abstract/Free Full Text]
40
40. Takada, H., H. Hirai, T. Fujiwara, T. Koga, T. Ogawa, and S. Hamada. 1990. Bacteroides lipopolysaccharides (LPS) induce anaphylactoid and lethal reactions in LPS-responsive and -nonresponsive mice primed with muramyl dipeptide. J. Infect. Dis. 162:428-434.[Medline]
41
41. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu. Rev. Immunol. 21:335-376.[CrossRef][Medline]
42
42. Tanamoto, K., S. Azumi, Y. Haishima, H. Kumada, and T. Umemoto. 1997. The lipid A moiety of Porphyromonas gingivalis lipopolysaccharide specifically mediates the activation of C3H/HeJ mice. J. Immunol. 158:4430-4436.[Abstract]
43
43. Tsai, C. M., and C. E. Frasch. 1982. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119:115-119.[CrossRef][Medline]
44
44. Underhill, D. M., and A. Ozinsky. 2002. Toll-like receptors: key mediators of microbe detection. Curr. Opin. Immunol. 14:103-110.[CrossRef][Medline]
45
45. Yoshimura, A., T. Kaneko, Y. Kato, D. T. Golenbock, and Y. Hara. 2002. Lipopolysaccharides from periodontopathic bacteria Porphyromonas gingivalis and Capnocytophaga ochracea are antagonists for human toll-like receptor 4. Infect. Immun. 70:218-225.[Abstract/Free Full Text]
46
46. Zhang, H., J. W. Peterson, D. W. Niesel, and G. R. Klimpel. 1997. Bacterial lipoprotein and lipopolysaccharide act synergistically to induce lethal shock and proinflammatory cytokine production. J. Immunol. 159:4868-4878.[Abstract]

---

Infection and Immunity, April 2005, p. 2157-2163, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.2157-2163.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Thakran, S., Li, H., Lavine, C. L., Miller, M. A., Bina, J. E., Bina, X. R., Re, F. (2008). Identification of Francisella tularensis Lipoproteins That Stimulate the Toll-like Receptor (TLR) 2/TLR1 Heterodimer. J. Biol. Chem. 283: 3751-3760 [Abstract] [Full Text]  
• Giacaman, R. A., Nobbs, A. H., Ross, K. F., Herzberg, M. C. (2007). Porphyromonas gingivalis Selectively Up-Regulates the HIV-1 Coreceptor CCR5 in Oral Keratinocytes. J. Immunol. 179: 2542-2550 [Abstract] [Full Text]  
• Hashimoto, M., Tawaratsumida, K., Kariya, H., Aoyama, K., Tamura, T., Suda, Y. (2006). Lipoprotein is a predominant Toll-like receptor 2 ligand in Staphylococcus aureus cell wall components. Int Immunol 18: 355-362 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Asai, Y. Articles by Ogawa, T. Search for Related Content PubMed PubMed Citation Articles by Asai, Y. Articles by Ogawa, T.