Altered Gingipain Maturation in vimA- and vimE-Defective Isogenic Mutants of Porphyromonas gingivalis
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感染与免疫杂志 2005年第3期
Department of Biochemistry and Microbiology, School of Medicine, Loma Linda University, Loma Linda, California
ABSTRACT
We have previously shown that gingipain activity in Porphyromonas gingivalis is modulated by the unique vimA and vimE genes. To determine if these genes had a similar phenotypic effect on protease maturation and activation, isogenic mutants defective in those genes were further characterized. Western blot analyses with antigingipain antibodies showed RgpA-, RgpB-, and Kgp-immunoreactive bands in membrane fractions as well as the culture supernatant of both P. gingivalis W83 and FLL93, the vimE-defective mutant. In contrast, the membrane of P. gingivalis FLL92, the vimA-defective mutant, demonstrated immunoreactivity only with RgpB antibodies. With mass spectrometry or Western blots, full-length RgpA and RgpB were identified from extracellular fractions. In similar extracellular fractions from P. gingivalis FLL92 and FLL93, purified RgpB activated only arginine-specific activity. In addition, the lipopolysaccharide profiles of the vimA and vimE mutants were truncated in comparison to that of W83. While glycosylated proteins were detected in the membrane and extracellular fractions from the vimA- and vimE-defective mutants, a monoclonal antibody (1B5) that reacts with specific sugar moieties of the P. gingivalis cell surface polysaccharide and membrane-associated Rgp gingipain showed no immunoreactivity with these fractions. Taken together, these results indicate a possible defect in sugar biogenesis in both the vimA- and vimE-defective mutants. These modulating genes play a role in the secretion, processing, and/or anchorage of gingipains on the cell surface.
INTRODUCTION
Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobic bacterium, is an important etiological agent of chronic periodontitis. Furthermore, accumulating data suggest that this organism is also associated with systemic diseases and complications including atherosclerosis, preterm births, and low-birth-weight babies (15). While several virulence factors are implicated in the pathogenicity of P. gingivalis, the proteolytic abilities of this organism have been considered to play the most significant role in its virulence (7, 11, 14, 15, 17). The major proteases, called gingipains, consist of an arginine-specific protease (Arg-gingipain [Rgp]) and lysine-specific protease (Lys-gingipain [Kgp]). The Rgp is encoded by two genes, rgpA and rgpB, while Kgp is encoded by a single gene, kgp. These proteases, which are both extracellular and cell associated, are required for growth of the organism and other housekeeping functions, which include processing enzymes for various cell surface proteins, including individual proteases (24). Moreover, the multiple pathogenic effects of the gingipains, such as degradation of complement and immunoglobulin, inactivation of cytokines and their receptors, aggregation of platelets, attenuation of neutrophil antibacterial activities, and increase in vascular permeability and blood clotting prevention, are well documented (14).
We have reported previously that the vimA and vimE genes can modulate the phenotypic expression of the gingipains in P. gingivalis (1, 23). While the vimA gene is part of the bcp-recA-vimA transcriptional unit, the downstream vimE can either be cotranscribed with vimA or independently expressed (23). The vimA- and vimE-defective mutant strains, designated P. gingivalis FLL92 and FLL93, respectively, were non-black pigmented and showed significant reductions in proteolytic, hemolytic, and hemagglutinating activities (1, 23). In in vivo experiments with a mouse model, the virulence of P. gingivalis FLL92 was dramatically reduced in comparison to that of the wild-type W83 strain (1).
While reductions in Arg-X- and Lys-X-specific proteolytic activities were observed in P. gingivalis FLL92 and FLL93, transcription of the gingipain genes was unaltered in these mutants compared to the wild-type strain (1, 23). A similar phenotype of the gingipain genes was also seen in P. gingivalis FLL32, a recA-defective isogenic mutant that had reduced Arg-X- and Lys-X-specific proteolytic activities (2). The 64-kDa RgpB partially processed proenzyme was secreted in both P. gingivalis FLL92 and FLL93 (19, 23). The distribution of the proteolytic activity in P. gingivalis FLL93 was similar to that of the wild-type strain and in contrast to P. gingivalis FLL92, the vimA-defective mutant, which had mostly soluble proteolytic activity with little or no cell-associated activity (23). While there was a unique late onset of Arg-X- and Lys-X-specific proteolytic activity in P. gingivalis FLL92, there were little or no observed changes of proteolytic activity in stationary phase in P. gingivalis FLL93, the vimE-defective mutant (23). Collectively, these observations have raised the question of whether the regulation of proteolytic activity in P. gingivalis occurs by multiple mechanisms. Furthermore, it is unclear if the vimA and vimE gene products are part of a common pathway for protease maturation and activation.
In this report, we have further characterized the vimE-defective P. gingivalis FLL93 strain in comparison to the vimA-defective strain FLL92 and the wild type. Our findings suggest an important role for these genes in gingipain secretion, processing, and/or anchorage of gingipains on the cell surface.
MATERIALS AND METHODS
Bacterial strains, growth conditions, and plasmids. P. gingivalis W83 and isogenic strains FLL92 and FLL93 were grown in brain heart infusion (BHI) broth (Difco Laboratories, Detroit, Mich.) supplemented with hemin (5 μg/ml), vitamin K (0.5 μg/ml), cysteine (0.1%), and yeast extract (0.5%). Unless otherwise stated, all cultures were incubated at 37°C and maintained in an anaerobic chamber (Coy Manufacturing, Ann Arbor, Mich.) in 10% H2, 10% CO2, and 80% N2. The growth rates for the P. gingivalis strains were determined spectrophotometrically (optical density at 600 nm).
Preparation of P. gingivalis extracellular fractions. One-liter cultures of P. gingivalis strains FLL92, FLL93, and W83 were grown from actively growing cells. Cells were harvested by centrifugation at 10,000 x g for 30 min. The cell-free culture fluid was precipitated with cold (–20°C), 37.5% or 60% acetone, and the protein pellet was resuspended in 7 ml of 100 mM Tris-HCl buffer (pH 7.4), dialyzed for 24 h against the same buffer, and then stored on ice or at 0°C.
Preparation of membrane fraction. One-liter cultures of P. gingivalis strains FLL92, FLL93, and W83 were grown from actively growing cells to the stationary phase (optical density at 600 nm of 1.3 to 1.4). Cells were harvested by centrifugation at 10,000 x g for 30 min. Membrane fractions were prepared with the French press (American Instrument Company, Silver Spring, Md.). The lysed cells were then centrifuged at 27,000 x g for 1 h. The supernatant was subjected to ultracentrifugation at 100,000 x g for 1 h. The pellet, designated the membrane fraction, was resuspended in 100 mM Tris-HCl, pH 7.4, containing 10 mM tosyl lysyl chloromethyl ketone (TLCK), a protease inhibitor. The remaining supernatant was considered the cytosolic fraction.
Membrane vesicle preparation. P. gingivalis W83, FLL92, and FLL93 were grown for 48 h in BHI supplemented with hemin and vitamin K. Membrane vesicles were prepared as previously described (19).
Protease assays. The presence of Arg-X- and Lys-X-specific cysteine protease activities was determined with a microplate reader (Bio-Rad, Hercules, Calif.) as previously reported (20).
Gel electrophoresis and immunoblot analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with a 4 to 12% Bis-Tris separating gel in MOPS (morpholinepropanesulfonic acid)-SDS running buffer according to the manufacturer's instructions (NuPAGE Novex gels; Invitrogen, Carlsbad, Calif.). Samples were prepared (65% sample, 25% 4x NuPAGE LDS sample buffer, 10% NuPAGE reducing agent), heated at 72°C for 10 min, and then electrophoresed at 200 V for 65 min in the XCell SureLock Mini-Cell system (Invitrogen, Carlsbad, Calif.). The protein bands were visualized by staining with Simply Blue Safe stain (Invitrogen, Carlsbad, Calif.). The separated proteins were then transferred to BioTrace nitrocellulose membranes (Pall Corporation, Ann Arbor, Mich.) and processed at 15 V for 25 min with a semidry Trans-blot apparatus (Bio-Rad, Hercules, Calif.). The blots were probed with gingipain-specific antibodies. Immunoreactive proteins were detected by the procedure described in the Western Lightning chemiluminescence reagent plus kit (Perkin-Elmer Life Sciences. Boston, Mass.). The secondary antibody was immunoglobulin G (heavy plus light chains)-horseradish peroxidase conjugate (Zymed Laboratories, Inc., South San Francisco, Calif..).
Activation of inactive gingipains with purified RgpB. Approximately 2.5 units of purified RgpB (Athens Research & Technology. Athens, Ga.) was incubated with approximately 750 μg of extracellular extracts of P. gingivalis FLL92 grown to the exponential phase (optical density at 600 nm of 0.7) or P. gingivalis FLL93 grown to the stationary phase. The reaction was performed in activated assay buffer (0.1 M Tris, pH 7.6, 0.2 M NaCl, 5 mM CaCl2, 10 mM NaOH, 9 mM L-cysteine). Samples were incubated at 37°C for 1 h under anaerobic conditions (10% H2, 10% CO2, 80% N2). Samples were then assayed for proteolytic activity and analyzed by immunoblot analysis.
Lipopolysaccharide and polysaccharide isolation and silver staining. Overnight cultures of P. gingivalis W83, FLL92, and FLL93 were grown to the stationary phase. Lipopolysaccharide isolation was done according to the manufacturer's protocol (Intron Biotechnology). Polysaccharides were separated from lipid A by treating the lipopolysaccharide preparations with 2% acetic acid for 2 h at 100°C. The soluble fraction containing the polysaccharides was purified with the Superdex 75 column (Amersham Biosciences, Piscataway, N.J.). Lipopolysaccharides were subjected to electrophoresis (SDS-PAGE) on 4 to 12% Bis-Tris gel with the MOPS running buffer. Polysaccharides were subjected to electrophoresis (SDS-PAGE) on 4 to 12% Bis-Tris gels with the morpholineethanesulfonic acid (MES) running buffer. Lipopolysaccharides and polysaccharides were visualized with the SilverQuest silver staining kit according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif.).
Mass spectrometry sequence analysis. Extracellular fractions of P. gingivalis FLL93 from the stationary phase were subjected to SDS-PAGE. Desired bands were excised from the gel and dried in a Speed Vac for 1 h. Dried gel bands were then reduced with 20 μl of 20 mM tri(2-carboxyethyl)phosphine at 56°C and then alkylated with 20 μl of 40 mM iodoacetamide for 30 min at 23°C. Alkylated samples were then washed twice with 100 mM ammonium bicarbonate and dried with the Speed Vac for 1 h. Digestion buffer (15 μl of 0.05 μg/μl trypsin stock diluted in 3% acetic acid) was added to the dried gel slice and incubated for 10 min on ice. Excess digestion buffer was removed, and 10 μl of 100 mM ammonium bicarbonate was added to cover the gel slice to prevent drying. The gel slices were then incubated for 16 h at 30°C. After 16 h, an additional 10 μl of 100 mM ammonium bicarbonate was added to the gel slices and then incubated for an additional 30 min at 30°C. Proteins were then trapped, washed and eluted with ZipTipC18 according to the manufacturer's instructions (Millipore, Bellerica, Mass.).
Eluted peptides were dried in the Speed Vac for 5 to 10 min and resuspended in 0.05% trifluoroacetic acid in mass spectrometry grade water. All buffers were prepared in 100 mM ammonium bicarbonate. Tryptic peptides were separated and analyzed on a Picoview model PV-500 Nanospray ESI unit (New Objective, Woodburn, Mass.) coupled to an LCQ Deca XP ion trap mass spectrometer (Thermo Electron, San Jose, Calif.) with a four-event program consisting of a full mass spectrometry scan followed by three mass spectrometry/mass spectrometry events for the most intense ions on full mass spectrometry. A capillary column (75 μm by 10 cm) packed with 5μC-18-coated silica was developed with a 40-min gradient elution program of 2 to 90% acetonitrile buffered with 0.5% acetic acid and 0.005% trifluoroacetic acid at a flow rate of 300 nl/min. Data were collected with the Xcalibur software (Thermo Electron) and screened with Bioworks 3.1 Turbosequest software (Thermo Electron) against a pgin.fasta database downloaded from the Los Alamos National Laboratory (www.oralgen.lanl.gov) website. Peptide tandem mass spectra were screened to filter out low and poor-quality spectra. Individual peptide matches were also confirmed with the BLAST database at www.oralgen.lanl.gov. Proteins were considered identified if at least two different peptides were identical matches.
Glycoprotein staining. Membrane and extracellular fractions from P. gingivalis FLL92, FLL93, and W83 at stationary phase were subjected to electrophoresis (SDS-PAGE). Glycoprotein staining was performed according to the manufacturer's Pro-Q Emerald glycoprotein gel and blot stain kit protocol (Molecular Probes, Eugene, Oreg.). Total protein was stained according to the manufacturer's SYPRO Ruby stain protocol (Molecular Probes, Eugene, Oreg.).
RESULTS
Extracellular proteins from P. gingivalis FLL93. Although there was a dramatic reduction in proteolytic activity in P. gingivalis FLL93, the gingipain genes appeared to be expressed (23). Furthermore, the RgpB proenzyme-specific antibodies revealed a 64-kDa immunoreactive band in P. gingivalis FLL93, which was similar in size to the partially processed RgpB proenzyme previously reported in the vimA-defective mutant FLL92 (19). This may suggest posttranscriptional regulation of gingipain maturation and activation.
To examine any changes in extracellular proteins from P. gingivalis FLL93 in comparison to the wild-type W83 and vimA-defective mutant FLL92, fractions from the exponential and stationary growth phases of all three strains were analyzed by SDS-PAGE. As shown in Fig. 1A, there were multiple protein bands, in contrast to P. gingivalis FLL92, that were unique to P. gingivalis FLL93. Also, in contrast to P. gingivalis FLL92, there were many high-molecular-weight bands that were unaffected by growth phase (Fig. 1B).
To determine the presence of the gingipains in P. gingivalis FLL93, culture supernatants from cells grown to the early stationary phase were evaluated with antibodies against RgpB, RgpA, and Kgp. As shown in Fig. 2, the extracellular proteins from P. gingivalis FLL93 showed multiple unique immunoreactive bands to RgpB, RgpA, and Kgp in contrast to the wild-type strain or P. gingivalis FLL92, the vimA-defective mutant. Although there was no detectable gingipain activity in this fraction, it is noteworthy that a 50-kDa band, which is similar to the expected size of the catalytic domain for RgpA (Fig. 2B) and Kgp (Fig. 2C), was present. A 48-kDa band representing the catalytic domain of RgpB was present in the wild-type strain and P. gingivalis FLL92 but was faintly detected in P. gingivalis FLL93. It is also noteworthy that in the vimE-defective mutant, there was no processing of any of the gingipains from exponential phase to stationary phase, unlike the vimA-defective mutant FLL92 (Fig. 2D, E and F).
Identification of the gingipain proenzymes. To confirm the identity of the 64-kDa band that immunoreacted with anti-RgpB-specific antibodies in P. gingivalis FLL93, this protein was isolated by SDS-PAGE and its sequence was determined by liquid chromatography-mass spectrometry. This 64-kDa protein was revealed to be the partially processed RgpB proenzyme with its attached propeptide. This protein was secreted in P. gingivalis FLL93 in both the exponential and stationary growth phases (Fig. 2A and 2D). A high-molecular-weight protein band of 185 kDa that immunoreacted with anti-RgpA antibody (Fig. 2D and 2E) was also confirmed by mass spectrometry to be the unprocessed proenzyme for RgpA. Also, immunoblot analysis with RgpB proenzyme antibodies (data not shown) and RgpB antibodies shows an 80-kDa immunoreacting band (Fig. 2A and 2D). This band may be the unprocessed, full-length form of RgpB.
Membrane protein profile of P. gingivalis FLL93. The gingipains can play a role in the processing of outer membrane proteins from P. gingivalis (23). Thus, a reduction in functional or active gingipains may result in unprocessed proteins on the membrane of P. gingivalis. As shown in Fig. 3A, SDS-PAGE analysis of membrane preparations from P. gingivalis strains W83, FLL92, and FLL93 shows that there are multiple unique protein bands in the membrane preparation of P. gingivalis FLL93 in comparison to the wild type and the vimA-defective mutant FLL92. Several of the protein bands in the vimE-defective mutant FLL93 were larger than 90 kDa.
Gingipain distribution in P. gingivalis FLL93. Similar to the vimA-defective mutant (1), the reduced membrane protease activity in P. gingivalis FLL93 (23) may indicate the absence and/or lack of activation of the cell-associated gingipains. To determine the presence of the cell-associated gingipains in P. gingivalis FLL93, membrane preparations of P. gingivalis W83, FLL92, and FLL93 were immunoreacted with antibodies against RgpA, RgpB and Kgp. As shown in Fig. 3B, antibodies against RgpB revealed immunoreactive bands in the membrane fractions from P. gingivalis W83, FLL92, and FLL93. There were no immunoreactive bands in the membrane preparations from P. gingivalis FLL92 with antibodies against RgpA (Fig. 3C) or Kgp (Fig. 3D). It is noted that in contrast to the wild-type strain, there were unique high-molecular-weight bands in P. gingivalis FLL93 that immunoreacted with antibodies against RgpA, RgpB, and Kgp. Taken together, these data suggest that gingipains RgpA and Kgp are not anchored in the membrane of the vimA-defective mutant P. gingivalis FLL92. In contrast, all the gingipains, although inactive, were present in the membrane fraction on the vimE-defective mutant P. gingivalis FLL93.
Analysis of membrane vesicles. Gingipain activity was assayed in membrane vesicle fractions from P. gingivalis FLL92, FLL93, and the wild-type strain. As shown in Fig. 4, there was gingipain activity in the membrane vesicle fraction from P. gingivalis FLL92 and the wild-type strain. However, most of the gingipain activity in P. gingivalis FLL92 (Fig. 4A) could be dialyzed away from the membrane vesicles, in contrast to the wild-type strain (Fig. 4B). Western blot analysis revealed the presence of less intensely immunoreactive bands to RgpA (Fig. 5A) and Kgp (Fig. 5B) in the membrane vesicle fraction from P. gingivalis FLL92 compared to the wild-type strain. In addition to the high-molecular-weight bands that may represent unprocessed gingipain species, immunoreactive bands similar in size to the catalytic and adhesin domains of the gingipains were observed in P. gingivalis FLL92. Immunoblot analysis of vesicles showed only unique high-molecular-weight bands in P. gingivalis FLL93 in contrast to W83. It is noteworthy that a protein band with a molecular mass greater than 200 kDa and identified by liquid chromatography-mass spectrometry as RagA (TonB-dependent outer membrane receptor protein) was also present in the vesicles of P. gingivalis FLL93 (Fig. 6).
Cell surface polysaccharide analysis. Lipopolysaccharides from P. gingivalis strains W83, FLL92, and FLL93 were isolated, purified, separated by SDS-PAGE, and subjected to silver staining. As shown in Fig. 7, the lipopolysaccharide bands for P. gingivalis FLL92 (Fig. 7A) and FLL93 (Fig. 7A) were similar but were truncated or of shorter length in comparison to that of the wild-type strain. Analysis of the polysaccharides from P. gingivalis after the removal of lipid A showed that the vimA-defective mutant had polysaccharides of shorter lengths than the wild type or FLL93 (Fig. 7B).
Glycosylation analysis. In P. gingivalis, posttranslational modification of the gingipains by the covalent attachment of sugars to the protein backbone has been shown to modulate the biological properties of these enzymes and may influence their cell membrane association (10). Because of an apparent defect in the maturation process of the gingipains in both the vimA- and vimE-defective mutants, the glycosylation profile of the membrane and extracellular proteins from these isogenic mutants was evaluated. Glycoprotein staining of membrane and extracellular fractions (Fig. 8) from P. gingivalis W83, FLL92, and FLL93 showed that the proteins in these mutants were being glycosylated or had carbohydrate moieties. However, the pattern of glycosylated proteins in P. gingivalis FLL93 appeared to be different than that of the wild type and P. gingivalis FLL92. Monoclonal antibody 1B5 has been demonstrated to immunoreact with membrane-associated Rgp and certain lipopolysaccharide modifications in P. gingivalis (9). Even though carbohydrates were present on the membrane proteins, as shown in Fig. 9, there was no detectable immunoreactivity in membrane preparations from P. gingivalis FLL92 or FLL93 compared to the wild type.
Activation of Rgp but not Kgp in extracellular fractions from P. gingivalis FLL92 and FLL93. Purified RgpB was incubated with extracellular fractions from FLL92 or FLL93. In Fig. 10, an increase in Rgp activity was observed in extracellular fractions from both P. ginigvalis FLL92 (Fig. 10A) and FLL93 (Fig. 10B). There was, however, no detectable activation of Kgp in either the P. gingivalis FLL92 (Fig. 10C) or FLL93 (Fig. 10D) fraction incubated with the purified RgpB gingipain. Immunoblot analysis of the activated fractions with antibodies against RgpB showed the disappearance of bands representing the partially processed and unprocessed RgpB proenzyme (data not shown).
DISCUSSION
Multiple nongingipain genes, including the vimA, vimE, porR, and gppX genes, can modulate proteolytic activity in P. gingivalis (1, 13, 21, 23). Collectively, these studies suggest that defects in posttranslational modification of the gingipains can affect their activation and distribution, which can also play a role in iron utilization and virulence. The identification of the partially processed RgpB proenzyme in the vimA-defective mutant or immunoreactive bands representing this proenzyme species in the vimE-defective mutant may indicate that both these genes are involved in the maturation pathway(s) of the gingipains in P. gingivalis (19, 23). While these proenzyme species have not yet been identified in the other P. gingivalis mutant strains, these observations have raised the possibility of multiple mechanisms for protease maturation and activation in P. gingivalis. Alternatively, although it is still unclear, these genes may be part of a common pathway for gingipain activation.
We have further characterized the vimE-defective mutant that was previously shown to exhibit reduced proteolytic activity. Similar to the vimA-deficient mutant P. gingivalis FLL92 reported previously (1), a 64-kDa band confirmed to be the partially processed RgpB proenzyme was secreted in P. gingivalis FLL93, the vimE-defective mutant. Consistent with the lack of protease activation in this isogenic mutant, this protein was present throughout the growth phases, unlike the vimA-defective mutant, which demonstrated a late onset of proteolytic activity. In contrast to the wild type and vimA-defective mutant, high-molecular-weight immunoreactive protein bands that could represent the unprocessed gingipains were also secreted in P. gingivalis FLL93. The protein band of 185 kDa was confirmed by mass spectrometry to be the unprocessed proenzyme for RgpA. Also, the 80-kDa band that immunoreacted with the RgpB proenzyme antibodies (data not shown) and RgpB antibodies may represent the unprocessed, full-length form of RgpB. In addition, the RgpB immunoreactive bands between 80 and 48 kDa may represent intermediates or partially processed RgpB species. Collectively, these data have confirmed a defect in the maturation of the gingipains in P. gingivalis FLL93.
Membrane-associated RgpA, RgpB, and Kgp were detected in P. gingivalis FLL93. Based on the high-molecular-weight immunoreacting bands to antibodies against these gingipains, the membrane-associated forms could represent the unprocessed or partially processed proteases. Although cross-reactivity can occur between RgpA, Kgp, and hemagglutinins with RgpA and Kgp antibodies, the lack of immunoreactivity to antibodies against RgpA and Kgp in membrane fractions from P. gingivalis FLL92, the vimA-defective mutant, could suggest that those gingipains are not present on the cell membrane in addition to the hemagglutinins. Alternatively, the antibodies may only be immunoreactive with the modified forms of the gingipains, and thus, the unmodified forms, if present on the cell membrane of the vimA-defective mutant, may be undetectable.
The presence of Kgp and RgpA in extracellular fractions of P. gingivalis FLL92 has been documented previously (19). In contrast to the absence of cell-associated RgpA and Kgp in P. gingivalis FLL92, RgpB was detected in this fraction. This may suggest that there are multiple mechanisms for posttranslational modification of the gingipains that will enable cell membrane association. This is consistent with emerging evidence that polysaccharide biogenesis plays a significant role in cell-associated gingipains, and the difference in their distribution among P. gingivalis strains might be related to the degree of production or maturation of cell surface polysaccharide. Recently, Rgp and Kgp were shown to be produced but not retained on the cell surfaces of a P. gingivalis porR mutant (21). The porR gene product is part of the polysaccharide biosynthetic pathway that can affect gingipain biogenesis and its anchorage and possible distribution on the cell surface (21).
There was no apparent defect in the production of membrane vesicles in either the vimA or vimE isogenic mutant. Proteolytic activity was more readily released from these vesicle fractions from P. gingivalis FLL92 in contrast to the wild type. Western blot analysis of the membrane vesicles from P. gingivalis FLL92 also showed less intense immunoreactive bands for RgpA and Kgp compared to the wild-type strain. These observations are consistent with the mostly soluble gingipain activity previously reported for P. gingivalis FLL92. They also raise questions about vesicle formation as a mechanism for gingipain secretion in the vimA-defective mutant.
Membrane vesicles contain components of the cell membrane, including outer membrane proteins and lipopolysaccharides. The formation of these vesicles is regulated by cell wall turnover and mechanical motions that may shear the blebs, facilitating their release into the culture medium (12, 22). Consistent with the lack of proteolytic activity in the membrane fraction from the vimE-defective mutant, little gingipain activity was detected in the membrane vesicles. Immunoblot analysis of vesicles, however, showed only unique high-molecular-weight bands in P. gingivalis FLL93 in contrast to W83. Furthermore, the unprocessed TonB-dependent outer membrane receptor protein RagA was identified in this fraction from P. gingivalis FLL93. It has been reported that RagA may be involved in virulence (5, 8) and may be regulated by temperature. This unprocessed high-molecular-weight RagA protein was not detected in P. gingivalis W83. The presence of this possible unprocessed RagA protein may further suggest that other factors involved in virulence (other than the gingipains) are affected by the inactivation of vimE. Thus, it is possible that its maturation pathway may require VimE, although we cannot rule out a role for the gingipains. Several reports have documented a role for the gingipains in the maturation of cell membrane proteins (6, 22).
There was an alteration in the glycosylation profile of the vimA- and vimE-defective isogenic mutants. While several proteins appeared to be glycosylated, the gingipain-specific oligosaccharide moiety detectable by monoclonal antibody 1B5 was missing in the mutants. These data may suggest that the carbohydrate polymers or residues present in the isogenic mutants are altered, which could affect the maturation of the gingipains. Collectively, these data further support a role for glycosylation in the modulation of gingipain activity in P. gingivalis.
Glycosylation has been demonstrated to play a crucial role in protein folding, its conformational stabilization, its activation and protection from enzyme degradation (10). While oligosaccharides, including arabinose, rhamnose, fucose, mannose, galactose, glucose, GalNAc, GlcNAc, and N-acetylneuraminic acid, have been shown to be associated with the gingipains in P. gingivalis (9), the specific alterations in the polysaccharide profile in the both the vimA- and vimE-defective mutants is unclear. A possible explanation for the alteration could be the downregulation of gene products, (such as glycosyltransferases) that play a role in the glycosylation of proteins and synthesis of lipopolysaccharide. A preliminary microarray analysis of the vimA-defective mutant has shown a downregulation of several glycosyltransferases and other genes involved in polysaccharide biogenesis (data not shown).
It has been shown that lipopolysaccharide modifications of the C terminus of proteins is needed for the stable interaction or attachment of the proteins to the cell surface (6, 21). Analysis of the cell surface polysaccharides isolated from the parent strain W83 and isogenic mutants grown under the same conditions showed that the lipopolysaccharides of FLL92, and FLL93 were truncated compared to that of the wild type. However, removal of the lipid A from the lipopolysaccharide resulted in polysaccharide fractions that had similar profiles for both the parent strain and FLL93 compared to the polysaccharide profile of FLL92, which was of shorter length, similar to that of the porR-defective mutant (21). The truncated polysaccharides of P. gingivalis FLL92 may be correlated with the absence of RgpA and Kgp on the membrane surface. These data may also suggest a difference or defect in the lipid A species or structure in the isogenic mutants in comparison to the wild type. A possible mechanism for the role of the vimA and vimE gene products in this process is under investigation in the laboratory.
Autoprocessing and/or the action of other proteolytic enzymes can generate active enzymes, including cysteine proteases, from a larger polypeptide (16). In P. gingivalis there is accumulating evidence that a multicomponent maturation pathway(s), including an autolytic mechanism, may be involved in the production of Arg-X- and Lys-specific proteases in P. gingivalis (16, 18, 23). In this study, purified RgpB could only activate Arg-specific gingipain activity. This is consistent with other observations that have demonstrated a role for Rgp in the maturation of Arg-specific activity in P. gingivalis (4). The lack of activation of Lys-specific activity in this study also confirms previous observations that generation of this specific activity in P. gingivalis is independent of Arg-gingipain activity in P. gingivalis (3).
While it is clear that proteolytic processing of the full-length gingipain precursors in P. gingivalis is required to produce the isoforms detected, the presence of the partially processed or unprocessed proenzyme forms in vimA and vimE, respectively, may suggest that the mechanism(s) of protease activation in this organism requires host-specific factors. It is unclear whether factors including the vimA and vimE gene products or products regulated by those genes, are involved in the maturation process. It is also unclear if the vimA and vimE genes may affect the phenotypic expression of other proteases in P. gingivalis. Furthermore, it is also possible that these gene products may interact with each other or other proteins to facilitate protease activation. Preliminary studies in the laboratory have provided some evidence that both the VimA and VimE proteins interact with the gingipains and other proteins that in other organisms are involved in protease maturation and for virulence. The effects of these proteins in gingipain maturation in P. gingivlis are being evaluated in the laboratory.
Finally, the data support a hypothesis that inactivation of both the vimA and vimE genes may affect a common pathway for protease biogenesis. The identification of the unprocessed and partially processed gingipain proenzymes from the vimE-defective mutant suggest that this gene product can act upstream of the VimA protein. Unlike the vimA-defective mutant, there was no growth phase-dependent activation of gingipain activity in the vimE-defective mutant. This implicates the vimE gene product in the initiation of gingipain biogenesis. We have presented evidence for posttranslational regulation of proteolytic activity in P. gingivalis. This model system will facilitate a more careful evaluation of gingipain biogenesis in P. gingivalis.
ACKNOWLEDGMENTS
This work was supported by the Loma Linda University School of Dentistry and by Public Health Service grants DE13664 and DE13664-S1 from the National Institute of Dental and Craniofacial Research (to H.M.F) and GM60507, a minority training grant from the National Institute of General Medicine.
We thank Jan Potempa for gingipain antibodies and Michael Curtis for antibody 1B5. We thank Shaun Sheets for her assistance with polysaccharide purification.
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ABSTRACT
We have previously shown that gingipain activity in Porphyromonas gingivalis is modulated by the unique vimA and vimE genes. To determine if these genes had a similar phenotypic effect on protease maturation and activation, isogenic mutants defective in those genes were further characterized. Western blot analyses with antigingipain antibodies showed RgpA-, RgpB-, and Kgp-immunoreactive bands in membrane fractions as well as the culture supernatant of both P. gingivalis W83 and FLL93, the vimE-defective mutant. In contrast, the membrane of P. gingivalis FLL92, the vimA-defective mutant, demonstrated immunoreactivity only with RgpB antibodies. With mass spectrometry or Western blots, full-length RgpA and RgpB were identified from extracellular fractions. In similar extracellular fractions from P. gingivalis FLL92 and FLL93, purified RgpB activated only arginine-specific activity. In addition, the lipopolysaccharide profiles of the vimA and vimE mutants were truncated in comparison to that of W83. While glycosylated proteins were detected in the membrane and extracellular fractions from the vimA- and vimE-defective mutants, a monoclonal antibody (1B5) that reacts with specific sugar moieties of the P. gingivalis cell surface polysaccharide and membrane-associated Rgp gingipain showed no immunoreactivity with these fractions. Taken together, these results indicate a possible defect in sugar biogenesis in both the vimA- and vimE-defective mutants. These modulating genes play a role in the secretion, processing, and/or anchorage of gingipains on the cell surface.
INTRODUCTION
Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobic bacterium, is an important etiological agent of chronic periodontitis. Furthermore, accumulating data suggest that this organism is also associated with systemic diseases and complications including atherosclerosis, preterm births, and low-birth-weight babies (15). While several virulence factors are implicated in the pathogenicity of P. gingivalis, the proteolytic abilities of this organism have been considered to play the most significant role in its virulence (7, 11, 14, 15, 17). The major proteases, called gingipains, consist of an arginine-specific protease (Arg-gingipain [Rgp]) and lysine-specific protease (Lys-gingipain [Kgp]). The Rgp is encoded by two genes, rgpA and rgpB, while Kgp is encoded by a single gene, kgp. These proteases, which are both extracellular and cell associated, are required for growth of the organism and other housekeeping functions, which include processing enzymes for various cell surface proteins, including individual proteases (24). Moreover, the multiple pathogenic effects of the gingipains, such as degradation of complement and immunoglobulin, inactivation of cytokines and their receptors, aggregation of platelets, attenuation of neutrophil antibacterial activities, and increase in vascular permeability and blood clotting prevention, are well documented (14).
We have reported previously that the vimA and vimE genes can modulate the phenotypic expression of the gingipains in P. gingivalis (1, 23). While the vimA gene is part of the bcp-recA-vimA transcriptional unit, the downstream vimE can either be cotranscribed with vimA or independently expressed (23). The vimA- and vimE-defective mutant strains, designated P. gingivalis FLL92 and FLL93, respectively, were non-black pigmented and showed significant reductions in proteolytic, hemolytic, and hemagglutinating activities (1, 23). In in vivo experiments with a mouse model, the virulence of P. gingivalis FLL92 was dramatically reduced in comparison to that of the wild-type W83 strain (1).
While reductions in Arg-X- and Lys-X-specific proteolytic activities were observed in P. gingivalis FLL92 and FLL93, transcription of the gingipain genes was unaltered in these mutants compared to the wild-type strain (1, 23). A similar phenotype of the gingipain genes was also seen in P. gingivalis FLL32, a recA-defective isogenic mutant that had reduced Arg-X- and Lys-X-specific proteolytic activities (2). The 64-kDa RgpB partially processed proenzyme was secreted in both P. gingivalis FLL92 and FLL93 (19, 23). The distribution of the proteolytic activity in P. gingivalis FLL93 was similar to that of the wild-type strain and in contrast to P. gingivalis FLL92, the vimA-defective mutant, which had mostly soluble proteolytic activity with little or no cell-associated activity (23). While there was a unique late onset of Arg-X- and Lys-X-specific proteolytic activity in P. gingivalis FLL92, there were little or no observed changes of proteolytic activity in stationary phase in P. gingivalis FLL93, the vimE-defective mutant (23). Collectively, these observations have raised the question of whether the regulation of proteolytic activity in P. gingivalis occurs by multiple mechanisms. Furthermore, it is unclear if the vimA and vimE gene products are part of a common pathway for protease maturation and activation.
In this report, we have further characterized the vimE-defective P. gingivalis FLL93 strain in comparison to the vimA-defective strain FLL92 and the wild type. Our findings suggest an important role for these genes in gingipain secretion, processing, and/or anchorage of gingipains on the cell surface.
MATERIALS AND METHODS
Bacterial strains, growth conditions, and plasmids. P. gingivalis W83 and isogenic strains FLL92 and FLL93 were grown in brain heart infusion (BHI) broth (Difco Laboratories, Detroit, Mich.) supplemented with hemin (5 μg/ml), vitamin K (0.5 μg/ml), cysteine (0.1%), and yeast extract (0.5%). Unless otherwise stated, all cultures were incubated at 37°C and maintained in an anaerobic chamber (Coy Manufacturing, Ann Arbor, Mich.) in 10% H2, 10% CO2, and 80% N2. The growth rates for the P. gingivalis strains were determined spectrophotometrically (optical density at 600 nm).
Preparation of P. gingivalis extracellular fractions. One-liter cultures of P. gingivalis strains FLL92, FLL93, and W83 were grown from actively growing cells. Cells were harvested by centrifugation at 10,000 x g for 30 min. The cell-free culture fluid was precipitated with cold (–20°C), 37.5% or 60% acetone, and the protein pellet was resuspended in 7 ml of 100 mM Tris-HCl buffer (pH 7.4), dialyzed for 24 h against the same buffer, and then stored on ice or at 0°C.
Preparation of membrane fraction. One-liter cultures of P. gingivalis strains FLL92, FLL93, and W83 were grown from actively growing cells to the stationary phase (optical density at 600 nm of 1.3 to 1.4). Cells were harvested by centrifugation at 10,000 x g for 30 min. Membrane fractions were prepared with the French press (American Instrument Company, Silver Spring, Md.). The lysed cells were then centrifuged at 27,000 x g for 1 h. The supernatant was subjected to ultracentrifugation at 100,000 x g for 1 h. The pellet, designated the membrane fraction, was resuspended in 100 mM Tris-HCl, pH 7.4, containing 10 mM tosyl lysyl chloromethyl ketone (TLCK), a protease inhibitor. The remaining supernatant was considered the cytosolic fraction.
Membrane vesicle preparation. P. gingivalis W83, FLL92, and FLL93 were grown for 48 h in BHI supplemented with hemin and vitamin K. Membrane vesicles were prepared as previously described (19).
Protease assays. The presence of Arg-X- and Lys-X-specific cysteine protease activities was determined with a microplate reader (Bio-Rad, Hercules, Calif.) as previously reported (20).
Gel electrophoresis and immunoblot analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with a 4 to 12% Bis-Tris separating gel in MOPS (morpholinepropanesulfonic acid)-SDS running buffer according to the manufacturer's instructions (NuPAGE Novex gels; Invitrogen, Carlsbad, Calif.). Samples were prepared (65% sample, 25% 4x NuPAGE LDS sample buffer, 10% NuPAGE reducing agent), heated at 72°C for 10 min, and then electrophoresed at 200 V for 65 min in the XCell SureLock Mini-Cell system (Invitrogen, Carlsbad, Calif.). The protein bands were visualized by staining with Simply Blue Safe stain (Invitrogen, Carlsbad, Calif.). The separated proteins were then transferred to BioTrace nitrocellulose membranes (Pall Corporation, Ann Arbor, Mich.) and processed at 15 V for 25 min with a semidry Trans-blot apparatus (Bio-Rad, Hercules, Calif.). The blots were probed with gingipain-specific antibodies. Immunoreactive proteins were detected by the procedure described in the Western Lightning chemiluminescence reagent plus kit (Perkin-Elmer Life Sciences. Boston, Mass.). The secondary antibody was immunoglobulin G (heavy plus light chains)-horseradish peroxidase conjugate (Zymed Laboratories, Inc., South San Francisco, Calif..).
Activation of inactive gingipains with purified RgpB. Approximately 2.5 units of purified RgpB (Athens Research & Technology. Athens, Ga.) was incubated with approximately 750 μg of extracellular extracts of P. gingivalis FLL92 grown to the exponential phase (optical density at 600 nm of 0.7) or P. gingivalis FLL93 grown to the stationary phase. The reaction was performed in activated assay buffer (0.1 M Tris, pH 7.6, 0.2 M NaCl, 5 mM CaCl2, 10 mM NaOH, 9 mM L-cysteine). Samples were incubated at 37°C for 1 h under anaerobic conditions (10% H2, 10% CO2, 80% N2). Samples were then assayed for proteolytic activity and analyzed by immunoblot analysis.
Lipopolysaccharide and polysaccharide isolation and silver staining. Overnight cultures of P. gingivalis W83, FLL92, and FLL93 were grown to the stationary phase. Lipopolysaccharide isolation was done according to the manufacturer's protocol (Intron Biotechnology). Polysaccharides were separated from lipid A by treating the lipopolysaccharide preparations with 2% acetic acid for 2 h at 100°C. The soluble fraction containing the polysaccharides was purified with the Superdex 75 column (Amersham Biosciences, Piscataway, N.J.). Lipopolysaccharides were subjected to electrophoresis (SDS-PAGE) on 4 to 12% Bis-Tris gel with the MOPS running buffer. Polysaccharides were subjected to electrophoresis (SDS-PAGE) on 4 to 12% Bis-Tris gels with the morpholineethanesulfonic acid (MES) running buffer. Lipopolysaccharides and polysaccharides were visualized with the SilverQuest silver staining kit according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif.).
Mass spectrometry sequence analysis. Extracellular fractions of P. gingivalis FLL93 from the stationary phase were subjected to SDS-PAGE. Desired bands were excised from the gel and dried in a Speed Vac for 1 h. Dried gel bands were then reduced with 20 μl of 20 mM tri(2-carboxyethyl)phosphine at 56°C and then alkylated with 20 μl of 40 mM iodoacetamide for 30 min at 23°C. Alkylated samples were then washed twice with 100 mM ammonium bicarbonate and dried with the Speed Vac for 1 h. Digestion buffer (15 μl of 0.05 μg/μl trypsin stock diluted in 3% acetic acid) was added to the dried gel slice and incubated for 10 min on ice. Excess digestion buffer was removed, and 10 μl of 100 mM ammonium bicarbonate was added to cover the gel slice to prevent drying. The gel slices were then incubated for 16 h at 30°C. After 16 h, an additional 10 μl of 100 mM ammonium bicarbonate was added to the gel slices and then incubated for an additional 30 min at 30°C. Proteins were then trapped, washed and eluted with ZipTipC18 according to the manufacturer's instructions (Millipore, Bellerica, Mass.).
Eluted peptides were dried in the Speed Vac for 5 to 10 min and resuspended in 0.05% trifluoroacetic acid in mass spectrometry grade water. All buffers were prepared in 100 mM ammonium bicarbonate. Tryptic peptides were separated and analyzed on a Picoview model PV-500 Nanospray ESI unit (New Objective, Woodburn, Mass.) coupled to an LCQ Deca XP ion trap mass spectrometer (Thermo Electron, San Jose, Calif.) with a four-event program consisting of a full mass spectrometry scan followed by three mass spectrometry/mass spectrometry events for the most intense ions on full mass spectrometry. A capillary column (75 μm by 10 cm) packed with 5μC-18-coated silica was developed with a 40-min gradient elution program of 2 to 90% acetonitrile buffered with 0.5% acetic acid and 0.005% trifluoroacetic acid at a flow rate of 300 nl/min. Data were collected with the Xcalibur software (Thermo Electron) and screened with Bioworks 3.1 Turbosequest software (Thermo Electron) against a pgin.fasta database downloaded from the Los Alamos National Laboratory (www.oralgen.lanl.gov) website. Peptide tandem mass spectra were screened to filter out low and poor-quality spectra. Individual peptide matches were also confirmed with the BLAST database at www.oralgen.lanl.gov. Proteins were considered identified if at least two different peptides were identical matches.
Glycoprotein staining. Membrane and extracellular fractions from P. gingivalis FLL92, FLL93, and W83 at stationary phase were subjected to electrophoresis (SDS-PAGE). Glycoprotein staining was performed according to the manufacturer's Pro-Q Emerald glycoprotein gel and blot stain kit protocol (Molecular Probes, Eugene, Oreg.). Total protein was stained according to the manufacturer's SYPRO Ruby stain protocol (Molecular Probes, Eugene, Oreg.).
RESULTS
Extracellular proteins from P. gingivalis FLL93. Although there was a dramatic reduction in proteolytic activity in P. gingivalis FLL93, the gingipain genes appeared to be expressed (23). Furthermore, the RgpB proenzyme-specific antibodies revealed a 64-kDa immunoreactive band in P. gingivalis FLL93, which was similar in size to the partially processed RgpB proenzyme previously reported in the vimA-defective mutant FLL92 (19). This may suggest posttranscriptional regulation of gingipain maturation and activation.
To examine any changes in extracellular proteins from P. gingivalis FLL93 in comparison to the wild-type W83 and vimA-defective mutant FLL92, fractions from the exponential and stationary growth phases of all three strains were analyzed by SDS-PAGE. As shown in Fig. 1A, there were multiple protein bands, in contrast to P. gingivalis FLL92, that were unique to P. gingivalis FLL93. Also, in contrast to P. gingivalis FLL92, there were many high-molecular-weight bands that were unaffected by growth phase (Fig. 1B).
To determine the presence of the gingipains in P. gingivalis FLL93, culture supernatants from cells grown to the early stationary phase were evaluated with antibodies against RgpB, RgpA, and Kgp. As shown in Fig. 2, the extracellular proteins from P. gingivalis FLL93 showed multiple unique immunoreactive bands to RgpB, RgpA, and Kgp in contrast to the wild-type strain or P. gingivalis FLL92, the vimA-defective mutant. Although there was no detectable gingipain activity in this fraction, it is noteworthy that a 50-kDa band, which is similar to the expected size of the catalytic domain for RgpA (Fig. 2B) and Kgp (Fig. 2C), was present. A 48-kDa band representing the catalytic domain of RgpB was present in the wild-type strain and P. gingivalis FLL92 but was faintly detected in P. gingivalis FLL93. It is also noteworthy that in the vimE-defective mutant, there was no processing of any of the gingipains from exponential phase to stationary phase, unlike the vimA-defective mutant FLL92 (Fig. 2D, E and F).
Identification of the gingipain proenzymes. To confirm the identity of the 64-kDa band that immunoreacted with anti-RgpB-specific antibodies in P. gingivalis FLL93, this protein was isolated by SDS-PAGE and its sequence was determined by liquid chromatography-mass spectrometry. This 64-kDa protein was revealed to be the partially processed RgpB proenzyme with its attached propeptide. This protein was secreted in P. gingivalis FLL93 in both the exponential and stationary growth phases (Fig. 2A and 2D). A high-molecular-weight protein band of 185 kDa that immunoreacted with anti-RgpA antibody (Fig. 2D and 2E) was also confirmed by mass spectrometry to be the unprocessed proenzyme for RgpA. Also, immunoblot analysis with RgpB proenzyme antibodies (data not shown) and RgpB antibodies shows an 80-kDa immunoreacting band (Fig. 2A and 2D). This band may be the unprocessed, full-length form of RgpB.
Membrane protein profile of P. gingivalis FLL93. The gingipains can play a role in the processing of outer membrane proteins from P. gingivalis (23). Thus, a reduction in functional or active gingipains may result in unprocessed proteins on the membrane of P. gingivalis. As shown in Fig. 3A, SDS-PAGE analysis of membrane preparations from P. gingivalis strains W83, FLL92, and FLL93 shows that there are multiple unique protein bands in the membrane preparation of P. gingivalis FLL93 in comparison to the wild type and the vimA-defective mutant FLL92. Several of the protein bands in the vimE-defective mutant FLL93 were larger than 90 kDa.
Gingipain distribution in P. gingivalis FLL93. Similar to the vimA-defective mutant (1), the reduced membrane protease activity in P. gingivalis FLL93 (23) may indicate the absence and/or lack of activation of the cell-associated gingipains. To determine the presence of the cell-associated gingipains in P. gingivalis FLL93, membrane preparations of P. gingivalis W83, FLL92, and FLL93 were immunoreacted with antibodies against RgpA, RgpB and Kgp. As shown in Fig. 3B, antibodies against RgpB revealed immunoreactive bands in the membrane fractions from P. gingivalis W83, FLL92, and FLL93. There were no immunoreactive bands in the membrane preparations from P. gingivalis FLL92 with antibodies against RgpA (Fig. 3C) or Kgp (Fig. 3D). It is noted that in contrast to the wild-type strain, there were unique high-molecular-weight bands in P. gingivalis FLL93 that immunoreacted with antibodies against RgpA, RgpB, and Kgp. Taken together, these data suggest that gingipains RgpA and Kgp are not anchored in the membrane of the vimA-defective mutant P. gingivalis FLL92. In contrast, all the gingipains, although inactive, were present in the membrane fraction on the vimE-defective mutant P. gingivalis FLL93.
Analysis of membrane vesicles. Gingipain activity was assayed in membrane vesicle fractions from P. gingivalis FLL92, FLL93, and the wild-type strain. As shown in Fig. 4, there was gingipain activity in the membrane vesicle fraction from P. gingivalis FLL92 and the wild-type strain. However, most of the gingipain activity in P. gingivalis FLL92 (Fig. 4A) could be dialyzed away from the membrane vesicles, in contrast to the wild-type strain (Fig. 4B). Western blot analysis revealed the presence of less intensely immunoreactive bands to RgpA (Fig. 5A) and Kgp (Fig. 5B) in the membrane vesicle fraction from P. gingivalis FLL92 compared to the wild-type strain. In addition to the high-molecular-weight bands that may represent unprocessed gingipain species, immunoreactive bands similar in size to the catalytic and adhesin domains of the gingipains were observed in P. gingivalis FLL92. Immunoblot analysis of vesicles showed only unique high-molecular-weight bands in P. gingivalis FLL93 in contrast to W83. It is noteworthy that a protein band with a molecular mass greater than 200 kDa and identified by liquid chromatography-mass spectrometry as RagA (TonB-dependent outer membrane receptor protein) was also present in the vesicles of P. gingivalis FLL93 (Fig. 6).
Cell surface polysaccharide analysis. Lipopolysaccharides from P. gingivalis strains W83, FLL92, and FLL93 were isolated, purified, separated by SDS-PAGE, and subjected to silver staining. As shown in Fig. 7, the lipopolysaccharide bands for P. gingivalis FLL92 (Fig. 7A) and FLL93 (Fig. 7A) were similar but were truncated or of shorter length in comparison to that of the wild-type strain. Analysis of the polysaccharides from P. gingivalis after the removal of lipid A showed that the vimA-defective mutant had polysaccharides of shorter lengths than the wild type or FLL93 (Fig. 7B).
Glycosylation analysis. In P. gingivalis, posttranslational modification of the gingipains by the covalent attachment of sugars to the protein backbone has been shown to modulate the biological properties of these enzymes and may influence their cell membrane association (10). Because of an apparent defect in the maturation process of the gingipains in both the vimA- and vimE-defective mutants, the glycosylation profile of the membrane and extracellular proteins from these isogenic mutants was evaluated. Glycoprotein staining of membrane and extracellular fractions (Fig. 8) from P. gingivalis W83, FLL92, and FLL93 showed that the proteins in these mutants were being glycosylated or had carbohydrate moieties. However, the pattern of glycosylated proteins in P. gingivalis FLL93 appeared to be different than that of the wild type and P. gingivalis FLL92. Monoclonal antibody 1B5 has been demonstrated to immunoreact with membrane-associated Rgp and certain lipopolysaccharide modifications in P. gingivalis (9). Even though carbohydrates were present on the membrane proteins, as shown in Fig. 9, there was no detectable immunoreactivity in membrane preparations from P. gingivalis FLL92 or FLL93 compared to the wild type.
Activation of Rgp but not Kgp in extracellular fractions from P. gingivalis FLL92 and FLL93. Purified RgpB was incubated with extracellular fractions from FLL92 or FLL93. In Fig. 10, an increase in Rgp activity was observed in extracellular fractions from both P. ginigvalis FLL92 (Fig. 10A) and FLL93 (Fig. 10B). There was, however, no detectable activation of Kgp in either the P. gingivalis FLL92 (Fig. 10C) or FLL93 (Fig. 10D) fraction incubated with the purified RgpB gingipain. Immunoblot analysis of the activated fractions with antibodies against RgpB showed the disappearance of bands representing the partially processed and unprocessed RgpB proenzyme (data not shown).
DISCUSSION
Multiple nongingipain genes, including the vimA, vimE, porR, and gppX genes, can modulate proteolytic activity in P. gingivalis (1, 13, 21, 23). Collectively, these studies suggest that defects in posttranslational modification of the gingipains can affect their activation and distribution, which can also play a role in iron utilization and virulence. The identification of the partially processed RgpB proenzyme in the vimA-defective mutant or immunoreactive bands representing this proenzyme species in the vimE-defective mutant may indicate that both these genes are involved in the maturation pathway(s) of the gingipains in P. gingivalis (19, 23). While these proenzyme species have not yet been identified in the other P. gingivalis mutant strains, these observations have raised the possibility of multiple mechanisms for protease maturation and activation in P. gingivalis. Alternatively, although it is still unclear, these genes may be part of a common pathway for gingipain activation.
We have further characterized the vimE-defective mutant that was previously shown to exhibit reduced proteolytic activity. Similar to the vimA-deficient mutant P. gingivalis FLL92 reported previously (1), a 64-kDa band confirmed to be the partially processed RgpB proenzyme was secreted in P. gingivalis FLL93, the vimE-defective mutant. Consistent with the lack of protease activation in this isogenic mutant, this protein was present throughout the growth phases, unlike the vimA-defective mutant, which demonstrated a late onset of proteolytic activity. In contrast to the wild type and vimA-defective mutant, high-molecular-weight immunoreactive protein bands that could represent the unprocessed gingipains were also secreted in P. gingivalis FLL93. The protein band of 185 kDa was confirmed by mass spectrometry to be the unprocessed proenzyme for RgpA. Also, the 80-kDa band that immunoreacted with the RgpB proenzyme antibodies (data not shown) and RgpB antibodies may represent the unprocessed, full-length form of RgpB. In addition, the RgpB immunoreactive bands between 80 and 48 kDa may represent intermediates or partially processed RgpB species. Collectively, these data have confirmed a defect in the maturation of the gingipains in P. gingivalis FLL93.
Membrane-associated RgpA, RgpB, and Kgp were detected in P. gingivalis FLL93. Based on the high-molecular-weight immunoreacting bands to antibodies against these gingipains, the membrane-associated forms could represent the unprocessed or partially processed proteases. Although cross-reactivity can occur between RgpA, Kgp, and hemagglutinins with RgpA and Kgp antibodies, the lack of immunoreactivity to antibodies against RgpA and Kgp in membrane fractions from P. gingivalis FLL92, the vimA-defective mutant, could suggest that those gingipains are not present on the cell membrane in addition to the hemagglutinins. Alternatively, the antibodies may only be immunoreactive with the modified forms of the gingipains, and thus, the unmodified forms, if present on the cell membrane of the vimA-defective mutant, may be undetectable.
The presence of Kgp and RgpA in extracellular fractions of P. gingivalis FLL92 has been documented previously (19). In contrast to the absence of cell-associated RgpA and Kgp in P. gingivalis FLL92, RgpB was detected in this fraction. This may suggest that there are multiple mechanisms for posttranslational modification of the gingipains that will enable cell membrane association. This is consistent with emerging evidence that polysaccharide biogenesis plays a significant role in cell-associated gingipains, and the difference in their distribution among P. gingivalis strains might be related to the degree of production or maturation of cell surface polysaccharide. Recently, Rgp and Kgp were shown to be produced but not retained on the cell surfaces of a P. gingivalis porR mutant (21). The porR gene product is part of the polysaccharide biosynthetic pathway that can affect gingipain biogenesis and its anchorage and possible distribution on the cell surface (21).
There was no apparent defect in the production of membrane vesicles in either the vimA or vimE isogenic mutant. Proteolytic activity was more readily released from these vesicle fractions from P. gingivalis FLL92 in contrast to the wild type. Western blot analysis of the membrane vesicles from P. gingivalis FLL92 also showed less intense immunoreactive bands for RgpA and Kgp compared to the wild-type strain. These observations are consistent with the mostly soluble gingipain activity previously reported for P. gingivalis FLL92. They also raise questions about vesicle formation as a mechanism for gingipain secretion in the vimA-defective mutant.
Membrane vesicles contain components of the cell membrane, including outer membrane proteins and lipopolysaccharides. The formation of these vesicles is regulated by cell wall turnover and mechanical motions that may shear the blebs, facilitating their release into the culture medium (12, 22). Consistent with the lack of proteolytic activity in the membrane fraction from the vimE-defective mutant, little gingipain activity was detected in the membrane vesicles. Immunoblot analysis of vesicles, however, showed only unique high-molecular-weight bands in P. gingivalis FLL93 in contrast to W83. Furthermore, the unprocessed TonB-dependent outer membrane receptor protein RagA was identified in this fraction from P. gingivalis FLL93. It has been reported that RagA may be involved in virulence (5, 8) and may be regulated by temperature. This unprocessed high-molecular-weight RagA protein was not detected in P. gingivalis W83. The presence of this possible unprocessed RagA protein may further suggest that other factors involved in virulence (other than the gingipains) are affected by the inactivation of vimE. Thus, it is possible that its maturation pathway may require VimE, although we cannot rule out a role for the gingipains. Several reports have documented a role for the gingipains in the maturation of cell membrane proteins (6, 22).
There was an alteration in the glycosylation profile of the vimA- and vimE-defective isogenic mutants. While several proteins appeared to be glycosylated, the gingipain-specific oligosaccharide moiety detectable by monoclonal antibody 1B5 was missing in the mutants. These data may suggest that the carbohydrate polymers or residues present in the isogenic mutants are altered, which could affect the maturation of the gingipains. Collectively, these data further support a role for glycosylation in the modulation of gingipain activity in P. gingivalis.
Glycosylation has been demonstrated to play a crucial role in protein folding, its conformational stabilization, its activation and protection from enzyme degradation (10). While oligosaccharides, including arabinose, rhamnose, fucose, mannose, galactose, glucose, GalNAc, GlcNAc, and N-acetylneuraminic acid, have been shown to be associated with the gingipains in P. gingivalis (9), the specific alterations in the polysaccharide profile in the both the vimA- and vimE-defective mutants is unclear. A possible explanation for the alteration could be the downregulation of gene products, (such as glycosyltransferases) that play a role in the glycosylation of proteins and synthesis of lipopolysaccharide. A preliminary microarray analysis of the vimA-defective mutant has shown a downregulation of several glycosyltransferases and other genes involved in polysaccharide biogenesis (data not shown).
It has been shown that lipopolysaccharide modifications of the C terminus of proteins is needed for the stable interaction or attachment of the proteins to the cell surface (6, 21). Analysis of the cell surface polysaccharides isolated from the parent strain W83 and isogenic mutants grown under the same conditions showed that the lipopolysaccharides of FLL92, and FLL93 were truncated compared to that of the wild type. However, removal of the lipid A from the lipopolysaccharide resulted in polysaccharide fractions that had similar profiles for both the parent strain and FLL93 compared to the polysaccharide profile of FLL92, which was of shorter length, similar to that of the porR-defective mutant (21). The truncated polysaccharides of P. gingivalis FLL92 may be correlated with the absence of RgpA and Kgp on the membrane surface. These data may also suggest a difference or defect in the lipid A species or structure in the isogenic mutants in comparison to the wild type. A possible mechanism for the role of the vimA and vimE gene products in this process is under investigation in the laboratory.
Autoprocessing and/or the action of other proteolytic enzymes can generate active enzymes, including cysteine proteases, from a larger polypeptide (16). In P. gingivalis there is accumulating evidence that a multicomponent maturation pathway(s), including an autolytic mechanism, may be involved in the production of Arg-X- and Lys-specific proteases in P. gingivalis (16, 18, 23). In this study, purified RgpB could only activate Arg-specific gingipain activity. This is consistent with other observations that have demonstrated a role for Rgp in the maturation of Arg-specific activity in P. gingivalis (4). The lack of activation of Lys-specific activity in this study also confirms previous observations that generation of this specific activity in P. gingivalis is independent of Arg-gingipain activity in P. gingivalis (3).
While it is clear that proteolytic processing of the full-length gingipain precursors in P. gingivalis is required to produce the isoforms detected, the presence of the partially processed or unprocessed proenzyme forms in vimA and vimE, respectively, may suggest that the mechanism(s) of protease activation in this organism requires host-specific factors. It is unclear whether factors including the vimA and vimE gene products or products regulated by those genes, are involved in the maturation process. It is also unclear if the vimA and vimE genes may affect the phenotypic expression of other proteases in P. gingivalis. Furthermore, it is also possible that these gene products may interact with each other or other proteins to facilitate protease activation. Preliminary studies in the laboratory have provided some evidence that both the VimA and VimE proteins interact with the gingipains and other proteins that in other organisms are involved in protease maturation and for virulence. The effects of these proteins in gingipain maturation in P. gingivlis are being evaluated in the laboratory.
Finally, the data support a hypothesis that inactivation of both the vimA and vimE genes may affect a common pathway for protease biogenesis. The identification of the unprocessed and partially processed gingipain proenzymes from the vimE-defective mutant suggest that this gene product can act upstream of the VimA protein. Unlike the vimA-defective mutant, there was no growth phase-dependent activation of gingipain activity in the vimE-defective mutant. This implicates the vimE gene product in the initiation of gingipain biogenesis. We have presented evidence for posttranslational regulation of proteolytic activity in P. gingivalis. This model system will facilitate a more careful evaluation of gingipain biogenesis in P. gingivalis.
ACKNOWLEDGMENTS
This work was supported by the Loma Linda University School of Dentistry and by Public Health Service grants DE13664 and DE13664-S1 from the National Institute of Dental and Craniofacial Research (to H.M.F) and GM60507, a minority training grant from the National Institute of General Medicine.
We thank Jan Potempa for gingipain antibodies and Michael Curtis for antibody 1B5. We thank Shaun Sheets for her assistance with polysaccharide purification.
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