VimA-Dependent Modulation of Acetyl Coenzyme A Levels and Lipid A Biosynthesis Can Alter Virulence in Porphyromonas gingivalis

Bioinformatic analysis.The DNA and amino acid sequences were aligned using Bioedit software (http://www.mbio.ncsu.edu/bioedit/bioedit.html). The phylogenetic relationships of these sequences between the oral pathogens were analyzed using the MEGA program, version 4.0 (57). The phylogenetic distance was calculated using the Kimura 2-parameter model (29). For clustering, the neighbor-joining method used bootstrap values based on 1,000 replicates (49). The amino acid sequences were analyzed using the ClustalW program, version 2.0 (http://www.ebi.ac.uk/). The secondary structure prediction and modeling of the protein were performed using the Modeler software package (13). The models were validated using the WHATIF program (65). The signal peptide and potential cleavage sites were predicted using both the neural network and hidden Markov models (25). Metabolic pathway analysis was carried out using the Kyoto Encyclopedia of Genes and Genomes (KEGG) (www.genome.jp/kegg/) (27), based on the information from the online databases Biosilico (24), BRENDA (7), and ExPASy Enzyme (3).

Bacterial strains and growth conditions.Strains and plasmids used in this study are listed in Table 1. P. gingivalis strains were grown in brain heart infusion (BHI) broth (Difco) supplemented with hemin (5 μg/ml), vitamin K (0.5 μg/ml), and cysteine (0.1%). Experiments with hydrogen peroxide were performed in BHI without cysteine. All cultures, unless otherwise stated, were incubated at 37°C. P. gingivalis strains were maintained in an anaerobic chamber (Coy Manufacturing) in 10% H₂, 10% CO₂, 80% N₂. Growth rates for P. gingivalis strains were determined spectrophotometrically (optical density at 600 nm [OD₆₀₀]). Antibiotics were used at the following concentrations: clindamycin, 0.5 μg/ml; erythromycin, 10 μg/ml.

DNA isolation and analysis.P. gingivalis chromosomal DNA was prepared as previously described (37). For plasmid DNA analysis, DNA extraction was performed by the alkaline lysis procedure as previously reported (14). For large-scale preparation, plasmids were purified using a Qiagen plasmid maxikit (Qiagen, Valencia, CA).

Construction of vimA chimera.Long PCR-based fusion of DNA fragments was done using overlapping extension PCR as previously described (23). The primers used in the development of the vimA chimera are listed in Table S1 in the supplemental material. Briefly, 1 kb of the flanking fragments upstream and downstream of the target vimA gene was PCR amplified from the chromosomal DNA of P. gingivalis W83. The ermF cassette was amplified from the pVA2198 plasmid (Table 1) using custom-made oligonucleotide primers containing overlapping nucleotides for the upstream and downstream fragments. The upstream fragment was fused with a His tag at the 3′ end, and the downstream fragment was fused with PG1866. The upstream fragment of vimA was fused with PG1864, and its downstream fragment was fused with the His tag (see Fig. S1C in the supplemental material). The fusion PCR program consisted of 1 cycle of 5 min at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at 55°C, and 4 min at 68°C, with a final extension of 5 min at 68°C. This PCR-fused fragment was used to transform P. gingivalis W83 by electroporation as previously described (1). The cells were plated on BHI agar containing 10 μg/ml of erythromycin and incubated at 37°C for 7 days. The correct gene replacement in the erythromycin-resistant mutants was confirmed by colony PCR and DNA sequencing.

Construction of vimA-defective mutant in ATCC 33277 strain.Construction of a P. gingivalis ATCC 33277 isogenic mutant defective in the vimA gene was carried out by long PCR-based fusion of several fragments using overlapping extension PCR as previously described (23). The primers used in the development of deletion mutants are listed in Table S1 in the supplemental material. The PCR-fused fragment was used to transform the P. gingivalis ATCC 33277 strain by electroporation as previously described (1). The cells were plated on BHI agar containing 10 μg/ml of erythromycin and incubated at 37°C for 7 days. The correct gene replacement in the erythromycin-resistant mutants was confirmed by colony PCR and DNA sequencing.

Complementation of vimA-defective isogenic mutants.DNA fragments containing an upstream regulatory region and open reading frame (ORF) for vimA were amplified from P. gingivalis ATCC 33277 chromosomal DNA using the appropriate primer set (Table S1 in the supplemental material). A BamHI restriction site was designed at the 5′ end of both primers to facilitate the subcloning of the PCR fragment into the BamHI-digested pTCOW plasmid (15). The purified recombined plasmid, designated pFLL451a, was used to electrotransform P. gingivalis FLL451 (vimA::ermF). The transformants were selected on BHI agar plates in the presence of erythromycin and tetracycline.

RT-PCR analysis.Total RNA was extracted from P. gingivalis strains grown to early stationary phase (OD₆₀₀s, 1.2 to 1.3) using a RiboPure kit (Ambion, Austin, TX). The primers used for the reverse transcription-PCR (RT-PCR) analysis are listed in Table S1 in the supplemental material. The reaction mixture (50 μl) contained 1 μg of template RNA in the Superscript One-Step RT-PCR mix (Invitrogen, Carlsbad, CA). RT-PCR cycling conditions were 1 cycle of 5 min at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at 54°C, and 1 min at 68°C, with a final extension of 5 min at 68°C.

Evaluation of protease activity.Non-gingipain protease estimation was carried out using a protease assay kit (Molecular Probes) as previously described (2). The presence of Arg-X- and Lys-X-specific gingipain activities was determined as reported elsewhere (34).

Sialidase assay.Sialidase estimation in the P. gingivalis mutants was carried out using an Amplex Red neuraminidase (sialidase) assay kit (Molecular Probes). The assay utilizes Amplex Red to detect H₂O₂ generated by galactose oxidase oxidation of desialylated galactose, the end result of neuraminidase action. The H₂O₂, then, in the presence of horseradish peroxidase (HRP), reacts with Amplex Red reagent to generate the red-fluorescent oxidation product resorufin. The assay was read at a wavelength of 492 nm using a BioTek FLx800 microplate reader (2).

BCAA assay.The presence of branched-chain amino acids (BCAAs) was determined using a BCAA kit (BioVision Research Products, CA) as per the manufacturer's instructions. Briefly, the amino acid standard supplied with the kit was diluted to generate 0, 2, 4, 6, 8, and 10 nmol/well in a 96-well plate. The OD₄₅₀ value at each concentration was used to establish a standard curve. The P. gingivalis strains were grown to an OD₆₀₀ of 0.9 and centrifuged at 10,000 × g for 10 min. The bacterial cells (2 × 10⁶) were homogenized with 100 μl of assay buffer. Aliquots of the homogenized samples (50 μl) were added to the wells in duplicate. Fifty microliters of reaction mix (assay buffer, 46 μl; enzyme mix, 2 μl; substrate, 2 μl) was added to each well, and the plate was incubated for 30 min at room temperature. A background control was performed by replacing the enzyme mix with 2 μl of the assay buffer. The OD₄₅₀ of the background was subtracted from the test sample readings and the result was expressed as percent activity.

Determination of acetyltransferase activity.The assay for determination of acetyltransferase activity was carried out using an acetyltransferase assay kit (Stressgen) as per the manufacturer's instruction. Briefly, in a 96-well plate, various acetyl-CoA acceptor substrates, including isoleucine, leucine, alanine, valine, cysteine, arginine, methionine, threonine, and glycine, were used at a 100 μM final concentration. The reaction mixture was made up of 25 μl of 1× transferase assay buffer, 25 μl of the acetyltransferase, 25 μl of the acceptor amino acids, and 25 μl of the 1× reaction mix (supplied in the kit). The plate was covered with a foil sealer and incubated for 30 min with constant shaking in an orbital shaker. Fifty microliters of the positive control (supplied in the kit) was added to appropriate wells, followed by the addition of 50 μl of ice-cold isopropyl alcohol to each well. Finally, 100 μl of the 1× detection solution (supplied in the kit) was added to each well. The plate was covered with a foil sealer and further incubated for 10 min at room temperature without shaking. The plate was then read at 380-nm excitation and 520-nm emission wavelengths. The test was performed in triplicate, and the mean of the relative fluorescence unit at 380-nm excitation and 520-nm emission versus the enzyme concentration was plotted. The signal-to-noise ratio was calculated from the relative fluorescence unit of a blank with buffer alone.

Quantification of intracellular acetyl-CoA level.Twenty milliliters of exponential-phase cultures (OD₆₆₀, 0.8) was harvested and washed with phosphate-buffered saline (PBS). Cell pellets were resuspended in 1 ml of PBS, lysed using a French press, and then centrifuged at 10,000 × g. The supernatant was neutralized with saturated KHCO₃ and centrifuged for 5 min at 10,000 × g at 4°C. The level of acetyl-CoA in the cell extract was quantified using a PicoProbe acetyl-CoA assay kit (BioVision Research Products, Mountain View, CA) according to the manufacturer's instructions. Experiments were repeated in triplicate using three independent samples. An acetyl-CoA standard curve was generated using various concentrations (0 to 1,000 pmol/μl) of the acetyl-CoA standard provided. The fluorescence was measured using excitation and emission wavelengths of 535 and 589 nm, respectively. The background values were subtracted from the standard, and the concentration of the samples was calculated using KC4 software and a Biotek-FLx 800 spectrophotometer.

Static biofilm formation assays.Static biofilm formation was assayed using the protocol of Hinsa and O'Toole (22) with slight modification. Briefly, an overnight culture was diluted with fresh BHI medium to obtain 5 × 10⁷ CFU/ml. The cells were aliquoted into the wells of a 96-well microtiter plate (260 μl per well) and incubated anaerobically at 37°C for 24 h. The supernatant of the culture was aspirated and then washed twice with phosphate-buffered saline (150 mM NaCl, 3 mM KCl, 10 mM Na₂HPO₄, and 1.5 mM KH₂PO₄, pH 7.4). The biofilms were stained by incubation of each well with 100 μl of 0.1% crystal violet for 5 min. The plate was then washed twice with distilled water and destained with 95% ethanol (200 μl per well) for 5 min. The solubilized crystal violet was transferred to a new microtiter plate, and the OD₅₄₀ was measured. Biofilm formation was qualitatively determined to be proportional to the absorbance of the crystal violet.

Standard antibiotic protection assay.Invasion was quantified using the standard antibiotic protection assay (11). Briefly, bacteria were harvested from solid agar plates, washed three times in PBS, and then adjusted to 10⁷ CFU/ml of bacteria (confirmed by colony count) in Dulbecco's modified Eagle's medium. Epithelial cell monolayers were washed three times with PBS, infected with bacteria at a multiplicity of infection of 1:100 (10⁵ epithelial cells), and then incubated at 37°C for 90 min under a 5% CO₂ atmosphere. Nonadherent bacteria were removed by washing with PBS, while cell surface-bound bacteria were killed with metronidazole (200 μg/ml, 60 min). After removal of antibiotic, the internalized bacteria were released by osmotic lysis in sterile distilled water. Lysates were serially diluted, plated (in duplicate) on BHI agar, and incubated for 6 to 10 days. The number of bacterial cells recovered was expressed as a percentage of the original inoculum. The number of adherent bacteria was obtained by subtracting the number of intracellular bacteria from the total number of bacteria obtained in the absence of metronidazole (6).

Production of rabbit polyclonal antibodies against VimA protein.Expression and purification of rVimA in Escherichia coli were done as previously reported (64). The purified rVimA (25 μg/lane) was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) using NuPAGE 4 to 12% bis-Tris gels. A total of approximately 12 mg of the VimA protein was excised from the gels, placed in 1× PBS, and stored at −80°C. The frozen gel slices were sent to Zymed Laboratories Inc. (South San Francisco, CA) for the production of polyclonal rabbit VimA antibodies by using the manufacturer's standard protocol. The dilutions and efficiencies of the antibodies were tested in the laboratory with the purified rVimA.

Immunofluorescence assay.The indirect immunofluorescence test was performed as previously reported (31) using anti-FimA (33) and anti-VimA antibodies. Briefly, coverslip cultures of HeLa cells were grown by seeding 10⁵ cells/ml in 6-well plates (Nunc). After incubation at 37°C for 2 days, the confluent monolayers on coverslips were fixed in chilled acetone. Slides were washed twice in 0.05 M PBS (pH 7.2) for 15 min. The cells were incubated with anti-VimA antibody (1:1,000) for 40 min. The coverslips were washed twice in 0.05 M PBS (pH 7.2) for 15 min and then further incubated for 1 h with 1:1,000 fluorescein-labeled goat anti-rabbit IgG (Sigma-Aldrich). The smears were washed again, and coverslips were mounted using 4′,6-diamidino-2-phenylindole. The cells were viewed using a Zeiss G42-110-Axioskop microscope equipped with incident light for fluorescence microscopy.

Purification of chimeric VimA His-tagged protein and pull-down assay.Purification of the His-tagged VimA chimera protein was carried out using nickel-nitrilotriacetic acid magnetic agarose beads (Invitrogen, Carlsbad, CA) as previously reported (21). The protein pull-down assay was performed using a Dynabeads His-tag isolation and pull-down kit (Invitrogen, Carlsbad, CA). Approximately 75 μg of the purified His-tagged VimA chimera was incubated with the Dynabeads. The beads with attached VimA chimera were washed with wash/interaction buffer (50 mM NaH₂PO₄, 300 mM NaCl, 50 mM imidazole, and 0.005% Tween 20) and incubated with cell lysate from P. gingivalis W83. As a negative control, the lysates from P. gingivalis were incubated with the magnetic beads without the attached His-tagged VimA chimera. After incubation, the unbound proteins were eliminated by repeated washing in wash/interaction buffer. Proteins were eluted off the beads using the His elution buffer.

SDS-PAGE and immunoblot analysis.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). Samples were prepared (65% sample, 25% 4× NuPAGE lithium dodecyl sulfate (LDS) sample buffer, 10% NuPAGE reducing agent), heated at 72°C for 10 min, and then electrophoresed at 200 V for 65 min in an XCell SureLock minicell system (Invitrogen, Carlsbad, CA). The protein bands were visualized by staining with Simply Blue Safe stain (Invitrogen, Carlsbad, CA). The separated proteins were then transferred to BioTrace nitrocellulose membranes (Pall Corporation) using a semidry Trans-Blot apparatus (Bio-Rad) at 15 V for 25 min. The blots were probed with VimA-specific antibodies. Immunoreactive proteins were detected by the procedure described in the Western Lightning chemiluminescence reagent plus kit (Perkin-Elmer Life Sciences, MA). The secondary antibody was the goat anti-rabbit IgG (heavy plus light chains)–horseradish peroxidase conjugate (Zymed Laboratories, CA).

2D-PAGE analysis.Two-dimensional (2D) gel electrophoresis was carried out using 2D gel strips (7 cm) of pI 3 to 10 in a Protean isoelectric focusing cell (Bio-Rad) following the method of Poznanovic et al. (46). Briefly, the protein concentration of the sample was measured using a Bio-Rad spectrophotometer. The protein samples were diluted to a final concentration of 350 μg protein, and 20 μl of the sample was added to solubilization buffer {7 M urea, 2 M thiourea, 2% [wt/vol] 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 65 mM dithiothreitol [DTT], 0.002% bromophenol blue, 1% [wt/vol] Zwittergent 3-10}. The first-dimension immobilized pH gradient (IPG) strip was run by adding 100 μl of the diluted sample in the rehydration buffer (2 ml, 730 mg DTT, 70 mg iodoacetamide) for 8 h at voltage gradients of 3 h at 300 V, 5 h at linear gradients of 300 to 3,500 V, and 18 h at 3,500 V. After equilibration, the IPG strips were loaded on the gel and the gel was electrophoresed at 200 V and 0.3 A for 4 to 5 h and then stained with Coomassie simply blue strain.

LPS isolation from whole bacterial cells.Isolation of lipopolysaccharide (LPS) was carried using the Tri Reagent isolation reagent (67). Lyophilized bacterial cells (1 to 10 mg) were suspended in 200 ml of the Tri Reagent. For complete cell homogenization, the cell suspension was incubated at room temperature for about 10 to 15 min. After incubation, 20 ml of chloroform per mg of cells was added to create a phase separation. The mixture was then vigorously vortexed and further incubated at room temperature for an additional 10 min. The resulting mixture was centrifuged at 12,000 × g for 10 min to separate the aqueous and organic phases. The aqueous phase was transferred into a new 1.5-ml centrifuge tube. The mixture was vortexed, further incubated at room temperature for 10 min, and then centrifuged at 12,000 × g for 10 min. The upper aqueous phases from both steps were combined. Two additional water extraction steps were repeated to ensure complete removal of LPS from the organic phase. The combined aqueous phases were dried using a SpeedVac concentrator (Savant Instruments Inc., NY).

Lipid A isolation.Lipid A was isolated from crude LPS by mild acid hydrolysis (8). LPS was dissolved in 500 ml of 1% SDS in 10 mM sodium acetate (pH adjusted to 4.5 with 4 M HCl) and then placed in an ultrasound bath until the sample was dissolved. It was then heated at 100°C for 1 h. The mixture was dried by use of the SpeedVac. SDS was removed by washing the mixture with 100 ml of distilled water and 500 ml of acidified ethanol (prepared by combining 100 ml 4 M HCl with 20 ml 95% ethanol), followed by centrifugation (2,000 × g for 10 min). The sample was then washed again with 500 ml of (nonacidified) 95% ethanol and centrifuged (2,000 × g for 10 min). The centrifugation and washing steps were repeated. Finally, the sample was lyophilized to give fluffy white solid lipid A. To the freeze-dried content, 10 to 14 ml of solvents was added in the sequence chloroform, methanol, and water to achieve a final chloroform/methanol/water ratio of 1:2:0.8 (vol/vol/vol). Samples were shaken for 15 s immediately following the addition of each solvent and allowed to stand for about 18 h, with occasional shaking by hand. Phase separation of the mass-solvent mixtures was achieved by adding chloroform and water to obtain a final chloroform/methanol/water ratio of 1:1:0.9 (vol/vol/vol), and the lipid A content was estimated (48).

Preparation of lipid A-associated proteins.The extracellular material was removed by suspending the bacteria in saline, the mixture was stirred gently for 1 h at 4°C, and the bacteria were harvested by centrifugation. The endotoxin was extracted from the surface-washed P. gingivalis by the method of Morrison and Leive (39). Briefly, bacterial cells were suspended in 0.15 M NaCl at 4°C, and an equal volume of butanol was added. The suspension was mixed thoroughly at 4°C for 10 min and centrifuged at 35,000 × g for 20 min. The aqueous phase was removed, and the butanol, together with the insoluble residue, was further extracted twice with approximately half of the initial volume of saline. The combined aqueous phases were centrifuged to remove any insoluble residues and dialyzed against distilled water at 4°C for 48 h, using a Slide-A-Lyzer dialysis cassette (Thermo Scientific, Rockford, IL). This crude preparation, which contained both lipid A-associated proteins and lipopolysaccharide, was resuspended in water and ultracentrifuged at 100,000 × g for 3 h. The lipid A-associated proteins were prepared by the method of Yi and Hackett (67). Briefly, 90% phenol was added to the endotoxin suspended in pyrogen-free distilled water, and the mixture was extracted at 68°C for 20 min. After cooling, the phenol phase, which contains the lipid A-associated proteins, was separated by centrifugation at 35,000 × g and the aqueous phase was removed and dialyzed against distilled water for 42 h. The protein content of the lipid A-associated protein was determined using bovine serum albumin as a standard in an Eppendorf biophotometer. The carbohydrate content was determined by the method of Dubois et al. (10) using glucose as the standard.

MS and data analysis.An LCQ Deca XP Plus system (Thermo Scientific) with nanoelectrospray technology (New Objective) was used to analyze the peptides extracted from each gel piece (21). The four-part protocol used for the mass spectrometry (MS) and MS/MS analyses included one full MS analysis (from 450 to 1,750 m/z), followed by three MS/MS events using data-dependent acquisition, where the most intense ion from a given full MS scan was subjected to collision-induced dissociation, followed by the second and third most intense ions. The nanoflow buffer gradient was extended over 45 min in conjunction with the cycle, repeating itself every 2 s, using a 0 to 60% acetonitrile gradient from buffer B (95% acetonitrile with 0.1% formic acid) developed against buffer A (2% acetonitrile with 0.1% formic acid) at a flow rate of 250 to 300 nl/min, with a final 5-min 80% bump of buffer B before equilibration. In order to move the 20-μl sample from the autosampler to the nanospray unit, flow-stream splitting (1:1,000) and a Scivex 10 port automated valve (Upchurch Scientific, Oak Harbor, WA), together with a Michrom nanotrap column, were used. The spray voltage and current were set at 2.2 kV and 5.0 μA, respectively, with a capillary voltage of 25 V in positive ion mode. The spray temperature used for peptides was 160°C. Data collection was achieved using Xcalibur software (Thermo Electron), and the data were then screened with Bioworks software, version 3.1. MASCOT software (Matrix Science) was used for each analysis to produce unfiltered data and out files. Statistical validation of peptide and protein findings was achieved using X TANDEM (www.thegmp.org) and SCAFFOLD (version 2; Proteome Software) meta-analysis software. The presence of two different peptides at a probability of at least 95% was required for consideration of a positive identification. Confirmation of individual peptide matches was achieved using the BLAST database (www.oralgen.lanl.gov).

Electron microscopy.Transmission electron microscopy was performed using an FEI Tecnai G² transmission electron microscope by the method of Harris (18). Briefly, Formvar-coated carbon grids were prepared; the Formvar support was removed by placing the grids in an atmosphere of solvent vapor. The grids were then placed on a wire mesh in a glass petri dish with carbon tetrachloride below the wire mesh. Five to 10 ml of the sample was placed under the carbon side of a 4- by 5-mm square of mica (approximately twice the size of an electron microscope grid). The grid was washed in 0.5% acetic acid and then acetone. The carbon film was broken to free the specimen grid, after which the grid was placed in stain solution–neutral 1% aqueous phosphotungstic acid for 30 s. After it was blotted dry, the grid was examined using the Tecnai transmission electron microscope.

Ultrathin sections were made by the method described by Massey (38). After a 2-h incubation with bacteria, the HeLa cell monolayers were washed four times in PBS and detached from the plastic surface with trypsin. The cell slurry was then immediately centrifuged for 4 min at 15,000 × g. The cell pellet was washed twice with PBS and fixed with 2.5% gluteraldehyde in 0.1 M sodium cacodylate. Cells were pelleted and postfixed in 1% OsO₄ in 0.1 M sodium cacodylate for 1 h, and the ultrathin sections were contrasted with lead citrate and uranyl acetate before examination using the FEI Technai G² transmission electron microscope.