Outer Membrane Proteome and Antigens of Tannerella forsythia

Outer Membrane Proteome and Antigens of Tannerella forsythia Paul D. Veith, Neil M. O’Brien-Simpson, Yan Tan, Deasy C. Djatmiko, Stuart G. Dashper, and Eric C. Reynolds* Cooperative Research Centre for Oral Health Science, Melbourne Dental School, Bio21 Institute, The University of Melbourne, 720 Swanston Street, Melbourne, Victoria, 3010, Australia Received April 23, 2009

Tannerella forsythia is a Gram-negative, anaerobic, fusiform bacterium implicated as a periodontal pathogen. With use of 2D PAGE, SDS PAGE, and LC-MALDI-TOF/TOF MS, 221 proteins of T. forsythia outer membrane preparations were identified, of which 197 were predicted to be localized to the cell envelope. Fifty-six proteins were reproducibly mapped by 2D PAGE and included several highly abundant proteins in the MW range 140-250 kDa that exhibited C-terminal sequence similarity to the CTD family of Porphyromonas gingivalis. Two-dimensional Western blot analyses revealed that these CTD family proteins together with several other outer membrane proteins were antigenic. The CTD family proteins exhibited a higher than expected MW, and were strongly reactive with the fluorescent glycoprotein stain, ProQ Emerald. This group included BspA and surface layer proteins A and B. TonBdependent receptors (TDRs) (46) were identified together with 28 putative lipoproteins whose genes are immediately downstream of a TDR gene. The major OmpA-like protein was found to be TF1331. Uniquely, it was found to exist as a homodimer held together by up to three disulfide bridges as demonstrated by MS/MS of a tryptic peptide derived from unreduced TF1331. Keywords: Tannerella forsythia • outer membrane • proteome • antigens • TonB-dependent receptor • OmpA • glycoprotein • MALDI-TOF/TOF • 2D PAGE

1. Introduction Tannerella forsythia is a Gram-negative, anaerobic, fusiform bacterium that is found associated with Porphyromonas gingivalis and Treponema denticola in human subgingival plaque. Together as a consortium they are strongly implicated as periodontal pathogens.1,2 The virulence factors of T. forsythia are as yet poorly understood, due in part to the difficulty in culturing this fastidious organism, and the lack of genetic tools for creating mutants. A gene inactivation system was not available for T. forsythia until 2001 when one was developed to construct a bspA mutant.3 For the study of virulence mechanisms, and the associated development of therapeutic strategies, an understanding of the surface structures of pathogens at the molecular level is necessary. For Gramnegative bacteria, this can be initiated at the protein level by proteomic studies of the outer membrane (OM). Such studies have been conducted for a diverse range of pathogens including P. gingivalis, Actinobacillus pleuropneumoniae, Pasteurella multocida, Bartonella henselae, and Dickeya dadantii.4-8 Typically about 30-60 outer membrane proteins (Omps) are identified from such studies employing standard proteomic techniques such as 2D PAGE, MALDI-TOF, and LC-MS. The proteomic approach has not yet been applied to T. forsythia and only few Omps or virulence factors have been described. A distinctive feature of SDS-PAGE profiles of whole cell extracts of T. forsythia is the presence of a major double * To whom correspondence should be addressed. Phone: +61-39-3411547. Fax: +61-39-3411596. E-mail: [email protected]. 10.1021/pr900372c CCC: $40.75

 2009 American Chemical Society

band greater than 200 kDa. These bands correspond to two proteins designated surface layer protein A (TfsA) and surface layer protein B (TfsB) which are the major constituents of the surface layer (S-layer), a distinct layer beyond the OM that can be clearly seen in electron micrographs.9,10 Recently, these proteins were reported to play an important role in the adherence of the bacteria to gingival epithelial cells with use of deletion mutants.11 Another well-studied protein is BspA, a cell surface antigen that contains a leucine-rich repeat (LRR) domain and binds to fibrinogen and fibronectin.12 BspA has been demonstrated to mediate coaggregation with other bacteria, invasion of epithelial cells, and induction of alveolar bone loss in mice.13-15 T. forsythia also possesses trypsin-like activity16 and a sialidase (neuraminidase)17 but the importance of these potential virulence factors or their corelation to specific protein sequences is not yet clear. In this study, the OM of T. forsythia was analyzed by 2DPAGE, LC-MS/MS, and Western blotting to identify a total of 221 proteins including 16 antigenic proteins. The proteome is dominated by high MW glycoproteins and a very large number of TonB-dependent receptors (TDRs) and their associated lipoproteins.

2. Methods 2.1. Growth of Tannerella forsythia. T. forsythia (ATCC 43037) was grown anaerobically in batch culture in a Don Whitley Mark 3 anaerobic workstation. Lyophilized T. forsythia (5 × 109 cells) was rehydrated in 20 mL of brain heart infusion Journal of Proteome Research 2009, 8, 4279–4292 4279 Published on Web 06/29/2009

research articles (BHI)/Trypticase media (BHI-T media) containing yeast extract (7.5 g/L), sodium thioglycate (0.5 g/L), asparagines (0.25 g/L), D-glucose (2 g/L), ascorbic acid (2 g/L), pyruvic acid (1 g/L), sodium chloride (2 g/L), cysteine (1 g/L), N-acetylmuramic acid (10 mg/L), ammonium sulfate (2 g/L), thiamine pyrophosphate (6 mg/L), sodium bicarbonate (2 g/L) heat-inactivated bovine serum (5% v/v), hemin (5 mg/L), and menadione (1 mg/L). After 7-14 days of incubation (anaerobically, 37 °C, no shaking) once the optical density (O.D 650 nm) reached 0.6 O.D, 2 mL was used to seed 200 mL of BHI-T media and the cells were incubated (anaerobically, 37 °C, no shaking) to an OD of 0.6 OD650. T. forsythia cells were immediately harvested by centrifugation (8000 g) and resuspended in Tris buffer (20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2) and used in the preparation of outer membranes. Outer Membrane Preparation. The OM of T. forsythia was prepared by using a modified method of Filip et al.18 T. forsythia cells (in 35 mL of Tris buffer) where sonicated on ice for 15 min at 50% power and a duty cycle of 5 in a Branson Sonifier. Undisrupted cells and cell debris were removed by centrifugation (8000 g, 4 °C, 30 min) and the supernatant collected. The collected supernatant was centrifuged (100 000 g, 4 °C, 40 min) and the pellet resuspended in 2% w/v N-lauroylsarcosine (Fluka, cat. no. 61743) in Tris-buffer (8 mL) and mixed (2 h, 25 °C). After incubation the OM was collected by centrifugation (100 000 g, 4 °C, 40 min) and the pellet was resuspended in Tris-buffer. The OM was washed (3 × 100 000 g, 4 °C, 40 min) in Tris-buffer and finally resuspended in 0.5 mL of Tris-buffer and stored at -70 °C. Polyacrylamide Gel Electrophoresis (PAGE). For SDS-PAGE 30 µg of T. forsythia OM was reduced with 50 mM DTT in LDS sample buffer at 100 °C for 5 min, centrifuged to remove insoluble particles, and loaded onto a 10% NuPAGE Bis-Tris gel and SDS-PAGE performed at 150 V for 50-60 min with NuPAGE MOPS SDS Running Buffer and the XCell SureLock Mini-cell system (Invitrogen, NSW, Australia). For 2D-PAGE the ZOOM IPGRunner system was used and the isoelectric focusing and SDS-PAGE was performed as per the manufacturer’s instructions (Invitrogen, NSW, Australia). Briefly, 50 µg of T. forsythia OM was solubilized with 2D protein solubilizer 2 containing 50 mM DTT, 2% v/v Zoom carrier ampholeytes pH 4-7, and a trace amount of bromophenol blue. IPG strips (pH 4-7) were rehydrated with the solubilized T. forsythia OM (25 °C, 18 h). After rehydration the IPG strips were assembled in the IPGRunner Min-Cell system and isoelectric focusing (IEF) was performed (175 V, 15 min, 175-2000 V ramp for 45 min, 2000 V for 120 min). After IEF the IPG strips were either used directly for SDS-PAGE for Western blotting and carbohydrate staining or alkylated with 125 mM iodoacetamide in NuPAGE LDS sample buffer (15 min, 25 °C) for protein identification by MALDI-TOF. The IPG strips were set by 0.5% w/v agarose in running buffer on top of the ZOOM gel and SDS-PAGE was performed as described above. Gels were stained for protein with colloidal Coomassie Blue19 or for carbohydrate with Pro-Q Emerald 300 fluorescent stain (Invitrogen, NSW, Australia) according to the manufacturer’s protocol and the gels were imaged with a LAS 3000 imaging system (Fuji, Tokyo, Japan). TF1331 Enrichment. OM sample from above was incubated in solubilizer 2 (Invitrogen) at 50 °C for 10 min and centrifuged at 50 000 g for 15 min. The pellet was resuspended in solubilizer 2 then centrifuged again, and the supernatant was discarded. The pellet containing mainly TF1331 was analyzed by SDSPAGE as described above. 4280

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Veith et al. Spot Picking and Digestion of 2D Gel Separated Proteins. Coomassie stained 2D gels were first thoroughly destained and equilibrated in water and then placed in an SP gel frame of cut-out size 76 × 70 mm2 and placed onto the scanner of the Proteineer SP spot picker (Bruker Daltonics) where it was submerged under water. Each gel was scanned and analyzed by using Proteomweaver software (Biorad) to enable spot detection and gel calibration with respect to pI and MW. The spot list was transferred to the spot picker and spot picking was performed with a 1 mm diameter cutting tool and the preset “pick pick” method for nonbacked gels. The gel plugs were transferred to 96 well digest adaptors (Bruker Daltonics) and transferred to a Class III cabinet (Laminar flow is sufficient) to minimize subsequent keratin contamination. The 96 well plates containing the gel plugs were removed from the digest adaptors and blotted onto lint-free tissue and alternately washed with 30 µL per well of solution A (8% CH3CN in 20 mM NH4HCO3) and 30 µL per well of solution B (50% CH3CN in 20 mM NH4HCO3) and finally dehydrated in solution C (100% CH3CN) for a total of eight washes (ABABABCC). Each wash was for 5 min duration after which the wash solution was removed by blotting. Immediately after the second acetonitrile wash was removed by blotting, 1.8 µL of trypsin solution (10 ng/µL sequencing grade modified porcine trypsin (Promega), 20 mM NH4HCO3, 1 mM CaCl2) was added to each gel plug with a 10 µL multichannel pipet, carefully ensuring contact between each gel plug and the trypsin solution. After 5 min of reswelling at room temperature, the gel plugs were rewetted with 5 µL of solution A and incubated at 30 °C in the digest adaptors for 4 h. Peptides were extracted with 4 µL of 1% TFA v/v at 20 °C for a minimum of 30 min. A 600 µm anchorchip MALDI target was manually prepared with HCCA matrix by using the thin layer technique according to the anchorchip manual (Bruker Daltonics) just prior to sample deposition. Digested peptides (4 µL) were deposited onto the target and after 10 min adsorption the samples were collectively washed by the rapid pouring of at least 100 mL of 0.1% TFA over the plate. Identification of 2D Gel-Separated Proteins by MALDI TOF/TOF Analysis. All MS related hardware and software used were from Bruker Daltonics. Automated TOF/TOF was performed on an Ultraflex TOF/TOF20 upgraded to a PAN source and Lift II, and controlled by FlexControl v2.4 software. MS was performed with a 25 kV positive reflectron method that was calibrated by using a peptide mix applied to the center of the target prior to automated analysis. MS spectra were then automatically acquired for each sample with the laser power set to a narrow range and under fuzzy logic control. A total of 300 spectra were accumulated for each sample in sets of 15 spectra, with each set having to exceed a S/N of 2 and resolution of 5000 in order to be included. Peaks within a mass range of 1200-2500 were used for this evaluation. At most, 75 spectra were recorded at each raster position. Peak detection and subsequent export to the ProteinScape v1.2 database were achieved by using FlexAnalysis v2.4. Monoisotopic peaks were automatically labeled with the SNAP algorithm within the range 800-4000 Da with a S/N threshold of 6, a quality factor of 50, and a maximum of 100 peaks. MS/MS spectra were acquired by using the Lift method on parent ions selected by ProteinScape (see below). A single set of 50 spectra were accumulated for the parent ion with a S/N threshold of 3 at a fixed laser power followed by 14 sets of 50 fragmentation spectra at a laser power elevated by 30%, each set being acquired at a different

Outer Membrane Proteome of T. forsythia raster position. The MS/MS spectra were smoothed, the baseline was subtracted, and peaks were detected as above except that the quality factor threshold was reduced to 30. Peak lists were automatically sent to ProteinScape as above. ProteinScape was used for internal calibration of MS spectra, removal of calibrants and contaminants from the MS peak lists, and submission of peak lists to Mascot 2.1 (Matrix Science) for both PMF and MS/MS ions searches.21 All searches were against the annotated T. forsythia protein sequence database as obtained from LANL (http://www.oralgen.lanl.gov) in May 2006 (the original source of this data is the J. Craig Venter Institute, http://www.jcvi.org). Searches were limited to fully tryptic peptides with carbamidomethyl-Cys and Met-Oxidation set as fixed and variable modifications, respectively. Two PMF searches were conducted for each MS spectrum, the first with a large mass tolerance of 300 ppm and zero partial cleavages allowed, and the second with 150 ppm and one partial cleavage allowed. Internal calibration with trypsin autolysis products and common contaminants such as keratins was applied for the second PMF search only. A third PMF search was conducted on positively identified MS spectra to enable further proteins to be identified. In this case internal calibration was applied using the peptides already identified and a mass tolerance of 20 ppm and zero partial cleavages were allowed. After the first two PMF searches, data-dependent MS/MS acquisition was triggered by ProteinScape. Providing that peaks had a “goodness for MS/ MS” value greater than 200, up to two peaks were chosen to verify PMF results, a further two to identify additional proteins, and two if no protein was identified from PMF. MS/MS ions searches were conducted with a mass tolerance of 300 ppm on the parent and 0.8 Da on fragments. One missed cleavage was allowed. PMF and MS/MS searches were deemed correct if the Mascot score was greater than 60 (p < 0.005) or 25 (p < 0.01), respectively. 2.2. LC-MALDI TOF/TOF of SDS-PAGE Bands. Fifty-five bands were excised from the SDS-PAGE gel from each of four identical lanes and pooled together. Each sample was cut into approximately 1 mm3 cubes and washed, alkylated and digested according to Mortz et al.22 The peptides were further extracted once with water and once with CH3CN, pooled with the original digest, dried in a vacuum centrifuge, and stored at -20 °C prior to LC-MALDI TOF/TOF analysis. HPLC of peptide extracts together with their deposition onto PAC target plates (Bruker Daltonics) was performed according to Ang et al.23 with the following modifications. Separation was achieved with use of a RP column (C18 Acclaim Pepmap100, 300 µm id × 15 cm, 5 µm, 100 Å, Dionex) and eluted with 0.1% TFA and a gradient of 0-64% CH3CN over 40 min followed by 64-80% CH3CN over 5 min. The flow rate was 6 µL/min through the column. Fractions were collected every 12 s from approximately 20% to 74% CH3CN. The target plate was analyzed with Bruker Ultraflex III MALDI TOF/TOF. All spectra acquisition was performed automatically with FlexControl version 3.0.151 and WARP-LC version 1.1 software. MS analysis was carried in reflectron mode measuring from 700 to 4000 Da, using an accelerating voltage of 25 kV. All MS spectra were produced from five sets of 100 laser shots using random movement. Calibration of the instrument was performed externally with ions of prespotted internal standards. MSMS analysis was carried in LIFT mode in which the ions were accelerated to 8 kV and subsequently “lifted” to 19 kV in the LIFT cell. MSMS spectra were produced from 750 laser shots using random movement.

research articles MS peak lists were generated by FlexAnalysis version 3.0.90, using SNAP algorithm with S/N threshold 4. The peak list was filtered to remove common contaminants such as Coomassie Blue and keratin peaks. Selection of parent precursors was determined by using WARP-LC software. The compounds separated by less than six fractions were considered the same and were selected as parent precursors if the SN was >25. The MSMS peak list was also generated by using the SNAP algorithm after the spectra were smoothed with use of the Savitsky-Golay algorithm (width 0.2 m/z) and baseline subtraction with the TopHat algorithm. WARP-LC generated a combined MS/MS peak list that was searched by using MASCOT version 2.2.04 (Matrix Science) via BioTools 3.1.0 software. MS/MS ion searches against the T. forsythia database (described above) were conducted with a mass tolerance of 100 ppm on the parent and 0.5 Da on fragments. One missed cleavage was considered with carbamidomethyl (C) as fixed modification and oxidation (MHW) as variable modifications. A decoy search was done automatically by MASCOT on a randomized database of equal composition and size. All of the results for each band were pooled together and all peptide assignments with scores less than 15 or E-values >0.2 were deleted. A protein assignment was accepted if it had two or more peptides assigned with a score greater than the Mascot identity threshold, which ranged from 15 to 22 (p < 0.05, false positive rate measured to be 0.047). Proteins identified from a single peptide were accepted only if the peptide score was greater than 25 (p < 0.02, false positive rate measured to be 0.014). Peptides with a score less than identity threshold were still included for unique peptide count. 2.3. Preparation of Formalin Killed T. forsythia. T. forsythia cell cultures were grown to an optical density of 0.6 OD650 and collected by centrifugation (8000 g, 30 min, 4 °C). The cells were then washed once in TC150 buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.4) and suspended in a 2-5% volume of formal saline (0.5% v/v formaldehyde in saline, 150 mM NaCl). Cells were incubated overnight at room temperature under agitation, then washed and suspended in TC150. After preparation, formalin-killed cells were stored at 4 °C. 2.4. Preparation of Antisera. The immunization experiments were approved by the University of Melbourne Ethics Committee for Animal Experimentation. BALB/c mice 6-8 weeks old (25 mice per group) were immunized (intraperitoneally (i.p. 100 µL)) with either 50 µg of the T. forsythia OM preparation (solubilized in 4 M urea in phosphate buffered saline, PBS) or 50 µg of formalin-killed T. forsythia in phosphate buffered saline (PBS), pH 7.4, emulsified in complete Freund’s adjuvant (IFA). After 30 days and 42 days the mice received 2° and 3° immunization, respectively, of the corresponding antigen (s.c. injection, emulsified in IFA), after 54 days the mice were bled and the sera collected and stored at -20 °C. 2.5. Western Blotting. Following the second dimension of the 2D gel electrophoresis procedure, Western blots were performed by using the method of Dashper et al.24 except that the gels were equilibrated for 15 min in transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3, 2% v/v methanol) before transfer of proteins onto a PVDF membrane. Primary antibodies were diluted 1:25 and incubated with the membrane for 16 h. The antigenic proteins determined by Western blot analysis were compared with the corresponding 2D gel by overlapping the images with use of Adobe Photoshop software. The images were aligned by linear stretches of one image with respect to the other image until the best fit was obtained. Journal of Proteome Research • Vol. 8, No. 9, 2009 4281

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Veith et al. Interestingly, in addition to having strong bands at positions consistent with the 2D gel profile (Figure 1), the 1D gel (Figure 2) also exhibited significant band density in the 70-100 kDa region. Fifty-five continuous gel segments were manually excised from the gel, in-gel digested with trypsin, and subjected to LC-MALDI TOF/TOF analysis. A total of 2473 peptides were identified that were above threshold (p < 0.05) and had a minimum Mascot score of 15. Of these, 1163 peptides were nonredundant corresponding to 210 different proteins of which 134 were identified with at least two peptides above threshold (p < 0.05), and 76 were identified on the basis of having a single high-scoring peptide (p < 0.02) (Table 1, Supporting Information). The MS data are available in the PRIDE database (www.ebi.ac.uk/pride) under accession numbers 9894-9942 inclusive.

Figure 1. Representative 2D gel of T. forsythia outer membrane. Spots are numbered 1-54 with each number corresponding to a unique protein. Adjacent spots corresponding to the same protein are joined by lines. Spots marked with an asterisk (*) indicate that only a fragment of the protein was identified. Spots are color-coded, with yellow representing the CTD family proteins, green representing the TDR-associated lipoproteins (LP-T), blue representing the other predicted lipoproteins, pink representing the predicted integral Omps including TDRs, and black representing the other proteins. Spots labeled “N” and “C” represent N- and C-terminal fragments of surface layer protein B, respectively.

3. Results and Discussion 3.1. Identification of Proteins by 2D-PAGE. OM preparations of T. forsythia were separated by 2D-PAGE in the pI 4-7 range resulting in a reproducible pattern of spots in both technical and biological replicates (Figure 1). Most striking was the presence of several groups of very intense spots in the 100-200+ kDa range. Also of note were several intense discrete spots between 30 and 70 kDa in the center of the gel, and some very intense spots that were usually poorly resolved around 40 kDa at the basic end of the gel. There was a surprising lack of spot intensity between 70 and 100 kDa where TonBdependent receptors (TDRs) are usually found. Gel spots were excised from the gels with a robot, manually digested and analyzed automatically with a MALDI-TOF/TOF mass spectrometer. In all, 56 nonredundant proteins were reproducibly identified and mapped to one or more spots, as shown on the master 2D gel and table (Figure 1, Table 1). For simplicity, multiple spots matching the same protein were labeled with the same number, shown in parentheses in Table 1, column 1. To be included in this list, each spot had to be identified from at least two 2D gels. A further 11 proteins were identified from a single 2D gel only (Table 1, labeled (-) in column 1). In total, 67 nonredundant proteins were identified by Mascot by using either PMF (p < 0.002) or MS/MS ions search (p < 0.03). At this level, the rate of false positive detection against random (PMF) and reverse (MS/MS) databases was undetectable and 0.2%, respectively. 2D gel images and MS data can be found at http://world-2dpage.expasy.org with the accession no. 0012. 3.2. Identification of Proteins by SDS-PAGE and LCMALDI. Due to inherent limitations of 2D-PAGE and the fact that we only analyzed proteins within a pI range of 4-7, we also separated the OM sample by SDS-PAGE (Figure 2). 4282


Considering proteins identified from both techniques, all except 24 were predicted to be localized to the inner membrane, periplasm, OM, or beyond on the basis of sequence similarity to proteins of known subcellular location, by the presence of predicted transmembrane helices, or by use of the prediction program CELLO25 (Table 1). The high proportion of cell envelope proteins identified suggests that the sample was a relatively clean membrane preparation with only minor cytoplasmic contamination. Of the 130 proteins with the annotation “outer membrane” in their definition, 71 were identified in this study. Similarly, 13 out of the 26 CTD family proteins were identified (see below) suggesting that approximately 50% of the theoretical outer membrane proteome was identified. The identified proteins were grouped into categories, namely, CTD family proteins, putative lipoproteins (LP), outer membrane located proteins (OM) and TDRs, putative inner membrane proteins (IMP), putative periplasmic proteins (PP), and proteins with neither N-terminal signal nor trans-membrane helices and therefore likely to be located in the cytoplasm (Cyt) (Table 1). The gel spots are color coded according to these categories (Figure 1). 3.3. CTD Family Proteins. All of the high molecular weight proteins identified from 2D gels (MW > 80 kDa) were found to share sequence similarity over approximately 60 amino acid residues at their extreme C-terminal end, which we designate the C-terminal domain (CTD) due to its similarity to the CTD of a family of proteins in P. gingivalis and certain other members of the Bacteroidetes.8,26,27 In P. gingivalis, the presence of the CTD has been demonstrated to be required for proper maturation of the CTD-containing proteinase, RgpB, together with its correct secretion and attachment to the cell surface.26-28 The CTD may therefore be a secretion signal for a novel Bacteroidetes secretion pathway. A search within the T. forsythia database for further T. forsythia proteins with this domain revealed the presence of 26 CTD family proteins, similar to the list provided by Nguyen et al.27 of which ten were identified from 2D gels in this study, and a further three by LC-MALDI. The 13 include surface-layer protein A (TfsA) and surface-layer protein B (TfsB),10 BspA,13 and a possible internalin-related protein (TF1032) while the remainder are annotated as hypothetical proteins. Of these, TF2339 and TF1741 and its homologue, TF2592, were particularly abundant (Figure 1). BspA, as previously sequenced,12 corresponds most closely to TF2998 with 97% sequence identity. The C-terminal ∼300 residues of BspA are almost identical with TF1843, which is also annotated as BspA. Five other proteins in the database but not identified in this study are also annotated as BspA due to extensive sequence similarity to the original BspA. As

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Outer Membrane Proteome of T. forsythia

Table 1. Identification Data for All Proteins Identified (the 2D Gel Data and LC-MALDI Data Are Merged and Sorted According to Category and Accession) band no. (spot no.)

accessiona

Protein descriptiona, abbreviatedb

categoryc

MW calcd (kDa)

PMF scored

best MS/MSe

10 (11) 52 47 31-32 36 9B 30 24 28 (29) 30 25B 27 9B 50-51 34 (41) 24 48-49 50-51 25A (28) 28 34 25B 37 22 (18) 29 24 33 48-49 34 (34) 9A 47 52 15 16 16 16 (30) 15 10 15 11B 21A 16 9B 16 31-32 13 25A 10 none 10 none 13 22 (20) 13 22 (22) 11B 25B (26) none 22 10 (24) 25B (25) 10 25B 11B 20

TF0071 TF0299 TF0324 TF0399 TF0436 TF0508 TF0706 TF0761 TF0773 TF0810 TF1015 TF1038 TF1059 TF1300 TF1331 TF1409 TF1441 TF1443 TF1444 TF1476 TF1793 TF1822 TF1959 TF2123 TF2450 TF2595 TF2613 TF2734 TF2852 TF2901 TF3007 TF3114 TF0041 TF0063 TF0064 TF0301 TF0318 TF0875 TF0980 TF2096 TF2124 TF2778 TF3087 TF0045 TF0044 TF0093 TF0092 TF0111 TF0112 TF0237 TF0238 TF0275 TF0277 TF0313 TF0312 TF0424 TF0425 TF0482 TF0483 TF0588 TF0587 TF0640 TF0641 TF0654 TF0655

HP-C HP HP-C HP conserved hypothetical protein HP-C possible OM transport protein HP-C OM efflux protein possible OM efflux protein HP-C HP-C possible xanthan lyase HP-C Omp Omp TolC HP-C HP HP-C; possible hemin receptor Omp P49 polyphosphate-selective porin O OM lipoprotein silC precursor HP-C HP-C; TPR-repeat protein Omp HP-C HP-C HP-C HP-C HP-C HP-C HP Omp, TDR HP-C HP-C Omp, TDR Omp, TDR OM receptor, TonB-linked OM TDR possible OmpA, OM-related protein HP-C; possible TDR Omp, TDR HP-C Omp, TDR HP-C Omp, TDR Omp Omp Omp Omp, TDR Omp Omp Omp Omp, TDR Omp OM receptor, TonB-linked Omp OM receptor OM receptor Omp Omp Omp, TDR Omp OM receptor, TonB-linked Omp

OM(4.6) OM(3.0) OM(1.5) OM(3.0) OM(2.9) OM(4.3) OM(4.5) OM(3.2) OM OM(3.2) OM(4.1) OM(2.6) OM(3.3) OM(1.6) OM OM(4.2) OM(2.8) OM(1.6) OM(4.4) OM(4.5) OM(2.2) OM(3.0) OM(2.2) OM(3.6) OM(4.2) OM(4.7) OM(2.5) OM(3.1) OM(4.0) OM(3.6) OM(2.6) OM(2.3) TDR TDR? TDR? TDR TDR TDR TDR TDR TDR TDR TDR? TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T

104 17.2 26.7 52.5 61.1 83.9 46.9 99.3 52.3 45.0 58.0 49.0 115 24.4 44.5 58.7 23.6 50.0 60.4 53.3 43.0 58.5 38.8 115 103 59.1 47.1 24.1 43.6 282 24.0 43.4 92.4 89.2 74.9 98.8 87.5 90.6 92.4 121 69.4 89.7 97.9 90.6 47.6 112 59.0 115 65.2 120 75.9 113 61.7 113 64.7 112 59.1 123 67.7 129 58.5 118 55.7 118 72.2

124

56

76

60

160

137

100

74

88

61

149

73

57

101

protein scoref

unique peptidesg

577 86 67 269 41 53 358 52 377 108 198 34 26 37 1252 23 36 32 1118 118 27 226 39 157 133 44 43 53 258 43 379 114 87 188 141 750 237 50 163 439 88 526 69 53 70 1104 783 33

17 1 2 6 2 1 9 2 8 3 4 1 2 1 18 3 2 2 15 5 2 3 1 5 3 2 2 1 5 2 6 4 3 2 of 6 1 of 5 16 8 1 6 7 3 11 4 2 2 17 13 1

not identified 31

2

312 292 666 725 571 365

4 5 10 14 7 11

317 1471 311 779 294 238 102

7 23 11 18 10 7 4

not identified 141

86

228

57

101 37 not identified 94 198

76 92

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Table 1. Continued band no. (spot no.)

accessiona


categoryc

MW calcd (kDa)

10 29 11B 26 (27) 11B 24 15 21A (45) 15 (47) 19 (14) 10 25B 11B none 11B 21B (21) 10 24 (19) 20 52 11B none 11B none 11B none 11B 19 (15) 10 none 16 24 (23) 9B none 13 25A 11B 25B 9B none none (40) 13 22 10 25B 10 18 10 21A 11B 33 (37) 47 (50) 48-49 24 (17) 35 (38) 18 (-)i 40-42 (-)i 37 24 52 52 52 11B 39 14

TF0682 TF0683 TF0778 TF0779 TF0976 TF0977 TF1053 TF1052 TF1057 TF1056 TF1207 TF1206 TF1318 TF1319 TF1415 TF1416 TF1506-7a TF1505 TF1535 TF1534 TF1605 TF1606 TF1989 TF1990 TF2032 TF2031 TF2193 TF2192 TF2301 TF2302 TF2347-8a TF2349 TF2403 TF2402 TF2412 TF2411 TF2417 TF2416 TF2597 TF2596 TF2605 TF2606 TF2725 TF2726-7a TF2728 TF2729 TF2801 TF2802 TF3011 TF3012 TF3104 TF3103 TF_extrah TF0015 TF0090 TF0091 TF0220 TF0304 TF0305 TF0322 TF0348 TF0365 TF0368 TF0447 TF0546 TF0652

Omp, TDR HP-C Omp, TDR possible Omp OM receptor, Ton-linked possible Omp OM receptor, TonB-linked HP-C possible OM receptor, TonB-linked HP-C OM receptor, TonB-dependent HP-C OM receptor Omp Omp, TDR Omp OM receptor, TonB-dependent HP-C possible OM receptor protein HP-C Omp, TDR Omp Omp, possible TDR Omp Omp, TDR HP-C Omp, TDR possible Omp Omp, TDR HP-C, possible Omp Omp, possibly involved in nutrient binding HP-C Omp, TDR Omp, possibly involved in nutrient binding Omp Omp Omp, TDR HP-C OM receptor protein; possible TDR HP-C, possible LP Omp, TDR HP-C Omp, TDR Omp, possibly involved in nutrient binding Omp, TDR possible Omp Omp, TDR possible Omp Omp, TDR possible Omp Omp, TDR Omp Not in LANL Omp (possible immunogenic lipoprotein) HP-C Omp HP-C peptidyl-prolyl cis-trans isomerase peptidyl-prolyl cis-trans isomerase possible YngK protein HP HP HP HP HP-C HP-C

TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T TDR LP-T LP LP LP LP LP LP LP LP LP LP LP LP LP LP

112 49.1 114 54.4 118 63.7 117 67.8 114 71.7 127 59.4 117 57.7 121 65.6 140 60.9 81.8 18.4 126 58.6 130 67.0 112 62.8 122 76.3 125 51.5 107 61.9 132 58.9 123 59.4 116 58.3 107 45.9 87.3 44.7 123 65.0 133 57.1 133 73.6 128 68.6 118 45.5 54.0 24.2 50.3 42.2 56.4 25.5 27.3 60.7 16.3 16.2 13.5 10.8 32.7 79.6

4284


PMF scored

best MS/MSe

138

49

148 184 257

89 137 114

protein scoref

unique peptidesg

60 114 309 141 363 179 1041 441 427 1232 41 56 79

1 2 6 2 6 3 19 of 20 12 18 of 19 19 1 1 4

886 234 >225 91 94 82 35

20 7 9 3 5 1 1

not identified 148

53

122

74

not identified 37

1

96

1

768 553 177

12 15 4

32 74 112

1 4 8 of 9

78 339 2453 768 162

1 of 2 6 25 7 3

80 >104 557 292 739 154 74 25 876 1,256 172 139 129 506 75 55 90 80 33 44 76 343 97 64

3 1 7 17 2 23 8 1 1 20 22 3 4 5 7 2 2 2 1 1 1 1 4 1 1

not identified not identified 323

89

not identified 154

74

not identified

not identified not identified 54 66

230

151 70 103 39

98 66

29 40

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Outer Membrane Proteome of T. forsythia Table 1. Continued band no. (spot no.)

accessiona


categoryc

(46) (-)i 40-42 45 (-)i 35 (38) (12) 39 (49) 52 (16) 31-32 (-)i 33 (51) 43-44 50-51 (-)i 35 28 (36) 52 (53) 35 (39) 40-42 28 (33) 50-51 22 (18) 40-42 (43) 47(48) 22 (-)i (13) 37 (42) 43-44 40-42 (44) 14 31-32 26 (32) 9B 18 3(1) 8(6) (8) 8(10) 2-4 3(2) 8(5) 9B(7) 7(4) 5(3) (8) (9) 1 31-32 45 40-42 40-42 37 36 36 43-44 24 29 52 6 25B 21A 50-51 52 52 52

TF0661 TF0749 TF0750 TF0765 TF0945 TF1033 TF1055 TF1158 TF1342 TF1404 TF1440 TF1525 TF1565 TF1733 TF1755 TF1940 TF2016 TF2035 TF2206 TF2207 TF2214 TF2327 TF2414 TF2415 TF2447 TF2531 TF2804 TF2806 TF2843 TF2925 TF3013 TF3024 TF3165 TF0955 TF1032 TF1259 TF1741 TF1843 TF2116 TF2320 TF2339 TF2592 TF2646 TF2661-2a TF2663 TF2998 TF3080 TF3163 TF1478 TF0454 TF1351 TF1970 TF2574 TF0477 TF0789 TF3036 TF0797 TF0813 TF1201 TF1245 TF2333 TF2924 TF3099 TF0334 TF0743 TF1039

HP protease II HP-C HP-C HP-C; possible surface protein endothelin converting enzyme, endopeptidase HP OM LP, NlpE involved in copper resistance possible lipoprotein HP-C HP HP-C polysaccharide export protein, BexD/CtrA/VexA family HP-C periplasmic protease TPR-repeat-containing protein HP HP-C HP-C; possible sugar phosphate isomerase/epimerase exoalpha-sialidase (neuraminidase) peptidyl-prolyl cis-trans isomerase HP-C; possible lipoprotein HP HP lipoprotein possible dipeptidyl-peptidase III HP-C HP-C HP-C; possible lipoprotein beta-N-acetylglucosaminidase HP-C periplasmic protease thiol:disulfide interchange protein HP-C possible internalin-related protein HP HP-C surface antigen BspA** HP-C; possible hemagglutinin/hemolysin HP HP HP-C HP-C surface layer protein A surface layer protein B surface antigen BspA** HP-C HP-C membrane fusion efflux protein xanthine/uracil permease family protein HP-C oxaloacetate decarboxylase, beta subunit preprotein translocase SecY dipeptide/tripeptide permease, POT family preprotein translocase, secDF family glucose/galactose transporter HP-C glycosyl hydrolase, secreted possible preprotein translocase LemA protein signal peptidase I DNA-binding response regulator/sensor histidine kinase HP-C HP HP-C HP-C

LP LP LP LP LP LP LP LP LP LP LP? LP LP LP LP LP LP LP LP LP LP LP LP LP LP LP LP LP LP LP LP? LP LP CTD CTD CTD CTD CTD CTD CTD CTD CTD CTD CTD CTD CTD CTD CTD Imp1 Imp10 Imp10 Imp10 Imp10 Imp11 Imp11 Imp12 Imp2 Imp2 Imp2 Imp2 Imp2 Imp2 Imp2 Imp3 Imp3 Imp3

MW calcd PMF best protein unique (kDa) scored MS/MSe scoref peptidesg

29.5 83.0 32.0 24.9 40.6 77.3 32.8 16.0 54.5 44.9 37.2 15.7 31.3 17.9 39.3 65.6 17.8 42.6 34.8 51.5 21.6 55.3 26.2 25.5 51.1 78.4 32.8 28.6 33.0 87.4 50.5 54.4 41.2 128 55.5 242 131 116 132 207 201 132 58.9 135 153 114 141 214 40.9 45.6 46.5 41.2 49.2 58.0 83.5 46.2 53.5 55.4 12.2 21.1 55.4 113 21.5 12.4 12.6 23.7

140 47

98 46

78 136 121 72

48 55 21 96

168 30

114 27

201 214 68 453 247 25 38

111 62

42

137 75 149

63 55 114

100

33

90 87 129 102 122 159

36 59 113 26 124

129

130

76

107

169 196 102 250 101 63 261 308 66

82 69 40 71 71 49 116 122 148 40 25

45 46 28 364 81 261 146 464 64 308 139 194 178 364 119 546 51 40 74 45 46 614 846 134 27 3531 586 20 2257 2309

36 41 25 25 112 62 31 143 82 30 252 69 57 291 26 34 49 28 34

4 1 4 3 1 5 10 3 5 1 1 1 1 1 2 11 1 7 4 8 3 8 2 5 3 2 5 3 8 1 3 1 2 1 1 18 of 23 6 of 15 0/1j 5 2 40 of 45 2 of 11 3 39 of 40 37 of 38 0/1j 1 2 1 1 2 1 1 1 2 1 1 5 3 2 4 1 1 1 1 1


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Table 1. Continued band no. (spot no.)

accessiona

11B (52) 52 31-32 50-51 50-51 10 29 31-32 10 30 27 (31) 52 (54) 52 52 28 45 (-)i 19 13 52 29 21A 33 37 (-)i 50-51 (-)i 45 52 52 45 48-49 48-49 48-49 19 (35) 45 (-)i 36 45

TF1101 TF1964 TF0405 TF2920 TF3137 TF1413 TF0959 TF1775 TF2330 TF1897 TF0421 TF2803 TF0183 TF0216 TF0217 TF0439 TF0841 TF1123 TF1150 TF1151 TF1193 TF1325 TF1575 TF1595 TF2190 TF2421 TF2551 TF2552 TF2560 TF2566 TF2569 TF2579 TF2649 TF2650 TF2838 TF3006


categoryc

ABC transporter, ATP-binding protein Imp3 MotA/TolQ/ExbB proton channel family Imp3 HP-C Imp4 HP-C Imp4 Na+-translocating NADH-quinone reductase, subunit E Imp6 possible transmembrane protein Imp7 periplasmic protease PP (2.1) oxidoreductase, Gfo/Idh/MocA family PP (2.1) HP-C PP (2.1) HP-C; possible aminopeptidase PP (2.3) PP (2.5) R-L-fucosidase possible NADH-dependent dehydrogenase PP (4.3) HP-C Cyt 50S ribosomal protein L20 Cyt 50S ribosomal protein L35 Cyt Na+-transporting NADH:ubiquinone oxidoreductase, subunit 1 Cyt NADH dehydrogenase/NAD(P)H nitroreductase Cyt glycosyltransferase Cyt pyruvate-formate lyase Cyt HP-C Cyt glycosyl transferase, group 1 family Cyt L-fucose isomerase Cyt (3.0) DNA-binding response regulator Cyt HP-C Cyt HP-C Cyt cytocidal toxin protein Cyt 30S ribosomal protein S10 Cyt 50S ribosomal protein L3 Cyt 30S ribosomal protein S3 Cyt 50S ribosomal protein L5 Cyt 50S ribosomal protein L6 Cyt 30S ribosomal protein S4 Cyt succinate dehydrogenase, flavoprotein subunit Cyt succinate dehydrogenase, iron-sulfur subunit Cyt HP-C Cyt (3.5) transcriptional regulator RprY Cyt

MW calcd PMF best protein unique (kDa) scored MS/MSe scoref peptidesg

32.4 26.4 17.5 44.1 22.5 23.5 122 52.4 42.7 106 51.1 55.2 19.2 13.3 7.4 47.6 30.0 48.4 103 10.2 47.9 66.6 24.2 38.0 20.8 60.8 11.4 22.2 28.8 21.2 20.3 22.9 72.9 27.9 26.1 28.0

25 27

34

203 80

104 22

66

65

30 89

30

88 58

43 67

37 70 36 51 402 119 195 31 239 1095 151 32 118 48 195 42 62 60 63 343 33 179 56 283 30 91 52 69 57 57 503 419 31 25

1 1 1 1 1 1 8 1 5 1 9 21 2 1 1 1 4 1 2 1 1 6 1 3 1 7 1 1 3 1 4 3 15 8 1 1

a Accession numbers and protein descriptions are from the Oralgen Web site (www.oralgen.lanl.gov). Hyphenated accession numbers are where two adjacent genes in the database correspond to a single protein as indicated by both proteomics and homology data. b Abbreviations: HP, hypothetical protein; HP-C, conserved HP; and others as already defined. c Cellular location or protein type as explained in the text. The number of predicted trans-membrane helices is provided for IMPs (from Oralgen), and a localization score from CELLO (http://cello.life.nctu.edu.tw/) is provided for many other proteins. d The PMF Mascot score provided is the highest obtained for the protein/2D gel spot combination indicated. e The MS/MS Mascot score provided is the best single peptide score obtained for the protein/2D gel spot combination indicated. f The protein score is the best total MS/MS score obtained for the indicated protein by LC-MALDI of SDS-PAGE bands. g The number of unique peptides identified by MS/MS is from the LC-MALDI data except for proteins identified from 2D gels only. Overlap of identical peptides is provided for TF0063 and TF0064, TF1053 and TF1057, TF2403 and TF2412, TF1259 and TF2339, TF1741 and TF2592, and TF2661-2 and TF2663. h The protein with accession TF_extra was identified from DNA sequence obtained from TIGR (www.tigr.org) and has not yet been annotated in the Oralgen database. i Proteins identified from 2D gel spot numbers labeled (-) were only identified from a single 2D gel, and are not included in Figure 1. j The BspA proteins TF1843 and TF2998 could not be distinguished as their identification was based on a single overlapping peptide.

mutants lacking a functional BspA gene (presumably TF2998) are highly attenuated with respect to binding to and invading KB epithelial cells,13 it appears that the other BspA-like proteins are unable to compensate for the lack of TF2998 suggesting they have a different function. Alternatively, TF2998 may be the only BspA protein produced at a sufficiently high enough level to allow binding and invasion to be reliably detected. The surface layer of T. forsythia has been shown to be composed primarily of TfsA and TfsB.10,11 As these proteins have a CTD, it is likely that the secretion of these proteins and hence the production of the surface layer is dependent on the presence of the CTD and the CTD secretion system.26,27 The observed 2D gel MW of eight CTD family proteins was higher than the theoretical mass raising the possibility that they may be glycosylated (Table 2). Therefore, replicate 2D gels were stained with a carbohydrate-specific flourescent dye (Figure 3). The spots which reacted strongly with the dye were indeed mostly the CTD family proteins, especially TF2646 (Table 2). 4286


The observed MW of this protein was 114 kDa as compared to the expected value of only 48 kDa, consistent with a high level of glycosylation. Furthermore, despite the significant intensity of this protein on 2D gels, only 5 different peptides could be recovered (Table 2). Non-CTD family proteins TF1342, TF2804, and TF2414 also appeared to be glycosylated (Table 3). The only CTD family proteins not to have a MW greater than that predicted were TF3080 and the BspA proteins TF1843 and TF2998 (Table 2). Each of these had an observed MW considerably less than expected suggesting proteolysis rather than nonglycosylation. The individual BspA proteins could not be distinguished, as their identification was based on a single peptide that is identical in both proteins. To assess whether any of the other CTD family proteins might be N- or C-terminally truncated, peptides close to the N- or C-termini in several of the CTD family proteins were specifically analyzed by MS/MS to confirm their identity (Table 2). In this way, the mature N-terminus of four proteins (TF1259,

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Figure 3. Carbohydrate staining and Western blot analysis. 2D gels of T. forsythia OM stained with Pro-Q Emerald fluorescent carbohydrate stain (B) prior to staining with Coomassie Blue (A). 2D-Western blots of T. forsythia OM were performed and probed with (C) antisera raised against the OM preparation and (D) antisera raised against formalin killed T. forsythia cells. E. Western blot image from panel C colored red and overlaid with Coomassie Blue stained 2D gel from panel A (blue). The scales of the images were manipulated by linear stretches only. The identities of numbered spots are presented in Tables 2 and 3. Table 2. Further Data for CTD Family Proteins Identified from 2D Gels

Figure 2. SDS-PAGE gel of T. forsythia outer membrane. Identical samples of T. forsythia OM were separated on a 10% PA BisTris gel with MOPs running buffer. The gel was stained with Coomassie Blue G250. Fifty-five contiguous gel segments were excised from four identical lanes. Two lanes are shown in order to clearly mark the center of each excised segment.

spot

1, 1* 2 3 4 5 6 7 8f

9 10

accession no. residues

CTD-Paralog % ID, % cova

Western OM/WCb

TF1259 2192 res TF2339 1816 res TF2663 1364 res TF2661-2 1179 res TF2592 1216 res TF1741 1197 res TF2646 540 res TF1843 1092 res TF2998 1087 res TF3080 1288 res TF2116 1252 res

TF2339 68, 60 TF1259 68, 60 TF2661-2 33, 65 TF2663 33, 65 TF1741 67, 99 TF2592 67, 99 TF2645 32, 51 TF2998 51, 99 TF1843 51, 99 TF1458 90, 100 TF1552 38, 52

CH20 stainc

most N-terminal peptide foundd

most C-terminal peptide foundd

+/+

+

+/+

+

+/+

+

+/+

+

+/+

+

+/+

+

+/+

+++

30-50 (pGlu) MS2 ) 82 26-32 (pGlu) MS2 ) 19 29-38 (pGlu) MS2 ) 32 25-33 (mature) LCMS2 ) 49 148-167 MS2 ) 49 63-78 MS2 ) 45 64-73 (PMF)

2069-2078 MS2 ) 28 1720-1739 LCMS2 ) 118e 1268-1284 MS2 ) 64 1085-1099 LCMS2 ) 130 1115-1129 MS2 ) 37 1096-1110 MS2 ) 61 406-421 (PMF)

?

+

531-546 (MS)

891-909 (MS)

?

+

330-343 (MS)

886-904 (MS)

?

+

461-475 (PMF)

1052-1065 (PMF)

?

+

73-82 (PMF)

1155-1174 (PMF)

sequence coverage

MW: obsd/ calcd

12%

248/230

28%

208/190

35%

191/143

40%

158/125

32%

144/120

33%

144/120

10% 5 peptides 4% 3 peptides 4% 3 peptides 7% 8 peptides 18%

114/48 83/108 83/106 95/141 158/122

a The closest relative in the same species (Paralog) containing the CTD domain is shown together with the % identical residues, and the corresponding % sequence coverage. b Data from Figure 3. OM ) antisera to OM sample (Figure 3C); WC ) antisera to whole cells (Figure 3D). c Data from Figure 3B. Reactivity to carbohydrate stain. d The data provided are from 2D gels except where indicated by LCMS.2 Residue numbers for each peptide are given together with the method of identification and Mascot score where relevant. e This peptide was identified as being 1 Da higher than expected, possibly due to deamidation. f The identity of TF1843 and TF2998 could not be distinguished as the matched peptides have identical sequence in both proteins.


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Table 3. Antigenic Proteins (non-CTD) Identified from Figure 3a spot no.

Accession

Category

Western OM/WC

CH20 Stain

41 42

TF1056 TF1342 TF0090 TF2123 TF0091 TF0945 TF1331 TF2804

LP-T LP LP OM LP LP-T OM LP

+/+ +/+ +/+ +/+ +/+ +/+ +/+/+

+ +

43

TF2414

LP

+/+

+

14 16 17 18 38

a

Comment

TDR-associated SusF, TDR-associated contains 8 TPR repeats SusE, TDR-associated TDR-associated OmpA-like protein “probable secreted glycosyl hydrolase” in operon with TDR

See Tables 1 and 2 for an explanation of the columns.

TF2339, TF2661-2, and TF2663) was determined from peptides identified by MS/MS that correspond to the predicted Nterminus, and were nontryptic at the N-terminal side. Three of the N-termini were found to be modified to pyroglutamine (Table 2). Besides TF3080 and the BspA proteins (see above), there was no indication that any of the other CTD proteins had undergone significant N-terminal truncation. Toward the Cterminus, however, there was a consistent lack of sequence coverage in the CTD region, with no peptides being found within the last 77 residues. Four peptides were found adjacent to this area with strong Mascot scores in TF2661-2, TF2663, TF2339, and TF1741 (Table 2). A lack of sequence coverage in the CTD was also observed in P. gingivalis. The CTD family proteins identified in P. gingivalis tended to be smaller, and diffused over a broad MW in SDS-PAGE or 2D-PAGE.8,26 This was not observed in T. forsythia, however, even for C-terminal fragments of surface layer protein B which still maintained sharp spots despite a higher than expected MW (Figure 1). This may suggest that the modification in T. forsythia has different properties or is less heterogeneous to that in P. gingivalis. As with P. gingivalis, the absence of CTD domain peptides suggests that the CTD is heavily glycosylated or that part or all of the CTD is removed by proteolytic processing. Comparison of the CTD sequences of T. forsythia to P. gingivalis reveals interesting differences (Figure 4). Motif E, consisting of an invariant Lys residue followed by hydrophobic residues is essentially the same in each species. Likewise, motif D characterized by an invariant GxY motif in P. gingivalis is also shared by T. forsythia with the notable exception of the surface layer proteins (TF2661-2, TF2663) which have Val in place of Tyr. Motif B also has a very similar character with invariant Gly and conserved hydrophobic, polar, and aromatic residues, again with the notable exception that the surface layer proteins lack conservation of the aromatic residue. The main difference in motif B is that in place of the highly conserved Asp in the P. gingivalis CTD, T. forsythia appears to prefer neutral hydrophilic residues such as Asn, Thr, or Ser. The overall length of the T. forsythia CTD is similar to the average length in P. gingivalis; however, in P. gingivalis the spacing of the motifs is more heterogeneous. Interestingly, the less well conserved N-terminal region of the CTD has a well conserved length between motif B and the most C-terminal peptide identified between the two species (Figure 4). In the apparent absence of a highly conserved motif in this region it is likely that its overall character and positioning allows it to be posttranslationally modified and or proteolytically processed. 3.4. Antigenic Proteins. Two 2D gels prepared in an identical manner and with the same sample to that shown in Figure 4288


1 were prepared and subjected to Western Blot analysis by using antisera raised to T. forsythia formalin-killed whole cells and outer membranes (Figure 3). The Western blot images were overlaid with images from Coomassie Blue stained gels in order to identify the antigenic proteins (Figure 3, Tables 2 and 3). Sixteen antigenic proteins were identified (Figure 3, Tables 2 and 3). All of the abundant CTD family proteins were easily identified in the Western images due to their characteristic spot patterns (Table 2). As these are also glycosylated it is uncertain whether the protein or the carbohydrate is being recognized by the antisera. The three non-CTD family proteins that appeared to be glycosylated, TF1342, TF2804, and TF2414, were also strongly antigenic (Table 3). Six further antigens that do not appear to be glycosylated were identified of which four are predicted lipoproteins (Table 3). Except for the surface layer proteins10 none of the other antigenic proteins have been previously identified in T. forsythia. TF1331 is similar to the Omp40 and Omp41 antigens (PG33, PG32) from P. gingivalis,29 and TF0090 and TF0091 are similar to TDR-associated starch binding proteins in B. thetaiotaomicron.30 Apart from TF1331, all of the identified antigens were reactive to sera raised against formalin killed whole T. forsythia cells indicating their likely cell-surface exposure. There are a considerable number of unidentified antigenic spots in the >65 kDa region, particularly in the Western probed with anti-OM sera. The spots that were identified in this region were primarily CTD proteins and CTD protein fragments. As all the identified CTD proteins were antigenic, it is likely that many of these unidentified antigens also correspond to CTD proteins or fragments thereof. There are also major antigenic spots close to spot no. 22 that could not be positively assigned to a Coomassie-stained spot. 3.5. Putative Lipoproteins. A total of 75 putative lipoproteins were identified based on the presence of a signal sequence suitable for cleavage by lipoprotein signal peptidase31 (Table 1). Many of the lipoproteins were annotated as hypothetical, or conserved hypothetical indicating that their function is as yet unknown. The signal sequences of the putative lipoproteins contained preferably serine, or alternatively glycine or alanine in the -1 position relative to the predicted cleavage site. Lipoproteins identified that have functional names include peptidyl-prolyl cis-trans isomerases (TF2214, TF0304, TF0305), proteinases (TF0749, TF1033, TF1755, TF2531, TF3024), a TPR domain protein (TF1940), a thiol:disulfide interchange protein (TF3165), a previously described β-N-acetylglucosaminidase (TF2925),32 and an exo-R-sialidase (or neuraminidase, TF2207).17 A basic protein (TF0447) with a predicted mass of 10.8 kDa (Table 1) was identified in almost every 1D gel band from 49 to 160 kDa, being most abundant in the 110 kDa band suggesting this predicted lipoprotein forms SDS-resistant complexes, with itself and or other proteins. The genes encoding 28 putative lipoproteins were found to be located adjacent to predicted TDR genes, suggesting that their products have a role in the transport of solutes together with their respective TDRs (see below). As shown in green in Figure 1, these TDRassociated lipoproteins (LP-Ts) represent some of the most intense spots on the 2D gel, particularly within the 39-64 kDa range. 3.6. TonB-Dependent Transport. As there are a very large number (>60) of predicted TDRs in the T. forsythia database, and we could only identify fragments of three from the 2D gel, we obtained the theoretical MW and isoelectric points of the TDRs whose genes are adjacent to the major LP-Ts that were


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Figure 4. Multiple alignment of CTD family proteins from T. forsythia and P. gingivalis. CTD family proteins from T. forsythia (TF) were identified by multiple BLAST searches against the T. forsythia database from the Oralgen Web site (www.oralgen.lanl.gov). To reduce redundancy, six CTD sequences TF2592, TF2339, TF1591, TF2998, TF3080, and TF0541 were excluded due to their high sequence identity to the exhibited CTD sequences TF1741, TF1259, TF1589, TF1843, TF1458, and TF0537, respectively. Multiple alignment was conducted separately for T. forsythia and P. gingivalis sequences using the ClustalW program. Invariant residues within the T. forsythia sequences are marked with an asterisk (*), strongly conserved positions are marked with a colon (:), and moderately conserved positions are marked with a period (.). Deletions of two residues and four residues from the P. gingivalis alignment are marked with triangles (1). Underlined residues represent the most C-terminal peptides identified by MS/MS (Table 2). Motifs B, D, and E are marked in accordance with Seers et al.26

Figure 5. Heat-modifiability and the effect of reduction on the MW of TF1331. TF1331 enriched sample was subjected to SDSPAGE: (A) 100 °C, +DTT; (B) 50 °C, +DTT; (C) 100 °C; (D) 50 °C. Arrows represent bands identified as TF1331 by MS. Benchmark protein standards were used containing proteins of MW 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 160, and 220 kDa. The 10, 15, and 25 kDa markers are not shown.

identified (see above). These TDR sequences typically predict a MW of 80-120 kDa and a basic pI, demonstrating why they were not observed in the pH 4-7 range used. LC-MALDI of 1D gel bands, however, resulted in the identification of 46 putative TDRs. Comparing the 46 identified TDRs with the 28 identified LP-Ts revealed that a total of 26 TDR/LP-T pairs were identified, 11 “lone” TDRs were identified that did not have an adjacent LP-T, nine TDRs were identified whose genes were adjacent to unidentified LP-Ts, and two LP-Ts were identified whose genes were adjacent to unidentified TDRs (Table 1). The

37 TDR/LP-T pairs (including unidentified proteins) are presented together in Table 1 below the “lone” TDRs. Interestingly, multiple sequence alignment of all identified TDRs revealed two major groupings (sequence alignment not shown). The larger group consists of large interrelated TDRs (108-140 kDa) with adjacent and interrelated LP-Ts. In contrast, the remaining TDRs exhibit poorer sequence conservation, are smaller (69-121 kDa), and are mostly “lone” TDRs. The exceptions are TF1535, TF0682, TF2597, TF0045, and the unidentified TDR, TF2606. Each of these has a downstream LP that is not significantly related to the other LP-Ts identified. A small number of TDR loci such as TF0094-TF0090, which is similar to the susB operon of Bacteroides thetaiotaomicron, contain multiple downstream genes that encode lipoproteins. The susB operon, which comprises susB through to susG, is involved in starch utilization.30 SusB and SusG are glycosidic enzymes, SusC is a TDR, and SusD, SusE, and SusF are OM lipoproteins. SusC and SusD are reported to form a complex that is essential for binding of cells to starch. SusE and SusF also appeared to interact with each other and the SusC-D complex; however, they were not essential for binding to starch.30,33 The susB operon in T. forsythia appears to be homologous to the B. thetaiotaomicron system except that the susG gene is absent. Interestingly, SusD (TF0092) was only identified from 1D gels in this study, which may be consistent with it forming a strong complex with SusC (TF0093) and therefore due to the high pI of SusC was not resolved on the pI 4-7 2D gels. SusD exhibits sequence similarity to many of the other LP-Ts identified whose genes are directly downstream of their respective TDR; however, the additional lipoproteins such as TF0090 and TF0091 whose genes are not directly downstream of a TDR are not well conserved. These results Journal of Proteome Research • Vol. 8, No. 9, 2009 4289

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Figure 6. LIFT-TOF/TOF data for m/z 3516.6 disulfide bonded peptide from TF1331. (A) Full spectrum. Boxed areas are presented enlarged in panels B and C. (B) Zoomed-in spectrum showing products resulting from cleavage of disulfide bonds. Symbols used to represent ˇ ) dehydroalanine; Cˇ ) thioaldehyde; X ) dithiocysteine. the chemical state of the three fragmented disulfides: C ) reduced cysteine; A Other combinations besides those that are shown are possible. (C) Zoomed-in spectrum showing backbone fragmentation pattern and its assignment to the RPEFCPECPKCPEVK peptide from TF1331.

suggest that the primary LP-Ts identified have a similar solute binding function associated with their respective TDRs. Most sequenced Gram-negative bacteria have up to nine TDRs encoded in their genomes and more than 80% have less than 25 TDRs and only 4% have more than 60.34 The 60 or more present in T. forsythia therefore is unusual. An overrepresentation of TDR genes was also reported for the B. thetaiotaomicron genome that encodes 106 predicted TDRs,34 and 57 LP-Ts (SusD paralogs). The majority of these are part of loci containing polysaccharide degrading enzymes and therefore the TDRs and LP-Ts of such loci are likely to be involved in binding to and transporting polysaccharides into the periplasm. T. forsythia, however, does not appear to share the same plethora of polysaccharide utilization enzymes and most TDR loci are devoid of them. Overrepresentation of TDR genes is also observed in various Proteobacteria, specifically those that share the ability to degrade a wide range of complex carbohydrates. The difference between TDR systems found in Proteobacteria compared to Bacteroidetes is that the former do not 4290


contain SusD homologues.35 This is the first time to our knowledge that TDR overrepresentation has been demonstrated at the protein level. As proteomic techniques are currently unable to detect all proteins expressed within a proteome, the results imply that T. forsythia prefers to express the majority of its TDRs at moderate to high levels rather than expressing a small range of receptors to suit the availability of specific nutrients. 3.7. Other Omps. Other proteins identified that are predicted to be integral to the OM include the P. gingivalis homologues, Omp41 (TF1331), P40 (TF2852), and P58 (TF1444),8 and the OM efflux proteins TF0773, TF0810, TF1409, TF1476, and TF1822 which are related to TolC.36 Due to the finding in P. gingivalis that Omp41 and its homologue Omp40 were heterodimeric under nonreducing conditions, held together by two disulfide bridges, and also exhibited heat-modifiability similar to OmpA,37 TF1331 was partially purified and examined by SDS-PAGE in both reducing and nonreducing conditions and at both 50 and 100 °C (Figure

research articles

Outer Membrane Proteome of T. forsythia 5). Under reducing conditions, TF1331 migrated at 42 kDa when fully denatured, or 33.5 kDa when partially denatured by heating at 50 °C, consistent with the heat modifiability demonstrated for Omp40/41 of P. gingivalis37 and of OmpA.38 Under nonreducing conditions, TF1331 migrated to a MW of 99 kDa when fully denatured, or 72 kDa at 50 °C, which is very similar to the MW estimated for heterodimeric Omp40/41 of P. gingivalis37 suggesting that the nonreduced form of TF1331 is a homodimer held together by disulfide bridges. To confirm this, the mass spectra of digests of the nonreduced forms were compared to digests from the reduced forms and also to the predicted masses of disulfide bonded tryptic peptides. A peak at m/z 3516.63 was found to be specific to the nonreduced TF1331 and its mass corresponded to the tryptic peptide 244RPEFCPECPKCPEVK258 triply disulfide bonded to itself to form a dimer of theoretical m/z 3516.57. To confirm this assignment, the digest was reduced with 10 mM DTT and reanalyzed by MS. Reduction caused the disappearance of the 3516.57 peak, and instead, a new peak at 1761.84 corresponding to the reduced peptide appeared (data not shown). MS/MS of this peak confirmed the assignment with a Mascot score of 57. MS/MS was also attempted on the m/z 3516.63 to directly confirm its identity. The MS/MS spectrum (Figure 6) was dominated by a cluster of symmetrical peaks centered at half the MW of the parent ion. The spacing between the isotopes was 1 Da, discounting the possibility of it representing a doubly charged parent ion. The central peak appeared to consist of three distinct peaks that correspond to the monomer peptides with each cysteine reduced (m/z 1761.8), and the presence of dehydroalanine and dithiocysteine (m/z 1759.8) and the presence of dehydroalanine, dithiocysteine, and thioaldehyde (m/z 1757.8) representing LIFT-TOF/TOF fragmentation patterns that have been previously reported for disulfide bonded peptides.39 The groups of peaks either side of the central peak correspond to a different mix of reduced cysteines, dithiocysteines, dehydroalanines, and thioaldehydes producing a difference of 32 Da (a sulfur atom) between clusters (Figure 6). Small peaks at approximately -17 Da relative to the large peaks are probably due to the loss of ammonia from the N-terminus. The intensity of other fragments was generally low; however, by comparing the fragmentation pattern with that of the reduced peptide it was possible to assign a significant series of a- and b-ions (Figure 6). The b-5 ion was 2 Da less than the b-5 ion of the reduced peptide, possibly due to the formation of a CdS bond, and also appeared to lose sulfur. An analogous phenomenon appeared to happen for the b-8 ion (Figure 6). Taken together, the MS/ MS data provide substantial evidence that the m/z 3516.63 peak corresponds to a disulfide-bonded dimer of the 244 RPEFCPECPKCPEVK258 peptide. It is not certain whether the main role of OmpA-like proteins such as TF1331 is simply to form a structural link between the OM and peptidoglycan wall40 or whether the ability of some to form a diffusion pore is biologically important.41

4. Conclusion This study defines and maps the major Omps of T. forsythia. The OM proteome is dominated by CTD family proteins, proteins involved with TonB dependent transport, and the OmpA-like protein, TF1331. TF1331 is a novel disulfide bonded homodimer that shares the OmpA-like properties of heat modifiability and high copy number. The CTD family members

include the surface layer proteins which are very abundant and also the more weakly expressed BspA protein that exhibits multiple virulence properties. Several other CTD-family proteins of very high abundance (e.g., TF2339 and TF1741) are implicated as being present on the surface of the cell, but with no clue as yet to their function. Forty-eight TDR loci were identified including 46 TDRs and 28 lipoproteins, most of which were very abundant. Fifteen proteins were found to be antigenic, and these could be useful for developing diagnostics and therapeutics against T. forsythia infection.

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Outer Membrane Proteome and Antigens of Tannerella forsythia

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