Identification of PorSS-dependently secreted proteins of P. gingivalis
We constructed an rgpA rgpB kgp porK mutant from an rgpA rgpB kgp strain and compared secreted proteins between the rgpA rgpB kgp and rgpA rgpB kgp porK strains to avoid degradation of secreted and surface proteins by gingipains as the wild-type strain secreted gingipains that had the ability to process both secreted and surface proteins, while the porK mutant secreted no gingipains. 2D-PAGE of the particle-free (membrane-free) culture supernatants from the kgp rgpA rgpB and kgp rgpA rgpB porK mutants was performed. As a control, three protein spots in each 2D gel, which exhibited the same amounts of proteins with the same molecular masses and isoelectric points, were subjected to MALDI-TOF mass analysis, resulting in the same proteins (PGN_0916, PGN_1367 and PGN_1587; Fig. 1). Their molecular masses and isoelectric points calculated from their amino acid sequences were 69 044 and 4.88 for PGN_0916, 49 199 and 5.99 for PGN_1367, and 30 130 and 5.30 for PGN_1587, respectively, which were consistent with their positions on the 2D gels. At least 16 protein spots, which were present in the particle-free culture supernatant of the kgp rgpA rgpB strain, were absent or faint in that of the kgp rgpA rgpB porK mutant (Fig. 1). Relative amounts (kgp rgpA rgpB porK versus kgp rgpA rgpB) of the protein spots were calculated (Table 2). The protein spots were then subjected to MALDI-TOF mass analysis. PMF analysis of the spots, in comparison with the genome database of P. gingivalis ATCC 33277T (Naito et al., 2008), allowed the identification of 10 proteins (Table 2). An immunoreactive 46-kDa antigen (PGN_1767) was identified in two different protein spots [spot 10 (33 kDa) and spot 8 (42 kDa)]. Both 33- and 42-kDa PGN_1767 proteins contained the D42-R66 fragment at the most N-terminal position, whereas the 42-kDa protein possessed the G403-R418 fragment in the CTD, but the 33-kDa protein did not, suggesting that the 42-kDa PGN_1767 protein was processed at the C-terminal end to yield the 33-kDa PGN_1767 protein. PGN_0659 (35-kDa hemin binding protein, HBP35) was identified in four different spots [one (spot 9) with a molecular mass of 36 kDa and three (spots 12, 13 and 14) with a molecular mass of 28 kDa] in 2D-PAGE. The three 28-kDa protein spots had different isoelectric points. All of the 28- and 36-kDa HBP35 proteins contained the A61-K87 fragment at the most N-terminal position, whereas the 36-kDa protein possessed the D244-R329 fragment at the C-terminal end, but the 28-kDa proteins had the E234-K273 or D244-K273 fragment, suggesting that the 36-kDa HBP35 protein was processed at the C-terminal end to yield the 28-kDa HBP35 proteins. HBP35 exhibits thioredoxin and hemin-binding activities and has an important role in heme acquisition for growth (Shoji et al., 2010). PGN_0898 (spot 15) is a bacterial peptidylarginine deiminase (PAD). Wegner et al. (2010) showed that deletion of the PAD (PGN_0898)-encoding gene resulted in complete abrogation of protein citrullination. Inactivation of Arg-gingipains, but not Lys-gingipain, led to decreased citrullination, suggesting that host peptides generated by proteolytic cleavage at Arg-X peptide bonds by Arg-gingipains were citrullinated at the C terminus by PAD. Citrullinated bacterial and host peptides may cause the autoimmune response in rheumatoid arthritis (Lundberg et al., 2010). CPG70 (PGN_0335, spot 4) exhibits Lys- and Arg-specific metallocarboxypeptidase activity. A previous study (Chen et al., 2002) suggested that CPG70 may have an important role in C-terminal processing of cell surface proteins containing Arg-gingipains, Lys-gingipain and adhesins of P. gingivalis. TapA (PGN_0152) was identified in two different protein spots [spot 7 (44 kDa) and spot 6 (48 kDa)]. TapA is associated with the periplasmic tetratricopeptide repeat protein TprA that is upregulated in host tissues of the subcutaneous chamber model, and is involved in the virulence of P. gingivalis W83 as mice infected with the tapA and tprA mutants showed higher survival rates than those infected with the wild-type (Yoshimura et al., 2008; Kondo et al., 2010). PGN_1416 (spot 3) is considered to be a lysyl endopeptidase in the P. gingivalis genome database, whereas PGN_0291 (spots 1 and 2), PGN_0654 (spot 11), PGN_0795 (spot 5) and PGN_1476 (spot 16) are hypothetical proteins (Naito et al., 2008). A number of proteins appeared to be more abundant in the particle-free supernatant of the rgpA rgpB kgp porK strain than that of the rgpA rgpB kgp strain, particularly in the pH 6–7/35- to 55-kDa region of the gel. However, it was not reproducible and the proteins included no CTD proteins (data not shown).
Table 2. Identification of protein spots shown in Fig. 1
|Spot||PGN number||Score||Protein||Relative amount of protein spota|
|3||PGN_1416||321||Probable lysyl endopeptidase precursor||0.13|
|4||PGN_0335||158||Conserved hypothetical protein with zinc carboxypeptidase domain (CPG70)||0.12|
|6||PGN_0152||210||Immunoreactive 61-kDa antigen (TapA)||0.12|
|7||PGN_0152||194||Immunoreactive 61-kDa antigen (TapA)||0.10|
|8||PGN_1767||165||Immunoreactive 46-kDa antigen||0.15|
|9||PGN_0659||125||35-kDa hemin binding protein (HBP35)||0.15|
|10||PGN_1767||55||Immunoreactive 46-kDa antigen||0.13|
|11||PGN_0654||90||Hypothetical protein (putative lipoprotein)||0.14|
|12||PGN_0659||105||35-kDa hemin binding protein (HBP35)||0.13|
|13||PGN_0659||97||35-kDa hemin binding protein (HBP35)||0.13|
|14||PGN_0659||88||35-kDa hemin binding protein (HBP35)||0.13|
|15||PGN_0898||134||Probable peptidylarginine deiminase (PAD)||0.20|
Figure 1. Comparative analysis of proteins in the particle-free culture supernatants of Porphyromonas gingivalis. 2D-PAGE analysis with IPG strips covering isoelectric points 4–7 was done using particle-free culture supernatants from the kgp rgpA rgpB and kgp rgpA rgpB porK mutants (a). Proteins were stained with CBB R250. No or faint protein spots corresponding to protein spots with red circles in the kgp rgpA rgpB mutant sample were observed in the kgp rgpA rgpB porK mutant sample. Two areas were magnified (b and c). Blue circles indicate positions of PGN_0916, PGN_1367, PGN_1587 and PGN_0180 (FimA; spots A1, A2 and A3). Numerals in parentheses are mascot scores of the proteins.
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Next, we compared a 2D-gel profile of the vesicle fraction of the rgpA rgpB kgp strain with that of the rgpA rgpB kgp porK strain (Fig. 2). We found two (spots 26 and 39) and seven (spots 45, 46, 48, 49, 53, 56 and 59) spots of CTD proteins in the vesicle fractions of the rgpA rgpB kgp and rgpA rgpB kgp porK strains, respectively. Five spots (spots 45, 46, 48, 56 and 59) were only observed in the rgpA rgpB kgp porK strain. CPG70 (PGN_0335) was identified in spots 45 and 46 (Table S2). PGN_1476, PAD (PGN_0898) and TapA (PGN_0152) were identified in spots 48, 56 and 59, respectively. An immunoreactive 46-kDa antigen (PGN_1767) and HBP35 (PGN_0659) were identified in both strains, but spot 49 (PGN_1767) and spot 53 (PGN_0659) of the rgpA rgpB kgp porK strain were clearly larger than spot 26 (PGN_1767) and spot 39 (PGN_0659) of the rgpA rgpB kgp strain, respectively. These six CTD proteins were also found in the particle-free supernatant of the rgpA rgpB kgp porK strain (Table 2). Molecular mass and PMF analyses revealed that the six CTD proteins found in the particle-free culture supernatant of the rgpA rgpB kgp strain were processed at the C terminus compared with those found in the vesicle fraction of the rgpA rgpB kgp porK strain (Table 3, Figs S1 and S2).
Table 3. Comparison of molecular masses of protein spots
|PGN number||Protein||Molecular mass of protein spot (kDa)|
|Particle-free culture supernatant of rgpA rgpB kgp in Fig. 1||Vesicle fraction of rgpA rgpB kgp porK in Fig. 2|
|PGN_0152||TapA||48 (6), 44 (7)||64 (59)|
|PGN_0335||CPG70||76 (4)||100 (45), 98 (46)|
|PGN_0659||HBP35||36 (9), 28 (12, 13, 14)||36 (53)|
|PGN_0898||PAD||48 (15)||56 (56)|
|PGN_1476||HP||42 (16)||51 (48)|
|PGN_1767||IR 46 kDa antigen||42 (8), 33 (10)||42 (49)|
Figure 2. Comparative analysis of proteins in the vesicle fractions of Porphyromonas gingivalis. 2D-PAGE analysis with IPG strips covering isoelectric points 4–7 was done using vesicle fractions from the kgp rgpA rgpB and kgp rgpA rgpB porK mutants. Protein spots with red circles were identified as CTD proteins.
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We also determined patterns of 2D-gels of the outer membrane fractions from the wild-type and porK strains (Fig. 3, Table S3). Spot 4 (PGN_1689), spot 5 (PGN_1366), spot 6 (PGN_0287), spot 8 (PGN_0293), spot 10 (PGN_1432), spot 11 (PGN_1808), spot 13 (PGN_0293), spot 14 (PGN_0293), spot 18 (PGN_0290), spot 19 (PGN_0293), spot 20 (PGN_0293) and spot 23 (PGN_0729) were present only in the wild-type. Judging from the molecular masses of the protein spots, the proteins appeared to be proteolytically processed products in the wild-type. None of them possessed CTD at the C-terminal region.
Figure 3. Comparative analysis of proteins in the outer membrane fractions of Porphyromonas gingivalis. 2D-PAGE analysis with IPG strips covering isoelectric points 4–7 was done using outer membrane fractions from the wild-type and porK strains. No or faint protein spots corresponding to protein spots with green circles in the wild-type sample were observed in the porK mutant sample.
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To examine the effect of the porK mutation on the expression of the 10 secreted protein-encoding genes at a transcriptional level, RT-PCR analysis was performed and relative amounts of mRNA were determined (Fig. 4). The genes encoding PGN_0291, PGN_0335 and PGN_0898 in the PorSS-deficient strain (kgp rgpA rgpB porK) were expressed at the same level as those in the PorSS-proficient strain (kgp rgpA rgpB), whereas the genes encoding PGN_0152, PGN_0654, PGN_0659, PGN_0795, PGN_1416 and PGN_1767 were about 50% downregulated in the PorSS-deficient strain compared with the PorSS-proficient strain. The gene encoding PGN_1476 in the PorSS-deficient strain was expressed about three times more than that in the PorSS- proficient strain. As the relative amounts of the protein spots were < 20% (Table 2), the results suggest that decrease of the 10 secreted proteins in the PorSS-deficient mutant are mostly dependent on the defect in the PorSS.
Figure 4. Relative quantities of mRNAs of genes for PorSS-dependent secretion proteins. Quantities of mRNAs of 10 genes for PorSS-dependent secretion proteins (PGN_0152, PGN_0291, PGN_0335, PGN_0654, PGN_0659, PGN_0795, PGN_0898, PGN_1416, PGN_1476 and PGN_1767) and genes for PGN_0293 (receptor protein A, RagA), PGN_0916 (DnaK), PGN_1296 (outer membrane protein A, OmpA) and PGN_1587 (elongation factor Ts, Tsf) were determined by real-time qPCR. The relative quantity of mRNA of each gene is indicated as the ratio of the amount of mRNA of each gene in the kgp rgpA rgpB porK mutant to that in the kgp rgpA rgpB mutant.
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The 10 PorSS-dependently secreted proteins as well as precursor forms of Arg-gingipains (RgpA and RgpB) and Lys-gingipain (Kgp) had CTDs in which the conserved DxxG and GxY motifs and the conserved Lys residue are located (Seers et al., 2006; Fig. 5). Seers et al. (2006) reported that 34 CTD family proteins with sequence similarity to the C-terminal region of the RgpB precursor were identified by a blast search with the P. gingivalis W83 genome, which include the 10 proteins identified in the present study. Slakeski et al. (2010) suggested that the CTD of RgpB is essential for covalent attachment to the cell surface by an A-LPS anchor containing anionic polysaccharide repeating units. In our previous studies (Kondo et al., 2010; Shoji et al., 2011), we demonstrated that HBP35 and TapA were modified by A-LPS and anchored on the bacterial cell surface. In addition, the green fluorescent protein–CTD fusion study revealed that the CTDs of CPG70, PAD and HBP35 as well as RgpB play roles in PorSS-dependent translocation and glycosylation (Shoji et al., 2011). We suggested in the study both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Cleaved CTD fragments of HBP35, CPG70, PAD, RgpB and PGN_1767 have recently been found in the culture supernatants of P. gingivalis (Glew et al., 2012), which is consistent with the present study and supports our model (Shoji et al., 2011).
Figure 5. Alignment of the C-terminal domains of the PorSS-dependent secretion proteins experimentally determined. Sequences spanning the last 80 residues of each protein were aligned with clustalw.
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Our results strongly indicate that the P. gingivalis secreted proteins with CTDs, which are responsible for colony pigmentation, hemagglutination, adherence and modification/processing of the bacterial surface proteins and host proteins, are translocated to the cell surface by the PorSS.