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The Journal of Immunology, 2008, 181, 5537 -5544
Copyright © 2008 by The American Association of Immunologists, Inc.
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Binding of Complement Inhibitor C4b-Binding Protein Contributes to Serum Resistance of Porphyromonas gingivalis1

Michal Potempa*,{dagger}, Jan Potempa{dagger},{ddagger}, Marcin Okroj*, Katarzyna Popadiak*,{dagger}, Sigrun Eick§, Ky-Anh Nguyen, Kristian Riesbeck|| and Anna M. Blom2,*

* Department of Laboratory Medicine, Section of Medical Protein Chemistry, University Hospital Malmö, Lund University, Malmö, Sweden; {dagger} Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; {ddagger} Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602; § Department of Medical Microbiology, University Hospital of Jena, Jena, Germany; Westmead Centre for Oral Health, Institute of Dental Research, Sydney, Australia; and || Department of Laboratory Medicine, Section of Medical Microbiology, University Hospital Malmö, Lund University, Malmö, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The periodontal pathogen Porphyromonas gingivalis is highly resistant to the bactericidal activity of human complement, which is present in the gingival crevicular fluid at 70% of serum concentration. All thirteen clinical and laboratory P. gingivalis strains tested were able to capture the human complement inhibitor C4b-binding protein (C4BP), which may contribute to their serum resistance. Accordingly, in serum deficient of C4BP, it was found that significantly more terminal complement component C9 was deposited on P. gingivalis. Moreover, using purified proteins and various isogenic mutants, we found that the cysteine protease high molecular weight arginine-gingipain A (HRgpA) is a crucial C4BP ligand on the bacterial surface. Binding of C4BP to P. gingivalis appears to be localized to two binding sites: on the complement control protein 1 domain and complement control protein 6 and 7 domains of the {alpha}-chains. Furthermore, the bacterial binding of C4BP was found to increase with time of culture and a particularly strong binding was observed for large aggregates of bacteria that formed during culture on solid blood agar medium. Taken together, gingipains appear to be a very significant virulence factor not only destroying complement due to proteolytic degradation as we have shown previously, but was also inhibiting complement activation due to their ability to bind the complement inhibitor C4BP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Periodontitis is an inflammatory condition with an infective etiology that leads to the loss of the supporting structures of the tooth. Together with Treponema denticola and Tannerella forsythia, Porphyromonas gingivalis is a part of the "red complex" of microorganisms most often associated with periodontitis (1). Although this periodontopathogen can be cultured occasionally from healthy sites, the bacteria proliferate in high numbers during active periodontal disease (2) despite the fact that there is a significant Ab response (3). Furthermore, several periodontal health indicators correlate inversely with the presence of P. gingivalis (4) and P. gingivalis is able to induce periodontal disease in animal models (5). More recently, there is accumulating evidence that P. gingivalis may also be associated with cardiovascular disease (6). Virulence factors produced by P. gingivalis include outer membrane vesicles, adhesins, LPS, hemolysins, and proteinases (7).

Among the proteinases, the gingipain cysteine proteinases are responsible for 85% of the general proteolytic activity displayed by the pathogen. There are three members of the gingipain family: lysine-gingipain (Kgp)3 is specific for the lysine-X peptide bond, whereas arginine-gingipains (RgpA and RgpB) are specific for the arginine-X peptide bond (8). RgpA, derived from the rgpA gene, is present in several molecular forms due to extensive posttranslational processing and glycosylation of the nascent polypeptide chain. These include the membrane-bound enzyme mt-RgpA and its two soluble forms, the 50-kDa catalytic domain alone (RgpA(cat)) and the 95 kDa, noncovalent complex composed of the catalytic domain and hemagglutinin/adhesin domains (HRgpA). In contrast to rgpA, rgpB lacks the sequence encoding hemagglutinin/adhesin domains and therefore its product, RgpB, may be encountered only in two different forms: either membrane-bound (mt-RgpB) or as a soluble 50-kDa RgpB. The hemagglutinin/adhesin domain responsible for binding to fibrinogen, fibronectin, and laminin as well as for hemagglutinin activity of P. gingivalis is also found in Kgp (9). Working in concert, gingipains are able to cleave not only constituents of periodontal tissues, including basement membrane structural protein collagen, but are also able to degrade host proteins used for protection, such as Abs and components of the complement system (10).

Complement is a major arm of the innate immune defense system and its main function is to recognize and destroy microorganisms (11). The three pathways of human complement ensure that virtually any non-host surface is recognized as hostile. The classical pathway is usually mediated by binding of the C1 complex to Igs recognizing invading pathogens. Thus, complement enhances the effectiveness of the existing "natural" or specifically generated Abs in pathogen clearance. The lectin pathway is able to recognize via mannose-binding lectin, "foreign" polysaccharide molecules normally present only on microbial surfaces. C4 is a crucial component of both pathways as it becomes covalently attached to the surfaces that activated C1 or mannose-binding lectin to form a part of the C3 convertase complex (C4bC2a), which activates C3. Finally, complement can also be activated through the alternative pathway, which can be directly initiated by properdin or due to a failure to appropriately regulate the constant low-level spontaneous activation of C3 (initiated due to inherent instability of this protein). All three pathways lead to opsonization of pathogen with C3b, which enhances phagocytosis while releasing anaphylatoxins C5a and C3a to attract phagocytes. Finally, the end result of the complement cascade is formation of the membrane attack complex (MAC) and lysis of the target cell. Host cells protect themselves from bystander damage following complement activation through the expression of membrane-bound or recruitment of soluble endogenous complement inhibitors.

C4b-binding protein (C4BP) is a circulating inhibitor of the classical and the lectin pathways of complement and inhibits the formation and accelerates the decay of C3 convertase. It also serves as a cofactor to factor I in the proteolytic degradation of C4b (12) and C3b (13). C4BP is a large plasma glycoprotein that exists in several forms with varying subunit composition. The major form consists of seven identical {alpha}-chains (70 kDa each) and one β-chain (45 kDa) (14). The {alpha}- and β-chains are composed of repeating domains of ~60 aa residues known as complement control protein (CCP) domains, with the {alpha}-chain having eight while the β-chain only three such domains (15). C4BP is also linked to the coagulation system since the β-chain is bound with high affinity to the vitamin K-dependent anticoagulant protein S (14).

Every successful human microbial pathogen must develop means to circumvent complement and we have found that many bacteria are able to capture either C4BP and/or factor H (FH), an inhibitor of the alternative pathway, and thereby decrease complement activation on their surface. This leads to a decrease in opsonization and ensuing phagocytosis. For example, binding of C4BP to M proteins of Streptococcus pyogenes appears to be responsible for the resistance of these bacteria to phagocytosis (16). We have shown previously that resistance to killing by serum of Neisseria gonorrhoeae correlates with the ability of gonococci to bind C4BP (17) and that human C4BP selectively interacts with N. gonorrhoeae, which results in species-specific infection (18). Pathogens known to bind C4BP include S. pyogenes (19), Moraxella catarrhalis (20), Escherichia coli strain K1 (21), Borrelia recurrentis (22), Candida albicans (23), and Haemophilus influenzae (24).

In the current study, we demonstrate that both culture collection strains and clinical isolates of P. gingivalis interact with C4BP, which contributes to the exceptional resistance to the complement system by this microorganism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Proteins

C4BP (in complex with protein S) was purified from human plasma (25) and labeled with FITC (26) as described previously. C4b was purchased from Complement Technologies. Recombinant C4BP was expressed in eukaryotic cells and purified by affinity chromatography (27). Arginine-specific (HRgpA and RgpB) and lysine-specific (Kgp) gingipains were purified from the P. gingivalis HG66 strain culture fluid as described previously (28, 29).

Bacterial strains and culture conditions

P. gingivalis strains listed in Tables I and II were grown in enriched tryptic soy broth medium (TSB) or on blood TSB agar at 37°C in an anaerobic chamber (Concept 400; Biotrace) with an atmosphere of 90% N2, 5% CO2, and 5% H2. For growth selection of mutants on solid medium, 1 µg/ml tetracycline or 5 µg/ml erythromycin was used. Clinical strains were obtained from patients with severe periodontitis (aggressive periodontitis, n = 3; chronic periodontitis, n = 9). Two paper points were inserted in each pocket for 20 s and subsequently placed in 2 ml of a transport medium (reduced buffered saline). After vigorous mixing for 30 s, the samples were serially diluted up to 10–5. Aliquots of 0.1 ml were plated on Schaedler-agar (Oxoid) supplemented with 8% sheep blood without antibiotics on the same agar plates with 100 µg/ml kanamycin. The Schaedler-agar plates were incubated anaerobically at 37°C for 7 days. After that the total number of CFU as well as the colonies typical for P. gingivalis were counted, the identity was confirmed by a biochemical test (rapid ID 32A identification system (bioMerieux) and 16S rDNA sequence analysis. The percentage of P. gingivalis was up to 57% of the total anaerobically cultivable flora. Strain PorT was constructed as described (30) and displayed the previously reported phenotype.


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  		Table I. Description of laboratory P. gingivalis strains used in this study

 

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  		Table II. Description of clinical P. gingivalis strains used in this study

 
Binding of C4BP to bacteria

P. gingivalis from 6-day-old agar plates (unless indicated otherwise) were harvested, washed twice in the binding buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 2 mM CaCl2 (pH 7.2)), and adjusted to an OD of 1.0 at 600 nm. FITC-labeled C4BP was diluted in the binding buffer to a concentration of 50 µg/ml and mixed with 6 x 105 cells followed by incubation for 60 min at RT. In competition experiments, samples also contained plasma-purified C4BP (20 µg/ml), recombinant C4BP (20 µg/ml), C4b (100 µg/ml), mAb 104 (100 µg/ml), mAb 67 (100 µg/ml), heparin (100 µg/ml), or BSA (100 µg/ml). mAb 104 and mAb 67 are directed against CCP1 and CCP4 of the C4BP {alpha}-chain, respectively (27). The concentration of C4BP used as competitor was chosen to give 50% inhibition on the basis of initial titration. Other competitors were added at five times higher concentrations than C4BP to compensate for possible avidity effects since C4BP is a polymeric molecule able to interact with most ligands via multiple binding sites. Thereafter, the cells were washed twice in the binding buffer and finally resuspended in flow cytometry buffer (50 mM HEPES, 100 mM NaCl, 30 mM NaN3, and 1% BSA (pH 7.4)). Flow cytometry analysis was performed using a FACSCalibur (BD Biosciences).

Bacterial binding of C4BP from serum

Normal human serum (NHS) was prepared from blood taken from six healthy volunteers and pooled. P. gingivalis from 6-day-old agar plates (unless indicated otherwise) were harvested, washed twice in the binding buffer, and adjusted to an OD of 1.0 at 600 nm. NHS was diluted in GVB++ (5 mM Veronal buffer, 140 mM NaCl, 0.1% gelatin, 1 mM MgCl2, and 0.15 mM CaCl2 (pH 7.3)) to a concentration of 5%, mixed with 6 x 105 cells, and incubated for 75 min at 37°C with shaking. Thereafter, the bacterial cells were washed twice and incubated with monoclonal mouse anti-C4BP Abs (mAb 104, 2 µg/ml) for 1 h at RT. Bacteria were washed twice and resuspended in goat anti-mouse FITC-conjugated polyclonal Abs (diluted 1/1000; DakoCytomation) and incubated for 1 h at RT. All washing and Ab-binding steps were performed in the binding buffer. Thereafter, flow cytometry buffer was added and flow cytometry analysis was performed. An aliquot of the bacteria was stained with a standard Gram staining procedure and photographs were taken using an LCD camera connected to a microscope (Nikon).

Binding of C4BP to purified gingipains by ELISA

Microtiter plates (Maxisorp; Nunc) were incubated overnight at 4°C with 50 µl of a solution containing 8 µg/ml HRgpA, RgpB, or Kgp in 75 mM sodium carbonate (pH 9.6). Plates were washed four times with 50 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20 (pH 7.5) between each of the following steps. The wells were blocked with the quenching solution (3% fish gelatin (Nordland) in the washing solution) for 1 h at RT. C4BP was diluted in TBS and used at concentrations ranging from 3 to 200 µg/ml and was thereafter incubated for 4 h at RT. When binding of C4BP and its mutants to HRgpA was tested, the recombinant proteins were diluted in GVB++ and used at 20 µg/ml. In the competition assay with prothrombin (purified from plasma) and fibrinogen (Sigma-Aldrich), C4BP was used at 15 µg/ml in TBS mixed together with up to 100 µg/ml competitors. Deposited C4BP was detected by mouse anti-C4BP Abs diluted in the quenching solution. Bound Abs were detected with HRP-labeled goat anti-mouse secondary Abs (DakoCytomation). Bound HRP-labeled polyclonal Abs were revealed using 1,2-phenylenediamine dihydrochloride tablets (DakoCytomation) and the absorbance was measured at 490 nm using a microtiter plate reader (Varian).

Deposition of C9 on bacteria

For C4BP depletion, fresh NHS was passed through a HiTrap column (GE Healthcare) coupled with mAb 104 (31). The flow through was analyzed by ELISA and the fractions lacking C4BP were pooled and frozen in –80°C. Plasma-purified C1q was added to the depleted serum to compensate for C1q that bound to the Ab-coupled column. The final concentration of C1q in NHS and C4BP-depleted NHS was then verified by ELISA. C4BP-depleted serum was supplemented with physiological concentrations of purified C4BP (0.2 mg/ml) to control that any effect exerted by C4BP-depleted serum was due to lack of C4BP and could be corrected in replete serum. P. gingivalis from 6-day-old agar plates were harvested, washed twice in the binding buffer, and adjusted to an OD of 1.0 at 600 nm. NHS was diluted in GVB++ to a concentration of 5%, mixed with 6 x 105 cells (final volume of 50 µl), and incubated for 60 min at 37°C with shaking. Heat-inactivated serum (56° C, 30 min) was used as a negative control. Thereafter, cells were washed twice in the binding buffer and C9 deposition was assessed by incubation of the cells for 1 h at RT with the goat anti-human C9 Abs (Complement Technologies) diluted 1/1000 in the binding buffer. Afterward, cells were washed twice and resuspended in FITC-conjugated rabbit anti-goat polyclonal Abs (DakoCytomation) diluted in the binding buffer and used at a 1/1000 dilution. Samples were thereafter incubated for 60 min at RT and analyzed by flow cytometry.

Western blotting

P. gingivalis (0.75–6 x 105 cells) harvested from 6-day-old agar plates were incubated with NHS (5%) for 60 min at 37°C with shaking in a final volume of 50 µl. Serum proteins (0.15 µl of NHS/well) were separated by gel electrophoresis under nonreducing conditions using 5% gel and transferred to polyvinylidene difluoride membrane using semidry blotting system. The membranes were blocked with 50 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl2, 0.1% Tween 20, and 3% fish gelatin (pH 8.0). C4BP was detected using the monoclonal MK104 Ab followed by goat anti-mouse Ab conjugated to HRP and 3,3'-diaminobenzidine tetrahydrochloride colorimetric substrate (Sigma-Aldrich). The amount of C4BP was quantified after digital scanning using ImageGauge software (Fuji Film).

Measurement of enzymatic activity of gingipains

The assay was performed as previously described (32). Briefly, purified HRgpA (ranging from 0.2 to 4 µg/ml) or the P. gingivalis strain J4261 collected from 9-day-old plates and washed twice with GVB++ (2.5 x 105 to 5 x 106 cells) were added to the wells of microtiter plates containing 100 µl of reaction buffer (200 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% NaN3 (pH 7.6) containing freshly added 20 mM L-cysteine-HCl). Total volume was adjusted to 200 µl for each sample and left to incubate for 10 min at 37°C. Thereafter, 20 µl of N{alpha}-benzoyl-L-arginine p-nitroanilide hydrochloride (L-BAPNA) was added to the wells yielding the final substrate concentration of 1 mM, and hydrolysis of the substrate was measured spectrophotometrically at 405 nm every 18 s for 5 min.

Statistical analysis

Student’s t test was used to calculate p values to estimate whether the observed differences were statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
C4BP binds to all tested strains of P. gingivalis

We started our investigation by testing whether the potent complement inhibitor C4BP can be captured by P. gingivalis. Using flow cytometric analysis, two widely used laboratory strains, W50 and W83, were found to bind plasma- derived FITC-labeled C4BP. The binding of C4BP was concentration dependent and saturable (Fig. 1A). Importantly, we found that all clinical isolates of P. gingivalis tested in this study bound C4BP to varying extent (Fig. 1B). The majority of the clinical strains bound more C4BP than the two laboratory strains analyzed. Notably, the ability to bind C4BP was specific for P. gingivalis, since two other anaerobic bacteria species (Bacteroides ureolyticus and Veillonella sp.) cultured in the same conditions, i.e., solid medium, did not bind C4BP (Fig. 1B). Interestingly, a P. gingivalis mutant lacking PorT, an integral outer membrane protein involved in the secretion of gingipains (K.-A. Nguyen et al., manuscript in preparation and Ref. 30) entirely lost the ability to bind C4BP in comparison to the parental strain W83. Importantly, P. gingivalis were able to bind C4BP from NHS as shown in Fig. 1C. The level of C4BP captured from NHS corresponded well with the binding experiment using purified C4BP (Fig. 1B) because the clinical strains J4261 and Ma RL both displayed the strongest binding.


Figure 1
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  		FIGURE 1. Binding of C4BP-FITC to clinical isolates and laboratory strains of P. gingivalis. Bacteria were grown for 6 days on TSB agar and suspended in the binding buffer at 6 x 105 cells/ml. A, Wild-type strains W83 and W50 were incubated with the indicated concentrations of C4BP-FITC for 1 h at RT, washed, and analyzed by flow cytometry. B, C4BP-FITC (50 µg/ml) was incubated with a number of strains of P. gingivalis for 30 min at RT, the cells washed, and analyzed by flow cytometry. C, The bacteria were incubated with 5% NHS diluted in GVB++ followed by detection of bound C4BP with mAb 104 and secondary FITC-labeled anti-mouse Ab. Binding to every strain was analyzed three times in duplicates. Shown are mean values ± SD.

 
C4BP binds to hemagglutinin/adhesin domain of HRgpA

Our next goal was to identify the C4BP ligand on the surface of P. gingivalis. To this end, we used P. gingivalis W83 mutants lacking various gingipains as described in Table I. We found that binding of C4BP was significantly decreased for both strains lacking HRgpA (Fig. 2A). Notably, the single rgpA gene mutant strain of P. gingivalis (W83/RgpB+) showed significantly higher binding in comparison to the double rgpA and rgpB gene-deficient strain (W83/RgpB{Delta}495). This observation may indicate that RgpB is also partially involved, directly or indirectly, through processing of other surface proteins (33) that may be involved in binding of C4BP. The mutant lacking only Kgp (W83/Kgp{Delta}Ig/HA) showed a slight increase in binding of C4BP compared with the parental W83 strain (Fig. 2A).


Figure 2
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  		FIGURE 2. C4BP binds mainly HRgpA. A, Wild-type strain W83 and its mutants lacking gingipains grown as described in Fig. 1 were incubated with (50 µg/ml) C4BP-FITC for 30 min at RT, washed, and analyzed by flow cytometry. Binding of C4BP to W83 in each experiment was set as 1. Statistical significance of differences between tested stains was estimated with Student’s t test; ***, p < 0.001. B, Purified HRgpA, RgpB, and Kgp were immobilized on microtiter plates and incubated with the indicated concentrations of C4BP, binding of which was detected with specific mAbs. In each experiment, the binding of highest concentration of C4BP to HRgpA was set as 1 and all values were normalized. C, C4BP (15 µg/ml) was incubated with HRgpA immobilized on microtiter plates in the presence of the indicated concentrations of fibrinogen and prothrombin and the binding of C4BP was detected with a mAb. Binding at every condition in A–C was analyzed three times in duplicates. Shown are mean values ± SD.

 
To test whether purified gingipains would also interact with C4BP, gingipains were immobilized on microtiter plates and the binding of purified C4BP was determined with specific Abs. C4BP bound avidly to HRgpA (Fig. 2B) and to a much lower extent to Kgp and RgpB (Fig. 2B). Taken together, our results show that HRgpA serves as a ligand for C4BP.

To further identify the binding site for C4BP on HRgpA, we performed a competition assay with fibrinogen that interacts with the hemagglutinin/adhesin domains of gingipains. HRgpA was immobilized on microtiter plates and incubated in a solution containing 25 µg/ml C4BP and increasing concentrations of fibrinogen or prothrombin that was included as a negative control. The binding of C4BP was detected using specific Abs. We found that fibrinogen strongly competed with C4BP for binding to HRgpA, whereas prothrombin had no significant effect (Fig. 2C). These findings imply that C4BP binds mainly to the hemagglutinin/adhesin domain of HRgpA.

Binding sites for P. gingivalis on C4BP are localized to CCP1 and CCP6 and 7 of the {alpha}-chains

To further determine details of the interaction between C4BP and its bacterial ligand and to elucidate which subunit of C4BP is responsible for binding to P. gingivalis, we incubated bacteria with FITC-labeled C4BP in the presence of various competitors. We found that the binding of C4BP-FITC was inhibited to the same degree by C4BP purified from plasma (composed of seven {alpha}-chains and one β-chain with bound protein S) as by recombinant C4BP (containing six {alpha}-chains), implying that binding is localized to {alpha}-chains (Fig. 3A). In addition, the C4BP-P. gingivalis interaction could be inhibited by C4b and mAb 104 but not mAb 67 or albumin (BSA). C4b interacts with CCP1–3 of the {alpha}-chains (27), while mAb 104 and mAb 67 bind CCP1 and CCP4, respectively. Furthermore, binding of immobilized HRgpA to recombinant C4BP mutants lacking one CCP domain at the time showed that mutants missing CCP1, CCP6, and CCP7 have significantly decreased binding capacity to HRgpA (Fig. 3B). The mutants lacking CCP3 and CCP5 bound better than the wild type. Moreover, similar results were obtained with whole bacteria and mutated recombinant C4BP using flow cytometry analysis (data not shown). Taken together, there appears to be two binding sites for P. gingivalis on C4BP and they are localized to CCP1 and CCP6 and 7 of the {alpha}-chains.


Figure 3
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  		FIGURE 3. The binding site for P. gingivalis is localized to the N terminus of C4BP. A, P. gingivalis strain W83 was incubated with 50 µg/ml C4BP-FITC in the presence of several competitors: plasma-purified C4BP (20 µg/ml), recombinant C4BP (20 µg/ml), C4b (100 µg/ml), mAb 104 (100 µg/ml), mAb 67 (100 µg/ml), heparin (100 µg/ml), and BSA (100 µg/ml). After 1 h of incubation at RT, the bacteria were washed and analyzed by flow cytometry. B, Recombinant C4BP and its mutants lacking single CCP domains were incubated with HRgpA and the binding was detected with a polyclonal Ab. Binding at every condition was analyzed three times in duplicates. Shown are mean values ± SD. Statistical significance of differences between experimental conditions without (set as 1) and with competitors was estimated with Student’s t test: *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

 
Binding of C4BP is related to the age of P. gingivalis cultures

Next, we tested whether binding of C4BP depends on stage of bacterial growth and maturity of colonies. Flow cytometry was used to assess binding of C4BP-FITC to strains W83, W50, J4261, and Ma RL cultured for 1–8 days on TSB- agar plates (solid medium) and the binding of C4BP was observed to be strongly related to the age of P. gingivalis culture (Fig. 4). In agreement with our previous data, the clinical strains Ma RL and J4261 were the strongest binders of C4BP and the binding increased proportionally to the time of culture (Fig. 4). The PorT mutant showed no ability to bind C4BP irrespective of the cultivation time.


Figure 4
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  		FIGURE 4. Binding of C4BP depends on the age of culture. Bacteria were grown for up to 8 days on TSB agar, harvested every day starting from day 1 after spreading on plates, washed twice, and suspended in binding buffer at 6 x 105 cells/ml. Afterwards, C4BP-FITC (50 µg/ml) was added for 30 min at RT, the cells washed, and analyzed by flow cytometry. Binding was investigated at least three times in duplicates for each time point.

 
Furthermore, analysis of flow cytometry data indicated that a new subset of bacteria (strain J4261) was formed during culture aging (Fig. 5B, gate R2). The bacterial cells in this new population appeared to be larger than single bacteria (Fig. 5B, gate R1) and was hypothesized to be aggregates of bacteria, which was confirmed microscopically after Gram staining (Fig. 5, G and H). We also found that dispersed bacterial cells of J4261 (Fig. 5B, gate R1) from 7-day-old colonies bound C4BP to a greater extent than dispersed cells (Fig. 5A, gate R1) derived from 3-day-old colonies (Fig. 5, C and D). Importantly, the bacterial population in gate R2 that appeared in colonies after 7 days of culture bound very large amounts of C4BP. These aggregates bound more C4BP (Fig. 5F) than dispersed cells derived from 3-day (Fig. 5C) and 7-day (Fig. 5D) cultures. Similar data were obtained for the W83 strain although the aggregates were not as pronounced as in the case of the J4261 strain. On the contrary, binding of C4BP to PorT mutant was constantly at background levels irrespective of the cultivation time (data not shown). Furthermore, the PorT mutant did not form any aggregates.


Figure 5
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  		FIGURE 5. Bacteria form aggregates during culture, which bind C4BP stronger than dispersed bacterial cells. Wild-type strain J4261 was harvested on the third and seventh day of culture and incubated with 50 µg/ml C4BP-FITC. The binding was detected by flow cytometry. After 7 days of culture, a subset of bacteria-forming aggregates appeared in gate R2 (B), which were not observed after 3 days of culture (A). These aggregates bound more C4BP (E and F) than single cells after both 3 (C) and 7 (D) days of culture. One representative flow cytometry graph of at least three independently performed is shown for each condition. The bacteria were also visualized using Gram (geometric mean) staining after 3 (G) and 7 (H) days of culture.

 
Binding of C4BP to P. gingivalis leads to decreased complement attack

To investigate the functional consequence of the binding of C4BP, we compared the amount of deposited C9, the final component of the complement cascade, on the bacterial surface upon incubation with NHS as well as serum from which C4BP was depleted using affinity chromatography. We chose the J4261 strain for these experiments because it was one of the best binders of C4BP. When P. gingivalis J4261 strain was incubated with C4BP-depleted serum, twice as much C9 was deposited on the bacterial surface as compared with those incubated with NHS (Fig. 6A). Notably, upon adding back C4BP at a physiological concentration of 0.2 mg/ml to the C4BP-depleted serum, the amount of deposited C9 did not differ from the one in NHS (Fig. 6A). This implies that binding of C4BP provides an increased level of protection from complement attack.


Figure 6
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  		FIGURE 6. In the absence of C4BP, P. gingivalis is more readily attacked by complement. A, P. gingivalis strain J4261 was harvested after 9 days of culture. Cells (6 x 105) were incubated in a total volume of 50 µl with 5% NHS in GVB++, 5% NHS lacking C4BP, and 5% C4BP-depleted NHS reconstituted with C4BP, respectively. Thereafter, deposition of C9 on bacteria was investigated using polyclonal Abs followed by flow cytometry analysis. Heat-inactivated serum (56°C, 30 min) was used as negative control and the background value was subtracted from the responses obtained in the sera experimentation. The values are presented relative to the signal obtained with NHS. Statistical significance of differences between depleted and reconstituted sera and NHS was estimated with Student’s t test: ***, p < 0.001; n.s., not significant. B, NHS incubated with the indicated amounts of the J4261 strain under the same conditions as in A was analyzed by Western blotting under nonreducing conditions and C4BP was detected with mAbs. Under these conditions, several isoforms of C4BP are separated. C, Indicated concentrations of purified HRgpA were incubated with 1 mM L-BAPNA and the kinetic reaction was followed by measurement of absorbance at 405 nm corresponding to the released product. The slopes of obtained linear curves were plotted against concentrations of HRgpA. The strain J4261 at the indicated concentrations was then incubated with L-BAPNA (inset).

 
Because gingipains are efficient proteinases, we investigated whether they would degrade C4BP in NHS in the conditions used for the C9 deposition assay. We found that at least 50% of C4BP remained intact at the end of the incubation period (Fig. 6B). C1q remained intact in the same samples while C3 was degraded with a similar efficiency as C4BP (data not shown). To test whether gingipains of the J4261 strain were active, we used a kinetic assay. The standard curve was obtained for purified HRgpA (Fig. 6C) and the slopes of kinetic curves obtained for the J4261 strain allowed us to estimate that 6 x 105 bacteria used in the experiment presented in Fig. 6A displayed an activity of arginine-specific gingipains corresponding to 67 ± 13 ng of the purified enzyme.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
All successful human bacterial pathogens must develop strategies to circumvent the complement system. Complement-mediated killing is relevant for P. gingivalis since complement components are present in the gingival crevicular fluid at 70% of the serum concentration (34) and that P. gingivalis have been reported to activate both the classical and alternative pathways of complement (35). It has also been demonstrated in vivo that there is a high level of complement activation in the gingival crevicular fluid of patients with periodontitis (36, 37). Furthermore, specific Ab responses against P. gingivalis are of importance because there is a relationship among Ab titers, opsonic activity, and accumulation of C3 (38), indicating the importance of the classical pathway in defense against this pathogen. However, P. gingivalis is able to override these host defense mechanisms because it is exceptionally resistant to bactericidal activity of human serum. One strategy of evasion depends on the production of large amounts of proteinases (gingipains) which are able to degrade several complement factors (39). Another strategy, as employed by many successful human bacterial pathogens, involves the capture of human complement inhibitors such as FH or C4BP to down-regulate complement attack. We could not detect interaction between FH and P. gingivalis (data not shown), but we did detect binding of C4BP. All tested clinical and laboratory strains of P. gingivalis bound C4BP. Notably, several clinical strains (J4261, Ma RL, J3741) bound more C4BP than the laboratory strains of P. gingivalis, whereas two other tested Gram-negative anaerobic bacteria showed no binding. The fact that clinical isolates bound more C4BP than laboratory strains may suggest that there is a correlation between the ability to bind C4BP and strain virulence. We also observed that P. gingivalis form aggregates during culture and that these bind more C4BP than dispersed bacterial cells. However, even dispersed cells of P. gingivalis bound C4BP better after a longer period of culture on agar plates. Taken together, it appeared that both aggregate formation (increased number of binding sites per particle) and accumulation of gingipains on the bacterial surface (mainly RgpA) resulted in an increase of C4BP binding to the bacteria.

Upon examining potential C4BP ligands on the bacterial surface, we found that C4BP bound to gingipains, mainly to HRgpA. Accordingly, P. gingivalis mutants lacking RgpA showed significantly lower binding of C4BP. Significantly, purified RgpB showed only a weak binding of C4BP despite the fact that RgpB is practically identical to the catalytic domain of HRgpA. This observation suggests that a C4BP binding site may be located within the hemagglutinin/adhesin domain of RgpA (40). The hypothesis is further supported by the fact that interaction between C4BP and HRgpA was inhibited by fibrinogen that binds to the hemagglutinin domains of gingipains (9). The mutant lacking only Kgp showed a slight increase in binding of C4BP compared with the parental W83 strain. Since Kgp and HRgpA exist as a complex on the surface of the wild-type strain (41), the absence of Kgp in the Kgp-null mutant may result in greater exposure of the C4BP binding site on the RgpA molecule resulting in higher binding in this mutant. Interestingly, deletion of both rgpA and rgpB genes did not entirely abolish the ability of the bacteria to bind C4BP, whereas there was absolutely no interaction with the PorT mutant lacking not only gingipains but also other cell surface- associated proteins carrying a specific C-terminal domain (42, 43). This suggests the presence of other surface ligand(s) in addition to HRgpA that may play a role in C4BP binding. One potential candidate for such a ligand is hemagglutinin A (HagA), in which some hemagglutinin/adhesin subdomains present in RgpA and Kgp are repeated several times (44) and exert the same hemagglutination, platelet aggregation, and hemoglobin-binding activities as gingipains (45).

So far, all known binding sites for various pathogens, including P. gingivalis as shown in this study, are localized to the {alpha}-chains of C4BP, which is in agreement with the fact that the β-chain of C4BP is always occupied by protein S which forms a high-affinity, hydrophobic interaction (46). However, various domains of {alpha}-chains are used for interaction by pathogens. N. gonorrhoeae (17) and S. pyogenes (19) bind the most N-terminal 70 aa, i.e., CCP1. Bordetella pertussis (47) and C. albicans (23) bind to a somewhat larger area covering CCP1 and 2, with C. albicans also interacting with CCP6. N. meningitidis (48) binds CCP2 and3, while M. catarrhalis (20) and H. influenzae (24) interact with CCP2 and CCP7 and E. coli K1 with CCP3 (the main site) and CCP8 (21). In the case of P. gingivalis, we found two major interaction sites in CCP1 and CCP6 and 7. The interaction with CCP1 is further supported by the fact that the binding was inhibited by addition of mAb 104 and C4b that both bind to this domain (27). Interestingly heparin, binding to CCP2 and 3 (27) and some positively charged amino acids on the interface between CCP1 and 2 (49) did not affect the binding, supporting the hypothesis that P. gingivalis does not extend its binding into CCP2. Somewhat surprisingly, the binding of C4BP lacking CCP3 and CCP5 to HRgpA was increased in comparison to the wild type. Perhaps the binding site on CCP1 and CCP6 and 7 becomes more adjacent or oriented in a more preferred conformation, which yields better interaction. We have observed such an effect for other ligands that have binding sites on both the N and C termini of the {alpha}-chains (our manuscript in preparation). Most importantly, irrespectively of the binding domain for a particular pathogen, C4BP always remains active when bound because of its polymeric nature. Even if several of its {alpha}-chains are engaged in interaction with pathogen, others are free to inhibit complement as we have shown numerous times previously (17, 20, 22, 23, 24, 48).

C4BP bound to the bacterial surface should inhibit complement activation by decreasing the level of C4 and C3 activation and subsequent downstream effects such as opsonization with C3b, release of anaphylatoxins, and formation of MAC. However, experiments proving that binding of C4BP to P. gingivalis impairs their destruction by complement proved to be challenging. For example, we could not compare complement deposition on the wild-type and mutant strains lacking gingipains since these proteases by themselves are strong inhibitors of complement (39) and their proteolytic activity could not be dissociated from the ability to bind C4BP. Subsequently, comparison of complement deposition on clinical strains of P. gingivalis was found to be highly variable due to the differing initial amounts of C1 deposition leading to large differences in activation of complement, thus precluding studies of the effect of C4BP binding. Finally, the PorT mutant lost entirely the ability to bind C1 but instead acquired the capacity to intensively activate the alternative pathway. However, we did show that C4BP binding to bacteria has functional importance since bacteria challenged with C4BP-depleted serum exhibited a 2-fold increase in C9 deposition on their surfaces in comparison to bacteria incubated with serum containing C4BP. Importantly, a large fraction of C4BP in NHS remained intact at the end of the incubation period with the bacteria harboring active gingipains, indicating that they are relatively resistant to degradation by these proteases. Taken together, our data suggest that binding to C4BP is another strategy P. gingivalis could employ to enhance survival in the host and the fact that gingipains act as ligands for C4BP further emphasize the role of these cysteine proteases in bacterial virulence.


   Acknowledgments
 
Margareta Pålsson is acknowledged for expert technical help.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Swedish Foundation for Strategic Research (INGVAR), Swedish Medical Research Council; the foundations of Österlund, Kock, King Gustav V’s 80th Anniversary, Knut and Alice Wallenberg, and Inga-Britt and Arne Lundberg; research grants from the University Hospital in Malmö (to A.M.B.); grants from the Ministry of Science and Higher Education (1642/B/P01/2008/35 Warsaw, Poland); and National Institutes of Health Grant DE 09761 (to J.P.). Back

2 Address correspondence and reprint requests to Dr. Anna M. Blom, Department of Laboratory Medicine, Division of Medical Protein Chemistry University Hospital Malmö entrance 46, The Wallenberg Laboratory floor 4; Lund University, S-205 02 Malmö, Sweden. E-mail address: Anna.Blom{at}med.lu.se Back

3 Abbreviations used in this paper: Kgp, lysine-gingipain; Rgp, arginine-gingipain; HRgp, high molecular weight arginine-gingipain; MAC, membrane attack complex; C4BP, C4b-binding protein; CCP, complement control protein (domain); FH, factor H; NHS, normal human serum; TSB, tryptic soy broth; RT, room temperature; L-BAPNA, N{alpha}-benzoyl-L-arginine p-nitroanilide hydrochloride. Back

Received for publication June 16, 2008. Accepted for publication August 6, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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