J Clin Periodontol 2013; 40: 924–932 doi: 10.1111/jcpe.12139

The role of RgpA in the pathogenicity of Porphyromonas gingivalis in the murine periodontitis model

Asaf Wilensky1, David Polak1, Yael Houri-Haddad2,* and Lior Shapira1,* 1

Department of Periodontology, School of Dental Medicine, Hebrew University and Hadassah, Jesusalem, Israel; 2Department of Prosthodontics, School of Dental Medicine, Hebrew University and Hadassah, Jesusalem, Israel

Wilensky A, Polak D, Houri-Haddad Y, Shapira L. The role of RgpA in the pathogenicity of Porphyromonas gingivalis in the murine periodontitis model. J Clin Periodontol 2013; 40: 924–932. doi: 10.1111/jcpe.12139.

Abstract Aim: To investigate the in vivo role of gingipains in Porphyromonas gingivalis’ virulence, and suggest a possible host mechanisms through which the bacteria cause alveolar bone loss. Materials and Methods: Mice were orally infected with P. gingivalis wild type, or the gingipains mutants (RgpA , Kgp , RgpA /Kgp ). Mice were analysed for alveolar bone loss using micro-computed tomography. The molecular effects of the proteases were evaluated using the subcutaneous chamber model. Mice were infected with P. gingivalis wild type or mutants. Exudates were analysed for cytokine and leukocytes levels, in vivo phagocytosis, P. gingivalis survival and serum anti-P. gingivalis IgG titres. Results: Only RgpA-expressing bacteria induced significantly alveolar bone loss, and suppressed phagocytosis resulting in increased survival of P. gingivalis in the chamber exudates. In addition, RgpA-expressing bacteria induced higher levels of leukocytes and cytokines 2 h post-infection, and reduced levels of serum anti-P. gingivalis IgG titres 7 days post-infection. Conclusions: Our findings showed that elimination of RgpA from P. gingivalis diminished inflammation, but augmented phagocytosis and antibody titres, coincidental with reduced alveolar bone loss. These findings support the hypothesis that RgpA is a critical virulence factor in the pathogenesis of experimental periodontitis in mice.

Periodontitis is a multi-factorial infectious disease characterized by inflammation, resulting in the Conflict of interest and source of funding statement The authors declare that they have no conflict of interests. This study was supported in part by the US-Israel Bi-National Science Foundation (US-Israel BSF). *LS and YHH had equal contribution to the study.

924

destruction of the periodontal tissues and tooth loss (Armitage 1996). Although there are many risk factors related to periodontal disease (Stabholz et al. 2010), bacterial plaque is considered the main aetiological factor. While human subgingival plaque harbours up to 700 bacterial species (Jenkinson & Lamont 2005, Paster et al. 2006), it is likely that only a small percentage of these are, in fact, involved in the aetiology of periodontal disease (Socransky et al. 1998). Evidence suggests that

Key words: alveolar bone loss; cytokines; experimental periodontitis; immunoglobulins; phagocytosis; Porphyromonas gingivalis Accepted for publication 27 June 2013

Porphyromonas gingivalis, a gramnegative anaerobic bacterium is strongly associated with periodontal disease (Slots et al. 1988, Holt & Bramanti 1991, Socransky et al. 1998, Byrne et al. 2009). Although P. gingivalis and its virulence factors are involved in the pathogenesis of periodontitis, the nature and magnitude of the periodontium’s response to this periodontal infection is modulated not only by the bacteria and its virulence factors, but by the host immune response and the interac-

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

RgpA important for Porphyromonas gingivalis pathogenicity tions between them (Van Dyke et al. 1993). The bacterial infection that exists in periodontal disease activates the innate immunity which provides the first line of defence against evading pathogens, in part through phagocytosis. This activation will initiate the emergence of the adaptive immunity which, in turn, results in secretion of cytokines and immunoglobulins (Ig). The pro-inflammatory cytokine response is characterized by the dominant secretion of interleukin (IL)-1b, IL-6, IL-11, IL-17, interferon (IFN)-c and tumour necrosis factor (TNF)-a (Cochran 2008). In contrast, an anti-inflammatory dominant response, which was thought to protect the periodontal tissues from destruction by bacterial infection (Eastcott et al. 1994), is characterized by IL-4, IL-12, IL-10, IL-13 and IL-18 secretion (Cochran 2008). The importance of the humoral response to the severity of human periodontal disease has been investigated in many studies (Gmur et al. 1986, Lopatin & Blackburn 1992, Lamster et al. 1998). Some studies have shown that elevated serum levels of anti-P. gingivalis IgG have been directly linked to disease severity. However, others have found that the levels of antibodies in the gingival crevicular fluid are inversely related to the number of organisms at the site of sampling (Kinane et al. 1993, Ebersole et al. 1995). In a clinical study, anti-P. gingivalis IgG levels at periodontitis sites were found to be lower than at gingivitis sites in the same subjects, suggesting that a failure of local antibody production may contribute to the transition from gingivitis to periodontitis (Mooney & Kinane 1997). To evade the host’s immune and defence responses, invading microorganisms developed various approaches such as evasion of recognition; subversion of antibacterial effectors and/or trespassing the mucosal surfaces; development of anti-humoral immunity; interference with cytokine/chemokine production; interruption of antigen presentation; exertion of immunosuppressive effects; inhibition of the effectors of the adaptive immunity; and escape from phagocytic capture. Among the virulent factors to be associated with the dysregulation of the host immune response are the cysteine proteases of P. gingivalis known as gingipains.

Gingipains consist of arginine-specific proteases [Arg-gingipain, (Rgp)] and lysine-specific protease [Lys-gingipain, (Kgp)]. The Rgp is encoded by two genes rgpA and rgpB and Kgp is encoded by a single gene kgp (Nakayama 1997). The mature forms of RgpA and Kgp proteins possess both a catalytic and a hemagglutinin domain, while RgpB possesses a catalytic domain but lacks the hemagglutinin domain (Potempa et al. 1998). Recent studies have shown that gingipains have the ability to neutralize/modulate the host defence response by affecting both arms of immune response. For example, they can efficiently degrade defensins (Bachrach et al. 2008, Carlisle et al. 2009) and modulate or even suppress all mechanisms of complement activation through proteolytic degradation of key complement components (Popadiak et al. 2007, Potempa & Pike 2009, Wang et al. 2010). In addition, gingipains can affect the adaptive immune response by cleaving several T-cell receptors, such as CD2, CD4 and CD8 (Kitamura et al. 2002, Yun et al. 2005), thereby hampering the cell mediated immune response. They can also stimulate expression of protease-activated receptors in T cells (Belibasakis et al. 2010), which are crucial for the induction of cytokine responses and the establishment of chronic inflammation in periodontitis (Holzhausen et al. 2010, Fagundes et al. 2011). Furthermore, gingipains have the ability to proteolytically inactivate both anti-inflammatory (IL-4, IL-5) and pro-inflammatory (IL-12, IFN-c, IL1b, IFNc and TNFa) cytokines (Calkins et al. 1998, Sharp et al. 1998, Yun et al. 2001, 2002, Tam et al. 2009). Gingipains can also stimulate IL-6 production by oral epithelial cells (Lourbakos et al. 1998) and IL-8 production by gingival fibroblasts (Oido-Mori et al. 2001), enhancing the inflammatory responses. Although gingipains considered as major virulence factors of P. gingivalis, most of the studies addressing their function were limited to ex vivo experiments using the native or recombinant forms of the protein. Moreover, the small amount of works investigating the role of gingipains during experimental periodontitis failed to provide a hypothetical mechanism by which

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

925

they influence periodontal destruction. The aim of this study is to elucidate the in vivo role of gingipains in P. gingivalis induced experimental periodontitis in mice. Materials and Methods Animals

Five- to six-week-old female BALB/c mice (Harlane, Jerusalem, Israel) were used. The study was carried out in the specific pathogen-free (SPF) unit of the animal facility. The mice were kept on a 12-h light/dark cycle and received distilled water and food ad libitum. The experimental protocols were approved by the Hebrew University Institutional Animal Care and Ethics Committee. Bacteria

Porphyromonas gingivalis wild type (W83), P. gingivalis RgpA , P. gingivalis Kgp-, P. gingivalis RgpA / Kgp strains were grown on blood agar plates in an anaerobic chamber with 85% N2, 5% H2 and 10% CO2. After incubation at 37°C for 2– 3 days, the bacterial cells were inoculated into Wilkins media (Oxoid, Basingstoke, England) for 2 days of incubation under the same conditions. The bacteria were washed three times with sterile phosphatebuffered saline (PBS) before use and the colony forming unit (CFU)/ml was determined using spectrophotometer (Houri-Haddad et al. 2000). Oral infection

Infection was carried out as described by Wilensky et al. (2009). Briefly, all animals (n = 8 for each group) were given sulfamethoxazole (0.80 mg/ml) and trimethoprim (0.16 mg/ml), ad libitum, in their drinking water for 10 days. Three days following the withdrawal of antibiotics (day 14), the animals were infected with the P. gingivalis strains or vehicle only (5 9 1010 CFU/ml in 0.2 ml of PBS and 2% carboxymethylcellulose). The process was repeated 3 times, once every other day. Forty-two days after the last infection, the mice were killed (using CO2) and the hemi-maxillae were collected and prepared for bone loss measurements using the micro-

926

Wilensky et al.

computerized tomography (lCT) technique (Wilensky et al. 2005). The subcutaneous chamber model

Two titanium coil chambers (length 1.5 cm, diameter 5.16  0.08 mm) were inserted subcutaneously via a mid-dorsal incision into the dorsolumbar region of each anesthetized female mice (n = 8 for each group) aged 5–6 weeks. One chamber was inserted to the left side of the body and the second to the right side, as previously described (Genco et al. 1991, Shapira et al. 1999). One week following chamber implantation, all three test groups were infected by means of intrachamber injection of the various live P. gingivalis strains (5 9 108 CFU in 100 ll of PBS), while saline was injected into the control, non-infected group. Exudates from both chambers of each mouse were collected at baseline (immediately before the intrachamber infection), at 2 h, 48 h and 4 days post-infection. Each chamber was sampled once and the individual mouse was chosen as the unit of analysis. The exudates were centrifuged for 10 min. at 290 g. The supernatants were collected and stored at 70°C until analysed.

three times in PBS to remove nonadherent bacteria. Trypan blue (0.2% in PBS) was added for quenching of any remaining extracellular bacteria. Trypan blue was removed by two additional washes and cells were re-suspended in 0.5% BSA/PBS. Phagocytosis was measured using a FACSCAN flow cytometer equipped with CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA).

Ten ll of chamber exudates were serially diluted in triplicate in PBS, and plated on tryptic soy agar containing sheep blood (Hylabs, Rehovot, Israel). Plates were grown under anaerobic conditions for 5–7 days at 37°C. P. gingivalis colonies were identified by their black pigment and by phase contrast microscopy (Burns et al. 2006). Analysis of anti- P. gingivalis IgG titres

Plates containing 96 wells were coated with P. gingivalis sonic extracts, and relative quantities of IgG were determined using commercially available antibody pairs as described previously (Houri-Haddad et al. 2005). Leukocyte counts

For quantitative three-dimensional analysis of the alveolar bone loss (ABL), the hemi-maxillae were examined by a desktop lCT system (lCT 40, Scanco Medical AG, Bassersdorf, Switzerland) as described previously (Wilensky et al. 2005). The results represent the residual bone in mm3.

In vivo leukocyte concentrations were determined as previously described (Houri-Haddad et al. 2001).

The evaluation of in vivo phagocytosis was performed as previously described (Burns et al. 2006). In brief, P. gingivalis wild type (n = 6 for each group) was labelled with 0.1 mg/ml FITC (Sigma, Rehovot, Israel) in carbonate buffer (pH 9.5) for 20 min. at room temperature. 5 9 108 CFU of FITC-labelled P. gingivalis in 100 ll were injected into the two implanted subcutaneous chambers (as described in the subcutaneous chamber model). Chamber exudates were drawn at 2, 24 and 48 h post-infection, and centrifuged. Cell pellets were washed

Results Alveolar bone loss

Viable bacterial counts

Quantification of alveolar bone loss

In vivo phagocytosis assay

inter-group differences were tested for significance by t-test with the Student–Newman–Keuls method correction for multiple testing. The level of significance was determined at p< 0.05. All the results are presented as mean values  the standard error of the mean.

Analysis of cytokines

The presence of IL-1b, IL-10 and TNFa in the chamber exudates were determined using ELISA, as previously described (Wilensky et al. 2009). The assays were based on antibody pairs matched for ELISA, obtained from Pharmingen (San Diego, CA, USA). Data analysis

Data analysis was performed using a statistical software package (SigmaStat, SPSS Science, Chicago, IL, USA). One-Way Analysis of Variance (ANOVA) was used to test the significance of the differences between the treated groups following passing an equal variance test. When significance was established, the

Since gingipains are thought to be major virulence factors of P. gingivalis, we were interested in evaluating their contribution to ABL during experimental periodontitis. For that, we used the oral infection model and measured the degree of ABL following repeated oral infections with the different mutants to the gingipains or wild-type P. gingivalis. Bone loss was measured using lCT to allow quantification of residual supportive bone volume (RSBV). Our data show that mice which were orally infected with the RgpA-expressing P. gingivalis strains (Kgp- and wild type) expressed significantly lower RSBV when compared with the noninfected group, while infection with the RgpA mutant strains (RgpA-, RgpA /Kgp ) did not (Fig. 1). These findings emphasize the important role RgpA plays in experimental periodontitis in mice. In vivo phagocytosis

Phagocytosis plays a crucial role in host defence by recognizing and eliminating invading pathogens (Soehnlein & Lindbom 2010). Due to the importance of phagocytosis for the host’s defence system, we hypothesized that the clinical effect off RgpAexpressing P. gingivalis is due to their ability to evade phagocytosis. We employed the subcutaneous chamber model and challenged the mice with FITC labelled P. gingivalis wild type and mutants (RgpA and Kgp ). Two hours post-infection, the percentage of leukocytes that engulfed the RgpA bacteria was significantly higher than that of the RgpA+ (wildtype and Kgp ) bacteria (Fig. 2a). In addition, the average number of engulfed bacteria in each leukocyte was significantly higher for the RgpA bacteria compared to the

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

RgpA important for Porphyromonas gingivalis pathogenicity RgpA+ bacteria (Fig. 2b). At 24 and 48 h, no significant differences were observed between the groups.

related with the previous results (In vivo phagocytosis), highlighting the central role of RgpA in the in vivo survival of P. gingivalis.

In vivo survival of P. gingivalis

To further confirm the importance of RgpA in enabling P. gingivalis to evade phagocytosis and thus survive, we evaluated the in vivo survival of P. gingivalis. At 2 h post-infection, significantly less of the RgpA P. gingivalis survived compared to the Kgp P. gingivalis and wild-type groups. No significant differences were observed at 24 and 48 h postinfection (Fig. 3). These results cor-

Antibody response

The above data demonstrated that RgpA helps P. gingivalis to evade phagocytosis. This may in turn affect antigen presentation by these cells, and consequently influence the humoral immune response. We thus sought to investigate whether the antibody response against P. gingivalis was altered in the presence of RgpA. Balb/c mice were infected with

927

P. gingivalis wild-type or gingipains mutants, and 7 days post-infection serum samples were obtained and analysed for anti-P. gingivalis IgG titres. The highest IgG titres were found in the RgpA P. gingivalis group, and the lowest for the wildtype group. Significant differences were found between all groups (Fig. 4). In vivo leukocytes infiltration

It has been shown that immune responses developed following P. gingivalis infection included leukocyte arrival at the infection site (HouriHaddad et al. 2002). As such, our goal was to investigate the impact of gingipains on this process. To this end, we employed the subcutaneous chamber model and challenged the mice with P. gingivalis, wild type and mutants, and examined the results at 2 h, 48 h and 4-day intervals. Two hours post-infection, significantly lower concentrations of leukocytes were found in the RgpA P. gingivalisinfected group compared to the wild-type infected group, whereas no significant differences were found with the control group (Fig. 5). No significant differences between the three infected groups were found at 48 h and 4 days. Intra-chamber cytokine levels

Fig 1. The impact of gingipains on the residual supportive bone volume (RSBV). Forty-two days following oral infection with Porphyromonas gingivalis wild type or mutants, mice were killed and hemi-maxillae were evaluated using a lCT. Bars represent mean values  standard error of the mean. *Significantly different compared to the control group. #Significantly different compared to the P. gingivalis RgpA/Kgp mutant group. ANOVA, p < 0.05.

TNF-a levels peaked at two hours post-infection in all three infected groups. Cytokine levels were significantly lower in the two mutant groups (RgpA and Kgp ) com-

% cells above autofluorescence

100

*

80 60

40

20 0

(a)

2h

24 h

48 h

(b)

Fig 2. The effect of gingipains on in vivo Porphyromonas gingivalis phagocytosis. FITC labelled P. gingivalis wild type or mutants were injected into the subcutaneous chambers. Chamber exudates were drawn 2, 24 and 48 h post-infection and were analysed using a flow cytometer. Bars represent mean values  standard error of the mean. *Significantly different compared to the other infected groups or control. ANOVA, p < 0.05. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

928

Wilensky et al. 500

*

Wild - type Kgp–

P.g CFU×108/ml

400

RgpA– Control

300

*

200 100 0

* 2h

24 h

48 h

Fig 3. The role of gingipains in the in vivo survival of Porphyromonas gingivalis. Two, 24 and 48 h post-infection with P. gingivalis (wild type or mutant) chamber exudates were collected and serially diluted in triplicate in PBS, and plated on tryptic soy agar containing sheep blood. Plates were grown under anaerobic conditions for 5–7 days at 37°C. Porphyromonas gingivalis colonies were identified by their black pigment and by phase contrast microscopy. Bars represent mean values  standard error of the mean. *Significantly different compared to all other groups. ANOVA, p < 0.05. Serum Anti P.gingivalis lgG levels #

Total lgG Titers Log2

13

* *#

12

Wild - type Kgp– RgpA– Control

11

#

* 10

9

7 days

Fig 4. The effect of gingipains on serum anti-Porphyromonas gingivalis IgG levels. IgG titres were evaluated using ELISA, 7 days following infection with P. gingivalis (wild type or mutant). Bars represent mean values  standard error of the mean. *Significantly different compared to the control group. #Significantly different compared to the other groups. ANOVA, p < 0.05.

pared to the wild type and control groups, whereas no significant difference were found between the mutant groups themselves. Cytokine levels dropped at 48 h and 4 days, with no differences between all groups (Fig. 6a). IL-1b levels peaked at two hours in the three infected groups, with the lowest levels found in the RgpA P. gingivalis-infected group, and the highest in the wild-type group. At 48 h and 4 days, IL-1b levels continued to be significantly higher in the three infected groups, but while the cytokine levels stayed the same in the two mutant groups, they

dropped significantly in the wild-type group (Fig. 6b). At two and 48 h, IL-10 levels in P. gingivalis-infected the RgpA group were found to be without significant differences compared to the control group, and significantly lower compared to the wild-type group. At 4 days, no significant differences were found between the groups (Fig. 6c). Discussion

In this study, we investigated the role of gingipains in P. gingivalis’ virulence, and suggested a hypotheti-

cal mechanism which specifies how the bacterium uses the gingipains in order to survive the hostile host environment. Previous data have shown that gingipains have the ability to dysregulate the host immune response in a number of ways: by helping P. gingivalis in colonization (Pike et al. 1996); by inactivating protease inhibitors (Carlsson et al. 1984); by increasing permeability and prostaglandin (PG) E2 levels through the elevation of bradykinine secretion (Rahman et al. 1992, Imamura et al. 1994, Ransjo et al. 1998); and by degrading a variety of cytokines (Calkins et al. 1998, Mezyk-Kopec et al. 2005, Stathopoulou et al. 2009), complement components (Jagels et al. 1996a,b), and Ig (Abe et al. 1998), as well as causing the cleavage of CD4 and CD8 from T cells (Kitamura et al. 2002, Yun et al. 2005). On the basis of this data, we wanted to investigate the role of gingipains in ABL during experimental periodontitis, using the oral infection model and P. gingivalis mutants (RgpA and Kgp ). Our data demonstrated that only the RgpA-expressing bacteria induce significant ABL (Fig. 1), and this led us to the conclusion that RgpA is a major virulent factor in the pathogenesis of experimental periodontitis in mice. These results are different from the results shown by Pathirana et al. (2007) who demonstrated that Kgp has a more significant role in ABL than RgpA (Pathirana et al. 2007). The dissimilar results could be explained by the different antibiotic treatment, P. gingivalis wild-type strains (W50 versus W83), the oral infection protocols used, and bone loss measuring techniques (two dimensional in the previous study versus 3 dimensional in the present one). While in this study, we administered sulfamethoxazole (0.80 mg/ml) and trimethoprim (0.16 mg/ml), in drinking water, ad libitum for 10 days Pathirana et al. used kanamycin (1 mg/ml) in deionized water ad libitum for 7 days. These two antibiotic protocols have different activity spectrum which could influence on the composition of the oral bacterial biofilm and hence on the ability of the different mutants to adhere/survive and generate damage. The use of different wild-type bacteria could affect on bone loss pattern (Evans et al. 1992). The use

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

RgpA important for Porphyromonas gingivalis pathogenicity of the morphometric technique by Pathirana et al. is able to measure horizontal bone loss only while ABL

pattern could include vertical and horizontal bone loss. It is possible that the different background of the

Fig 5. The impact of gingipains on in vivo leukocyte counts: Two, 48 and 4 days postinfection with Porphyromonas gingivalis (wild type or mutant) chamber exudates were collected and leukocyte counts were evaluated. Bars represent mean values  standard error of the mean. *Significantly different compared to the wild-type group. #Significantly different compared to the control group. ANOVA, p < 0.05.

929

mutants (P. gingivalis W50 versus W83) may induce different bone loss patterns which could be measured accurately only by using the lCT technique. Other differences which could account for the opposite results might be the fact that in the current study mice were infected three times every other day with 5 9 1010 CFU/ml in 0.2 ml of P. gingivalis or mutants in PBS and 2% carboxymethylcellulose, while Pathirana et al. infected the mice four times 2 days apart with 1 9 1010 of P. gingivalis W50 or mutants in PG buffer and re-infected the mice the same way 2 weeks following the first infection. An essential prerequisite for the survival and virulence of bacteria in the host is their ability to evade the host’s immune system, with phagocytosis being one of the initial defence mechanisms. Phagocytosis contributes to host defence by recognizing and eliminating invading

TNF- α Levels

25 000

Wild - type Kgp–

20 000

Pg/ml

RgpA– Control

*

15 000

*

10 000 5000

* 0 (a)

Base Line

2h

48 h

4 days

(b) IL- 10 Levels

1800 1600 1400

Pg/ml

1200

*

1000 800

#

600

*

400 200 0 (c)

#

*

0h

2h

* * 48 h

4 days

Fig 6. The effect of gingipains on in vivo cytokine levels. Two, 48 and 4 days post-infection with Porphyromonas gingivalis (wild type or mutant) chamber exudates were collected and evaluated using ELISA for TNF-a (a), IL-1b (b) and IL-10 (c). Bars represent mean values  standard error of the mean. *Significantly different compared to the wild-type group. #Significantly different compared to the Kgp mutant group. ANOVA, p < 0.05. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

930

Wilensky et al.

pathogens (Soehnlein & Lindbom 2010). Recently, it was shown that in vitro phagocytic activity of crevicular and peripheral blood neutrophils, obtained from localized aggressive periodontitis patients, is reduced (Asif & Kothiwale 2010). To determine the ability of gingipains to disrupt phagocytosis, we used the in vivo phagocytosis assay. The results revealed that significantly higher percentages of leukocytes engulfed the RgpA P. gingivalis compared to the wild type or Kgp P. gingivalis (Fig. 2a). These results could be explained by the ability of Arg-gingipains to degrade components of the complement system such as C3, C4, C5 (Popadiak et al. 2007, Potempa & Pike 2009, Wang et al. 2010) and consequently to prevent phagocytosis of the bacterium. Similar results were found when we analysed the mean fluorescence index, suggesting that not only higher percentages of leukocytes were engulfing the RgpA bacteria, but that each leukocyte was engulfing higher numbers of RgpA bacteria (Fig. 2b). These results correspond to our findings in the microbiological cultures (in vivo survival assay) which show that significantly fewer RgpA bacteria survived inside the chambers (Fig. 3). Taken together, these results emphasize the importance of RgpA in the survival and virulence of the bacterium. A possible explanation for our findings could be the ability of RgpA to inactivate complement components such as C3 and thus decreasing opsonization of bacteria and leukocyte function (Wingrove et al. 1992, Schenkein et al. 1995, Discipio et al. 1996). The fact that RgpA helps the bacterium to evade phagocytosis by leukocytes and antigen presenting cells (APC’s), can affect the ability of these cells to process and present antigens to T cells. This situation may in turn interfere with antibody production by B cells. In consideration of this hypothesis, we evaluated the effect of gingipains on serum anti-P. gingivalis IgG levels. The results demonstrated that a week following infection, the mice infected with RgpA-expressing bacteria (wild type and Kgp ) showed significantly lower IgG titres compared to the mice infected with RgpA P. gingivalis (Fig. 4). In a recent study, Pathirana et al. showed that follow-

ing oral infection with P. gingivalis wild type or mutants, serum IgG titres in the RgpA P. gingivalisinfected mice were not found to be significantly different from the wildtype group, while IgG titres in the Kgp P. gingivalis-infected group were significantly lower compared to the wild-type group (Pathirana et al. 2007). The differences between the two studies could be explained by the different strains used, and/or by the different oral infection protocol. The fact that IgG titres were significantly lower in the wild-type group compared to the RgpA P. gingivalis-infected group may explain the higher ABL levels found in the former group. The inverse correlation between antibody titres and ABL levels was previously described by Wilensky et al. (2009). To further evaluate the effect of gingipains on the cellular immune response, we tested the effect of gingipains on leukocyte arrival at the local inflammatory site. Our data (Fig. 5) demonstrate that RgpA has a positive role in recruiting leukocytes at the local inflammatory site. These results could be explained by the ability of soluble gingipains to degrade cytokines such as IL-8 into peptides which are 2–3 times more potent than the original cytokine, and in so doing, elevate neutrophil arrival (Mikolajczyk-Pawlinska et al. 1998). Another explanation could be the ability of gingipains to induce elevated C5a levels and thus contribute to neutrophil recruitment into the inflammatory tissues (Wingrove et al. 1992, Discipio et al. 1996). RgpA evidently increases neutrophil accumulation and inflammation on one hand, but compromises the host’s ability to eliminate infection on the other, as shown in Fig. 3. This situation can lead to chronic inflammation, and subsequently, ABL (Fig. 1). Cytokines, secreted mainly by T cells, are another important factor in the development and control of the immune response through synchronization of the innate and adaptive immune systems. The fact that IL-10, TNF-a and IL-1b levels peaked significantly 2 h post-infection in the wild-type group compared to the RgpA group (Fig. 6) underscores the virulent role of RgpA in initiating the inflammatory response to P.

gingivalis. One explanation for our findings could be the ability of RgpA to increase the accumulation of leukocytes at the inflammatory site. Another explanation could be related to the fact that gingipains are virulent factors and, once engulfed by APCs, they will induce cytokine secretion. This hypothesis may be supported by Fitzpatrick et al. (2009), who have shown that the expression of IL-1b and IL-10 genes in human monocytic cell line following exposure to gingipains is significantly elevated (Fitzpatrick et al. 2009). Our results indicate that gingipains, and RgpA in particular, are important factors to the initiation of the early inflammatory response. Similar results demonstrating the presence of Th1 and Th2 cytokines at the inflammatory site were published by Lappin et al. who demonstrated the presence of cytokines in the gingiva and granular tissue taken from patients with periodontitis (Lappin et al. 2001). In summary, this study describes the in vivo role of RgpA in the pathogenesis of experimental periodontitis and local inflammation in mice. Our data indicate that RgpA can suppress in vivo phagocytosis by leukocytes and probably could influence the capacity of these cells to present antigens. This may explain the lower serum anti-P. gingivalis IgG titres, which may have in turn led to the higher ABL that was observed in the mice infected with the RgpAexpressing bacteria. Furthermore, RgpA was shown to have a positive role in the recruitment of leukocytes at the local inflammatory site and to the higher cytokine levels found at the site, which in turn may collectively contribute to the higher ABL. Acknowledgement

The authors thank CA Genco for kindly providing the P. gingivalis mutants and for her help with the oral infection model. In addition, we thank Amal Halabi, MSc for excellent technical assistance. References Abe, N., Kadowaki, T., Okamoto, K., Nakayama, K., Ohishi, M. & Yamamoto, K. (1998) Biochemical and functional properties of lysine-specific cysteine proteinase (Lys-gingi-

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

RgpA important for Porphyromonas gingivalis pathogenicity pain) as a virulence factor of Porphyromonas gingivalis in periodontal disease. Journal of Biochemistry 123, 305–312. Armitage, G. C. (1996) Periodontal diseases: diagnosis. Annuals of Periodontology 1, 37–215. Asif, K. & Kothiwale, S. V. (2010) Phagocytic activity of peripheral blood and crevicular phagocytes in health and periodontal disease. Journal of Indian Society of Periodontology 14, 8–11. Bachrach, G., Altman, H., Kolenbrander, P. E., Chalmers, N. I., Gabai-Gutner, M., Mor, A., Friedman, M. & Steinberg, D. (2008) Resistance of Porphyromonas gingivalis ATCC 33277 to direct killing by antimicrobial peptides is protease independent. Antimicrobial Agents and Chemotherapy 52, 638–642. Belibasakis, G. N., Bostanci, N. & Reddi, D. (2010) Regulation of protease-activated receptor-2 expression in gingival fibroblasts and Jurkat T cells by Porphyromonas gingivalis. Cell Biology International 34, 287–292. Burns, E., Bachrach, G., Shapira, L. & Nussbaum, G. (2006) Cutting Edge: TLR2 is required for the innate response to Porphyromonas gingivalis: activation leads to bacterial persistence and TLR2 deficiency attenuates induced alveolar bone resorption. Journal of Immunology 177, 8296–8300. Byrne, S. J., Dashper, S. G., Darby, I. B., Adams, G. G., Hoffmann, B. & Reynolds, E. C. (2009) Progression of chronic periodontitis can be predicted by the levels of Porphyromonas gingivalis and Treponema denticola in subgingival plaque. Oral Microbiology and Immunology 24, 469–477. Calkins, C. C., Platt, K., Potempa, J. & Travis, J. (1998) Inactivation of tumor necrosis factoralpha by proteinases (gingipains) from the periodontal pathogen, Porphyromonas gingivalis. Implications of immune evasion. Journal of Biological Chemistry 273, 6611–6614. Carlisle, M. D., Srikantha, R. N. & Brogden, K. A. (2009) Degradation of human alpha- and beta-defensins by culture supernatants of Porphyromonas gingivalis strain 381. Journal of Innate Immunity 1, 118–122. Carlsson, J., Herrmann, B. F., Hofling, J. F. & Sundqvist, G. K. (1984) Degradation of the human proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis. Infection and Immunity 43, 644–648. Cochran, D. L. (2008) Inflammation and bone loss in periodontal disease. Journal of Periodontology 79, 1569–1576. Discipio, R. G., Daffern, P. J., Kawahara, M., Pike, R., Travis, J., Hugli, T. E. & Potempa, J. (1996) Cleavage of human complement component C5 by cysteine proteinases from Porphyromonas (Bacteroides) gingivalis. Prior oxidation of C5 augments proteinase digestion of C5. Immunology 87, 660–667. Eastcott, J. W., Yamashita, K., Taubman, M. A., Harada, Y. & Smith, D. J. (1994) Adoptive transfer of cloned T helper cells ameliorates periodontal disease in nude rats. Oral Microbiology and Immunology 9, 284–289. Ebersole, J. L., Cappelli, D., Sandoval, M. N. & Steffen, M. J. (1995) Antigen specificity of serum antibody in A. actinomycetemcomitansinfected periodontitis patients. Journal of Dental Research 74, 658–666. Evans, R. T., Klausen, B., Ramamurthy, N. S., Golub, L. M., Sfintescu, C. & Genco, R. J. (1992) Periodontopathic potential of two strains of Porphyromonas gingivalis in gnotobiotic rats. Archives of Oral Biology 37, 813–819.

Fagundes, J. A., Monoo, L. D., Euzebio Alves, V. T., Pannuti, C. M., Cortelli, S. C., Cortelli, J. R. & Holzhausen, M. (2011) Porphyromonas gingivalis is associated with protease-activated receptor-2 upregulation in chronic periodontitis. Journal of Periodontology 82, 1596–1601. Fitzpatrick, R. E., Aprico, A., Wijeyewickrema, L. C., Pagel, C. N., Wong, D. M., Potempa, J., Mackie, E. J. & Pike, R. N. (2009) High molecular weight gingipains from Porphyromonas gingivalis induce cytokine responses from human macrophage-like cells via a nonproteolytic mechanism. Journal of Innate Immunity 1, 109–117. Genco, C. A., Cutler, C. W., Kapczynski, D., Maloney, K. & Arnold, R. R. (1991) A novel mouse model to study the virulence of and host response to Porphyromonas (Bacteroides) gingivalis. Infection and Immunity 59, 1255– 1263. Gmur, R., Hrodek, K., Saxer, U. P. & Guggenheim, B. (1986) Double-blind analysis of the relation between adult periodontitis and systemic host response to suspected periodontal pathogens. Infection and Immunity 52, 768–776. Holt, S. C. & Bramanti, T. E. (1991) Factors in virulence expression and their role in periodontal disease pathogenesis. Critical Reviews in Oral Biology and Medicine 2, 177–281. Holzhausen, M., Cortelli, J. R., da Silva, V. A., Franco, G. C., Cortelli, S. C. & Vergnolle, N. (2010) Protease-activated receptor-2 (PAR(2)) in human periodontitis. Journal of Dental Research 89, 948–953. Houri-Haddad, Y., Soskoine, W. A. & Shapira, L. (2001) Immunization to Porphyromonas gingivalis enhances the local pro-inflammatory response to subcutaneous bacterial challenge. Journal of Clinical Periodontology 28, 476–482. Houri-Haddad, Y., Soskolne, W. A., Halabi, A., Barak, V. & Shapira, L. (2000) Repeat bacterial challenge in a subcutaneous chamber model results in augmented tumour necrosis factor-alpha and interferon-gamma response, and suppression of interleukin-10. Immunology 99, 215–220. Houri-Haddad, Y., Soskolne, W. A., Halabi, A. & Shapira, L. (2005) The effect of immunization on the response to P. gingivalis infection in mice is adjuvant-dependent. Journal of Clinical Periodontology 32, 933–937. Houri-Haddad, Y., Soskolne, W. A., Shai, E., Palmon, A. & Shapira, L. (2002) Interferongamma deficiency attenuates local P. gingivalisinduced inflammation. Journal of Dental Research 81, 395–398. Imamura, T., Pike, R. N., Potempa, J. & Travis, J. (1994) Pathogenesis of periodontitis: a major arginine-specific cysteine proteinase from Porphyromonas gingivalis induces vascular permeability enhancement through activation of the kallikrein/kinin pathway. Journal of Clinical Investigation 94, 361–367. Jagels, M. A., Ember, J. A., Travis, J., Potempa, J., Pike, R. & Hugli, T. E. (1996a) Cleavage of the human C5A receptor by proteinases derived from Porphyromonas gingivalis: cleavage of leukocyte C5a receptor. Advances in Experimental Medicine and Biology 389, 155–164. Jagels, M. A., Travis, J., Potempa, J., Pike, R. & Hugli, T. E. (1996b) Proteolytic inactivation of the leukocyte C5a receptor by proteinases derived from Porphyromonas gingivalis. Infection and Immunity 64, 1984–1991. Jenkinson, H. F. & Lamont, R. J. (2005) Oral microbial communities in sickness and in health. Trends in Microbiology 13, 589–595.

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

931

Kinane, D. F., Mooney, J., MacFarlane, T. W. & McDonald, M. (1993) Local and systemic antibody response to putative periodontopathogens in patients with chronic periodontitis: correlation with clinical indices. Oral Microbiology and Immunology 8, 65–68. Kitamura, Y., Matono, S., Aida, Y., Hirofuji, T. & Maeda, K. (2002) Gingipains in the culture supernatant of Porphyromonas gingivalis cleave CD4 and CD8 on human T cells. Journal of Periodontal Research 37, 464–468. Lamster, I. B., Kaluszhner-Shapira, I., HerreraAbreu, M., Sinha, R. & Grbic, J. T. (1998) Serum IgG antibody response to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis: implications for periodontal diagnosis. Journal of Clinical Periodontology 25, 510– 516. Lappin, D. F., MacLeod, C. P., Kerr, A., Mitchell, T. & Kinane, D. F. (2001) Anti-inflammatory cytokine IL-10 and T cell cytokine profile in periodontitis granulation tissue. Clinical and Experimental Immunology 123, 294–300. Lopatin, D. E. & Blackburn, E. (1992) Avidity and titer of immunoglobulin G subclasses to Porphyromonas gingivalis in adult periodontitis patients. Oral Microbiology and Immunology 7, 332–337. Lourbakos, A., Chinni, C., Thompson, P., Potempa, J., Travis, J., Mackie, E. J. & Pike, R. N. (1998) Cleavage and activation of proteinaseactivated receptor-2 on human neutrophils by gingipain-R from Porphyromonas gingivalis. FEBS Letters 435, 45–48. Mezyk-Kopec, R., Bzowska, M., Potempa, J., Jura, N., Sroka, A., Black, R. A. & Bereta, J. (2005) Inactivation of membrane tumor necrosis factor alpha by gingipains from Porphyromonas gingivalis. Infection and Immunity 73, 1506–1514. Mikolajczyk-Pawlinska, J., Travis, J. & Potempa, J. (1998) Modulation of interleukin-8 activity by gingipains from Porphyromonas gingivalis: implications for pathogenicity of periodontal disease. FEBS Letters 440, 282–286. Mooney, J. & Kinane, D. F. (1997) Levels of specific immunoglobulin G to Porphyromonas gingivalis in gingival crevicular fluid are related to site disease status. Oral Microbiology and Immunology 12, 112–116. Nakayama, K. (1997) Domain-specific rearrangement between the two Arg-gingipain-encoding genes in Porphyromonas gingivalis: possible involvement of nonreciprocal recombination. Microbiology and Immunology 41, 185–196. Oido-Mori, M., Rezzonico, R., Wang, P. L., Kowashi, Y., Dayer, J. M., Baehni, P. C. & Chizzolini, C. (2001) Porphyromonas gingivalis gingipain-R enhances interleukin-8 but decreases gamma interferon-inducible protein 10 production by human gingival fibroblasts in response to T-cell contact. Infection and Immunity 69, 4493–4501. Paster, B. J., Olsen, I., Aas, J. A. & Dewhirst, F. E. (2006) The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontology 2000 42, 80–87. Pathirana, R. D., O’Brien-Simpson, N. M., Brammar, G. C., Slakeski, N. & Reynolds, E. C. (2007) Kgp and RgpB, but not RgpA, are important for Porphyromonas gingivalis virulence in the murine periodontitis model. Infection and Immunity 75, 1436–1442. Pike, R. N., Potempa, J., McGraw, W., Coetzer, T. H. & Travis, J. (1996) Characterization of the binding activities of proteinase-adhesin

932

Wilensky et al.

complexes from Porphyromonas gingivalis. Journal of Bacteriology 178, 2876–2882. Popadiak, K., Potempa, J., Riesbeck, K. & Blom, A. M. (2007) Biphasic effect of gingipains from Porphyromonas gingivalis on the human complement system. Journal of Immunology 178, 7242–7250. Potempa, J., Mikolajczyk-Pawlinska, J., Brassell, D., Nelson, D., Thogersen, I. B., Enghild, J. J. & Travis, J. (1998) Comparative properties of two cysteine proteinases (gingipains R), the products of two related but individual genes of Porphyromonas gingivalis. Journal of Biological Chemistry 273, 21648–21657. Potempa, J. & Pike, R. N. (2009) Corruption of innate immunity by bacterial proteases. Journal of Innate Immunity 1, 70–87. Rahman, S., Bunning, R. A., Dobson, P. R., Evans, D. B., Chapman, K., Jones, T. H., Brown, B. L. & Russell, R. G. (1992) Bradykinin stimulates the production of prostaglandin E2 and interleukin-6 in human osteoblast-like cells. Biochimica et Biophysica Acta 1135, 97– 102. Ransjo, M., Marklund, M., Persson, M. & Lerner, U. H. (1998) Synergistic interactions of bradykinin, thrombin, interleukin 1 and tumor necrosis factor on prostanoid biosynthesis in human periodontal-ligament cells. Archives of Oral Biology 43, 253–260. Schenkein, H. A., Fletcher, H. M., Bodnar, M. & Macrina, F. L. (1995) Increased opsonization of a prtH-defective mutant of Porphyromonas gingivalis W83 is caused by reduced degradation of complement-derived opsonins. Journal of Immunology 154, 5331–5337. Shapira, L., Houri-Haddad, Y., Frolov, I., Halabi, A. & Ben-Nathan, D. (1999) The effect of stress on the inflammatory response to Porphyromonas gingivalis in a mouse subcutaneous chamber model. Journal of Periodontology 70, 289–293. Sharp, L., Poole, S., Reddi, K., Fletcher, J., Nair, S., Wilson, M., Curtis, M., Henderson, B. & Tabona, P. (1998) A lipid A-associated protein of Porphyromonas gingivalis, derived from the

Clinical Relevance

Scientific relevance for the study: Although gingipains considered as major virulence factors of P. gingivalis, most of the studies addressing their function were limited to ex vivo experiments. In this study, we investigated the in vivo role of

haemagglutinating domain of the RI protease gene family, is a potent stimulator of interleukin 6 synthesis. Microbiology 144 (Pt 11), 3019–3026. Slots, J., Rams, T. E. & Listgarten, M. A. (1988) Yeasts, enteric rods and pseudomonads in the subgingival flora of severe adult periodontitis. Oral Microbiology and Immunology 3, 47–52. Socransky, S. S., Haffajee, A. D., Cugini, M. A., Smith, C. & Kent, R. L. Jr (1998) Microbial complexes in subgingival plaque. Journal of Clinical Periodontology 25, 134–144. Soehnlein, O. & Lindbom, L. (2010) Phagocyte partnership during the onset and resolution of inflammation. Nature Reviews Immunology 10, 427–439. Stabholz, A., Soskolne, W. A. & Shapira, L. (2010) Genetic and environmental risk factors for chronic periodontitis and aggressive periodontitis. Periodontology 2000 53, 138–153. Stathopoulou, P. G., Benakanakere, M. R., Galicia, J. C. & Kinane, D. F. (2009) The host cytokine response to Porphyromonas gingivalis is modified by gingipains. Oral Microbiology and Immunology 24, 11–17. Tam, V., O’Brien-Simpson, N. M., Chen, Y. Y., Sanderson, C. J., Kinnear, B. & Reynolds, E. C. (2009) The RgpA-Kgp proteinase-adhesin complexes of Porphyromonas gingivalis Inactivate the Th2 cytokines interleukin-4 and interleukin-5. Infection and Immunity 77, 1451–1458. Van Dyke, T. E., Lester, M. A. & Shapira, L. (1993) The role of the host response in periodontal disease progression: implications for future treatment strategies. Journal of Periodontology 64, 792–806. Wang, M., Krauss, J. L., Domon, H., Hosur, K. B., Liang, S., Magotti, P., Triantafilou, M., Triantafilou, K., Lambris, J. D. & Hajishengallis, G. (2010) Microbial hijacking of complementtoll-like receptor crosstalk. Science Signaling 3, ra11. Wilensky, A., Gabet, Y., Yumoto, H., HouriHaddad, Y. & Shapira, L. (2005) Three-dimensional quantification of alveolar bone loss in Porphyromonas gingivalis-infected mice using

micro-computed tomography. Journal of Periodontology 76, 1282–1286. Wilensky, A., Polak, D., Awawdi, S., Halabi, A., Shapira, L. & Houri-Haddad, Y. (2009) Straindependent activation of the mouse immune response is correlated with Porphyromonas gingivalis-induced experimental periodontitis. Journal of Clinical Periodontology 36, 915–921. Wingrove, J. A., DiScipio, R. G., Chen, Z., Potempa, J., Travis, J. & Hugli, T. E. (1992) Activation of complement components C3 and C5 by a cysteine proteinase (gingipain-1) from Porphyromonas (Bacteroides) gingivalis. Journal of Biological Chemistry 267, 18902–18907. Yun, P. L., Decarlo, A. A., Chapple, C. C., Collyer, C. A. & Hunter, N. (2005) Binding of Porphyromonas gingivalis gingipains to human CD4(+) T cells preferentially down-regulates surface CD2 and CD4 with little affect on costimulatory molecule expression. Microbial Pathogenesis 38, 85–96. Yun, P. L., Decarlo, A. A., Collyer, C. & Hunter, N. (2001) Hydrolysis of interleukin-12 by Porphyromonas gingivalis major cysteine proteinases may affect local gamma interferon accumulation and the Th1 or Th2 T-cell phenotype in periodontitis. Infection and Immunity 69, 5650–5660. Yun, P. L., DeCarlo, A. A., Collyer, C. & Hunter, N. (2002) Modulation of an interleukin-12 and gamma interferon synergistic feedback regulatory cycle of T-cell and monocyte cocultures by Porphyromonas gingivalis lipopolysaccharide in the absence or presence of cysteine proteinases. Infection and Immunity 70, 5695–5705.

gingipains in the pathogenicity of P. gingivalis. Principal finding: RgpA gingipain is a major virulent factor which augmented the host’s pro-inflammatory process and consequently alveolar bone loss.

Practical implications: As a major virulent factor, RgpA could be used as a tool for better understanding the mechanisms standing behind P. gingivalis virulence and as a target for the development of an efficient vaccine.

Address: Asaf Wilensky Department of Periodontology, Dental Faculty, Hadassah and the Hebrew University Medical Center, P.O. Box 12272, Jerusalem 91120, Israel E-mail: [email protected]

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd