JAC Advance Access originally published online on February 10, 2006
Journal of Antimicrobial Chemotherapy 2006 57(4):685-690; doi:10.1093/jac/dkl031
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Inhibition of periodontopathogen-derived proteolytic enzymes by a high-molecular-weight fraction isolated from cranberry
Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval, Quebec City, Quebec, Canada G1K 7P4
* Corresponding author. Tel: +1-418-656-7341; Fax: +1-418-656-2861; E-mail: Daniel.Grenier{at}greb.ulaval.ca
Received 15 December 2005; returned 8 January 2006; revised 20 January 2006; accepted 21 January 2006
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Abstract |
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Background: Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are three major aetiological agents of chronic periodontitis. The strong proteolytic activities of these bacteria are critical to their survival since their energy source is obtained from peptides and amino acids derived from proteins. In addition, proteases are important factors contributing to periodontal tissue destruction by a variety of mechanisms, including direct tissue degradation and modulation of host inflammatory responses.
Objectives: The aim of this study was to investigate the effect of non-dialysable material (NDM) prepared from cranberry juice concentrate on the proteolytic activities of P. gingivalis, T. forsythia and T. denticola.
Methods: The effect of NDM on gingipain and dipeptidyl peptidase IV (DPP IV) activities of P. gingivalis, trypsin-like activity of T. forsythia and chymotrypsin-like activity of T. denticola was evaluated using synthetic chromogenic peptides. In addition, the capacity of P. gingivalis to degrade fluorescein-labelled type I collagen and fluorescein-labelled transferrin in the presence of NDM was evaluated by fluorometry.
Results: NDM dose-dependently inhibited the proteinases of P. gingivalis, T. forsythia and T. denticola as well as type I collagen and transferrin degradation by P. gingivalis.
Conclusions: These results suggest that NDM has the potential to reduce either the proliferation of P. gingivalis, T. forsythia and T. denticola in periodontal pockets or their proteinase-mediated destructive process occurring in periodontitis.
Keywords: Porphyromonas gingivalis , Tannerella forsythia , Treponema denticola , periodontitis , protease inhibitors
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Introduction |
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Periodontal diseases are multifactorial infections caused by a specific group of Gram-negative anaerobic bacteria leading to destruction of the tooth-supporting tissue, including the alveolar bone and the periodontal ligament. Two major factors contribute to the pathogenesis of periodontitis. First, periodontopathogens cause direct damage to periodontal tissue through the secretion of toxic products. Second, the host response to periodontopathogens, which results in release of inflammatory mediators (pro-inflammatory cytokines, matrix metalloproteinases and prostanoids), is also involved in the progression of periodontitis. Specific bacterial species and bacterial complexes occur more frequently in diseased sites, while others are associated with healthy or stable periodontal tissues.1 Among the suspected periodontopathogens, the red complex, which includes Porphyromonas gingivalis, Tannerella forsythia (formerly Bacteroides forsythus) and Treponema denticola, is strongly related to clinical measures of periodontitis, particularly pocket depth and bleeding on probing.1,2 These three major periodontopathogens produce a broad array of virulence factors, such as proteolytic enzymes, that allow them to colonize subgingival sites, resist host defences and cause tissue destruction.3–5 The strong proteolytic activities exhibited by bacteria of the red complex are an important factor contributing to periodontal tissue destruction through a variety of mechanisms, including direct tissue degradation and modulation of host inflammatory responses.4–8 It has been proposed that inhibitors of periodontopathogen proteinases reduce the bacterial pathogenicity and they are considered to be potentially new therapeutic agents for periodontal diseases.9–11
The cranberry is a polyphenolic-rich fruit exhibiting various beneficial properties for human health, notably by interfering with microbial pathogens. Indeed, cranberry juice fractions were found to inhibit the adhesion to host cells of bacteria such as Escherichia coli12 and Helicobacter pylori13 and the coaggregation of many oral bacteria.14 Cranberry juice compounds were also reported to promote Streptococcus sobrinus desorption from artificial biofilms15 and to affect influenza virus adhesion and infectivity.16 In addition, it was reported that a high-molecular-weight fraction of cranberry juice inhibits glucosyltransferase and fructosyltransferase activities of oral bacteria.17 Previously, we reported that this fraction exhibits anti-inflammatory properties.18 In this study, we investigated the capacity of a high-molecular-weight cranberry fraction to inhibit the proteolytic activities of three major periodontopathogens: P. gingivalis, T. forsythia and T. denticola.
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Cranberry fraction
Juice concentrate from the American cranberry Vaccinium macrocarpon was kindly provided by Ocean Spray Cranberries (Lakeville-Middleboro, MA, USA). The juice was exhaustively dialysed (5 days) in 14 000 mol. wt cut-off dialysis bags at 4°C against distilled water and then lyophilized. This non-dialysable material was named NDM. The cranberry powder was dissolved in distilled water before use. Chemical analysis of NDM was carried out by Robin Roderick (Ocean Spray Cranberries) and revealed that this fraction is devoid of sugars and acids and contains 0.35% anthocyanins (0.055% cyanidin-3-galactoside, 0.003% cyanidin-3-glucoside, 0.069% cyanidin-3-arabinoside, 0.116% peonidin-3-galactoside, 0.016% peonidin-3-glucoside and 0.086% peonidin-3-arabinoside) and 65.1% proanthocyanidins.
Bacterial strains and growth conditions
The bacterial strains used were P. gingivalis ATCC 33277, T. denticola ATCC 35405 and T. forsythia ATCC 43037. P. gingivalis was grown in Todd–Hewitt broth (THB; BBL Microbiology Systems, Cockeysville, MD, USA) supplemented with haemin (10 µg/mL) and vitamin K (1 µg/mL). T. denticola was grown in oral spirochete medium as described previously.19 T. forsythia was grown in brain heart infusion broth (BHI; BBL Microbiology Systems) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Sigma Chemical Co., St Louis, MO, USA) and 0.001% N-acetyl muramic acid (10 µg/mL; Sigma). The bacterial cultures were incubated at 37°C under anaerobic conditions (80% N2, 10% H2, 10% CO2) for 24 h (P. gingivalis) or 4 days (T. denticola and T. forsythia).
Hydrolysis of synthetic chromogenic peptides
Bacterial cells were harvested by centrifugation (10 000 g for 10 min), washed and suspended in 50 mM PBS (pH 7.4) to an optical density at 660 nm (OD660) of 2 for P. gingivalis Arg-gingipains A and B activities and of 1 for P. gingivalis Lys-gingipain and dipeptidyl peptidase IV (DPP IV) activities, T. forsythia trypsin-like activity and T. denticola chymotrypsin-like activity. Bacterial cells were incubated in PBS with or without NDM at various final concentrations (10, 25, 50, 75, 100 and 150 µg/mL), synthetic chromogenic peptides (2 mM in distilled water) and 10 mM DTT as a reducing agent. The substrates used were glycyl-proline-p-nitroanilide (specific for P. gingivalis DPP IV; Bachem Bioscience, King of Prussia, PA, USA), benzoyl-arginine-p-nitroanilide (specific for P. gingivalis Arg-gingipains A and B and T. forsythia trypsin-like activity; Sigma), N-p-tosyl-glycine-proline-lysine-p-nitroanilide (specific for P. gingivalis Lys-gingipain; Sigma) and N-succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide (specific for T. denticola chymotrypsin-like activity; Sigma). The assay mixtures were incubated for 1 h at 37°C. Cells were removed by centrifugation (10 000 g for 10 min) and hydrolysis of the chromogenic substrates was determined by measuring the absorbance at 405 nm of the supernatants using a microplate reader (model 680; Bio-Rad Laboratories, Mississauga, ON, Canada). Chromogenic substrates alone or with NDM were used as control.
Degradation of fluorescein-labelled transferrin and type I collagen
The capacity of the NDM cranberry fraction to inhibit the degradation of fluorescein-labelled human transferrin (Molecular Probes, Eugene, OR, USA) by P. gingivalis was assessed. Bacterial cells (100 µL) suspended in 50 mM PBS to an OD660 of 2 were mixed with 67 µL of DTT (30 mM), 20 µL of fluorescein-labelled transferrin (1 mg/mL) and 13 µL of PBS with or without the cranberry NDM fraction at a final concentration up to 150 µg/mL. Assay mixtures were incubated at 37°C for 3 h in the dark before removing cells by centrifugation (10 000 g for 10 min). The fluorescence of the supernatant was measured using a fluorometer (VersaFluor model; Bio-Rad Laboratories) at excitation and emission wavelengths of 490 and 520 nm, respectively. Fluorescein-labelled human transferrin alone or with NDM was used as control. The effect of the cranberry NDM fraction on the degradation of fluorescein-labelled type I collagen by P. gingivalis was also evaluated. The reaction mixtures contained 20 µL of bacterial cells (OD660 = 1.5 in PBS), 10 µL of self-quenched bovine skin type I collagen labelled with fluorescein (Molecular Probes; 1 mg/mL), 137 µL of PBS with or without the cranberry fraction and 33 µL of 30 mM DTT. The reaction mixtures were incubated for 2 h in the dark at 25°C. Cells were then removed by centrifugation (10 000 g for 10 min) and the fluorescence of the supernatants was measured using a fluorometer at excitation and emission wavelengths of 490 and 520 nm, respectively. Fluorescein-labelled type I collagen alone or with NDM was used as control.
Statistical analyses
Each experiment was repeated three times. Data are expressed as the means ± SD. Statistical analysis was performed by one-way analysis of variance (ANOVA) for repeated measurements followed by Dunnett's test. A difference was considered significant if P < 0.05.
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Effect of the cranberry NDM fraction on proteolytic activities of P. gingivalis, T. denticola and T. forsythia
The effect of the cranberry NDM fraction on proteolytic activities of three major periodontopathogens was investigated. NDM efficiently inhibited the proteolytic activities of P. gingivalis (Figure 1). Arg-gingipain and DDP IV activities were significantly reduced (P < 0.05) by NDM at concentrations ranging from 50 to 150 µg/mL (Figure 1a and c). These activities were reduced by 50% at an NDM concentration of 75 µg/mL for Arg-gingipain and of 150 µg/mL for DDP IV. Lys-gingipain was significantly inhibited by a lower concentration of NDM, a 50% inhibition being obtained with 25 µg/mL (Figure 1b). T. denticola chymotrypsin-like and T. forsythia trypsin-like activities were also affected by NDM (Figure 2). The chymotrypsin-like activity of T. denticola was highly sensitive to NDM at all concentrations tested (Figure 2a). Indeed, the lowest NDM concentration of 10 µg/mL caused 
30% inhibition of this proteolytic activity, while 60% inhibition was obtained with an NDM concentration of 25 µg/mL. NDM at concentrations of 100 and 150 µg/mL significantly inhibited the trypsin-like activity of T. forsythia (Figure 2b).
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Effect of the cranberry NDM fraction on degradation of type I collagen and transferrin by P. gingivalis
The capacity of P. gingivalis to degrade type I collagen and transferrin was affected by NDM from cranberry (Figure 3). NDM at a concentration of 50 µg/mL significantly reduced the collagenase activity of P. gingivalis by 30% (Figure 3a). At a high concentration of NDM (100 µg/mL), the type I collagen degradation was completely inhibited. The degradation of transferrin by P. gingivalis was also significantly reduced by an NDM concentration of 50 µg/mL (Figure 3b). A 95% inhibition was obtained with NDM at a concentration of 150 µg/mL.
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Discussion |
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Accumulated evidence points to the members of the red complex, P. gingivalis, T. denticola and T. forsythia, as three major aetiological agents of chronic periodontitis.1,2,5 These bacteria produce proteases that are able to degrade a broad range of connective tissue proteins and to disrupt host defence mechanisms.5,8 In addition, these proteases are probably critical for their survival since most periodontopathogens derive their energy from protein sources. Therefore, it has been proposed that proteases produced by P. gingivalis, T. forsythia and T. denticola may participate in the destructive process occurring in periodontitis.8 As suggested by various studies,9–11,20 agents that inhibit protease activities of periodontopathogens may be potentially useful for the prevention or treatment of periodontal disease. The aim of this study was to investigate the effect of a cranberry high-molecular-weight fraction on proteolytic activities exhibited by three major periodontopathogens.
Gingipains, which can be both cell bound and secreted, are the main endopeptidases produced by P. gingivalis and contribute to the virulence properties of this bacterium since they degrade a large variety of host proteins.4,5 These proteases appear to be essential for P. gingivalis by providing iron, peptides and amino acids from environmental proteins.21,22 They also contribute to processing/maturation of several cell-surface proteins of P. gingivalis.23 Gingipains can degrade various extracellular matrix proteins,4 as well as activate pro-matrix metalloproteinases,24 the kallikrein/kinin pathway and the blood coagulation system.4 Gingipains also contribute to the evasion of host defence mechanisms by modulating cytokine functions25,26 and degrading immunoglobulins, complement proteins27 and cell-membrane proteins such as interleukin-6 receptor28 or CD14.29 Interestingly, it was reported that gingipain inhibitors reduce the pathogenicity of P. gingivalis.9 In this study, we showed that cranberry NDM efficiently inhibits both Arg- and Lys-gingipain activities of P. gingivalis. Other polyphenolic compounds, including the green tea catechins, have been previously reported to also strongly inhibit the gingipain activities of P. gingivalis.20 On the basis of the critical roles suggested previously for gingipains in the pathogenesis of periodontitis, our results suggest that cranberry compounds found in the NDM fraction may contribute to the reduction of periodontal tissue destruction.
Iron plays an important role in the growth and virulence of P. gingivalis.22 The ability of P. gingivalis to multiply under iron-limiting conditions has been correlated with its pathogenicity in an animal model.30 One major source of iron for periodontopathogens is human transferrin, which is present in high amounts in the gingival crevicular fluid. Cleavage of human transferrin by gingipains promotes growth and formation of hydroxyl radicals that may contribute to tissue destruction during periodontitis.31 In our study, NDM exhibited an inhibitory effect on transferrin degradation by P. gingivalis. This suggests that NDM can contribute to the reduction in vivo of the growth and virulence of P. gingivalis.
T. denticola chymotrypsin-like activity has been shown to degrade transferrin, fibrinogen, gelatin, immunoglobulins, 
1-antitrypsin and various basement membrane proteins.32,33 This activity may thus play a major role in the invasion and destruction of basement membrane by T. denticola33 as well as in epithelial cell layer penetration by this organism.34 The chymotrypsin-like activity of T. denticola was strongly inhibited by NDM, suggesting that NDM may limit the pathogenicity of T. denticola.
Collagen degradation enzymes produced by periodontopathogens may be involved in gingival connective tissue destruction.35 Since type I collagen is the predominant protein of periodontal tissues, representing 60% of this tissue volume, degradation of this major constituent of the gingival matrix would lead to tissue destruction. P. gingivalis can degrade type I collagen, and the collagenolytic activity of this bacteria has been attributed to various proteases.36–38 A pathological role of P. gingivalis DPP IV in the progression of periodontitis was suggested recently.39 Indeed, DPP IV was reported to act in conjunction with collagenase. NDM by inhibiting both the collagenolytic and DPP IV activities of P. gingivalis can reduce the type I collagen disruption and the subsequent connective tissue fragilization in periodontitis patients. Other compounds, such as tetracycline and doxycycline, which reduce the severity and progression of periodontitis in animal models and humans, also inhibit the collagenase activity of P. gingivalis.11 In addition, proteases from T. denticola and P. gingivalis can activate host collagenases and plasminogen, and destroy host protease inhibitors that would enhance host-enzyme-mediated tissue destruction.6,40 NDM from cranberry, by inhibiting these proteolytic activities, may also contribute to the reduction of the impact of host-enzyme-mediated destructive processes occurring in periodontitis.
To prevent periodontal disease progression, mechanical procedures are used to remove the dental biofilm. Although these procedures are effective in managing the majority of periodontitis patients, there are situations in which conventional therapy does not always achieve the desired clinical outcome. Control of disease in individuals with significantly increased risk for periodontitis (smokers, diabetics or individuals with a genetic predisposition) or who do not respond to conventional therapy may require adjunctive treatments, such as use of antimicrobials or host modulators. Indeed, systemic administration of subantimicrobial doses of doxycycline, which down-regulates matrix metalloproteinase activity, is indicated as an adjunctive treatment for periodontitis and confers clinical benefits to patients with periodontitis.41 In regard to developing additional alternatives to conventional therapy for individuals with a substantially increased risk for periodontitis, local application of cranberry NDM may provide a new practical approach based on attenuation of periodontopathogen virulence.
Our results showed that NDM efficiently inhibits proteases of periodontopathogens, which are major factors for their pathogenicity and are required for their growth and survival. These results suggest that NDM may contribute to the reduction of the proliferation of periodontopathogens in periodontal pockets and the proteinase-mediated destructive process occurring in periodontitis. NDM exhibits interesting properties for periodontal health and thus appears promising for the development of new therapeutic approaches for adjunctive treatment of periodontitis.
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None to declare.
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This work was supported by the Cranberry Institute (East Wareham, MA, USA). We thank Robin Roderick and Marge Leahy (Ocean Spray Cranberries) for chemical analyses and for providing cranberry juice concentrate.
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  J. Labrecque, C. Bodet, F. Chandad, and D. Grenier Effects of a high-molecular-weight cranberry fraction on growth, biofilm formation and adherence of Porphyromonas gingivalis J. Antimicrob. Chemother., August 1, 2006; 58(2): 439 - 443. [Abstract] [Full Text] [PDF]
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