Characterization of protease activities in Capnocytophaga spp., Porphyromonas gingivalis, Prevotella spp., Treponema denticola and Actinobacillus actinomycetemcomitans

Protease activities in cell sonicates of denned bacterial strains were examined using peptide substrates and class-specific inhibitors. Capnocytophaga spp. all produced serine dipeptidyl peptidase activity and arginine/lysine, elastase- and chymotrypsin-like enzymes with some metalloprotease characteristics. The elastase-like activity was strongest in Capnocytophaga sputigena , but the others were greatest in Capnocytophaga gingivalis. The latter also had a separate arginine-specific enzyme which appeared not to be present in the other two species. Porphyromonas gingivalis showed serine dipeptidyl peptidase activity and very strong arginine and lysine cysteine protease activities. Prevotella spp. had inhibitor-resistant dipeptidyl peptidase activity and arginine cysteine protease activity that was much weaker but biochemically similar to P. gingivalis. Treponema denticola possessed a strong trypsin-like serine protease activity as well as very weak dipeptidyl peptidase and chymotrypsin-like activities that were sensitive to some cysteine protease reagents. Actinobacillus actinomycetemcomitans showed a novel ala-nine- and lysine-specific activity, but its nature was unclear.     

Acquisition of iron from human transferrin by Porphyromonas gingivalis: a role for Arg- and Lys-gingipain activities

Porphyromonas gingivalis, a key causative agent of adult periodontitis, is known to produce a variety of virulence factors including proteases. The aim of this study was to evaluate the participation of Arg- and Lys-gingipain activities of P. gingivalis in the acquisition of iron from human transferrin and its subsequent utilization in growth. Iron-saturated transferrin was found to support the long-term growth of P. gingivalis. Our results indicated that P. gingivalis does not produce siderophore and does not possess ferric reductase and transferrin-binding activities. Incubating transferrin with P. gingivalis resulted in degradation of the protein, a step that may be critical for the acquisition of iron from transferrin. Spontaneous and site-directed mutants of P. gingivalis deficient in one or several proteases were used to demonstrate the key role of specific enzymes in degradation of transferrin and subsequent utilization for growth. The lack of both Arg- and Lys-gingipain activities (mutants M1 and KDP128) was associated with an absence of degradation of transferrin and the incapacity of bacteria to grow in the presence of transferrin as the sole source of iron. It was also found that the Lys-gingipain activity is more critical than the Arg-gingipain activity since the mutant KDP112 (deficient in Arg-gingipain A and B) could grow whereas the mutant KDP129 (deficient in Lys-gingipain) could not. The fact that growth of mutant KDP112 was associated with a lower final optical density and a generation time much longer compared with the parent strain suggests that the Arg-gingipain activity also participates in the acquisition of iron from transferrin. Selected inhibitors of cysteine proteases (TLCK, leupeptin and cathepsin B inhibitor II) were tested for their capacity to reduce or inhibit the growth of P. gingivalis under different iron conditions. All three inhibitors were found to completely inhibit growth of strain ATCC 33277 in a medium supplemented with transferrin as the source of iron. The inhibitors had no effects when the bacteria were grown in a medium containing hemin instead of transferrin. The ability of P. gingivalis to cleave transferrin may be an important mechanism for the acquisition of iron from this protein during periodontitis.     

Inhibitory effect of cranberry polyphenol on biofilm formation and cysteine proteases of Porphyromonas gingivalis

BACKGROUND AND OBJECTIVE: The purpose of this study was to investigate the effects of cranberry polyphenol fraction on biofilm formation and activities of Arg-gingipain and Lys-gingipain in Porphyromonas gingivalis. MATERIAL AND METHODS: The polyphenol fraction was prepared by using a glass column packed with Amberlite XAD 7HP and 70% aqueous ethanol as an elution solvent. RESULTS: Synergistic biofilm formation by P. gingivalis and Fusobacterium nucleatum was significantly inhibited by the polyphenol fraction at a concentration of 250 microg/mL compared with untreated controls (p < 0.01). Arg-gingipain and Lys-gingipain activities in P. gingivalis ATCC 33277 and FDC 381 were inhibited significantly at a polyphenol fraction concentration of > or = 1 microg/mL (p < 0.05). CONCLUSION: These findings indicate that the polyphenol fraction inhibits biofilm formation and the Arg-gingipain and Lys-gingipain activities of P. gingivalis.     

Genetic evidence for the relationship of Porphyromonas gingivalis cysteine protease and hemagglutinin activities

Cysteine protease and hemagglutinin activities of Porphyromonas gingivalis have been implicated as virulence factors in periodontitis. In addition, a close structural relationship between these factors has been suggested. In order to examine the molecular basis for such a relationship, we constructed an isogenic mutant. G-102, of P. gingivalis 381 deficient in Arggingipain cysteine protease activity. The mutant displayed not only reduced protease activity but also significantly reduced hemagglutination activity compared with the wild-type strain. Therefore, this investigation provided genetic evidence for the recently proposed structural relationship between Arg-gingipain and the hemagglutinin activity of P. gingivalis strains.     

Participation of the secreted dipeptidyl and tripeptidyl aminopeptidases in asaccharolytic growth of Porphyromonas gingivalis

Background and Objective:  Porphyromonas gingivalis secretes gingipains, endopeptidases essential for the asaccharolytic growth of this bacterium. P. gingivalis also secretes dipeptidyl aminopeptidases (DPPIV and DPP-7) and a tripeptidyl aminopeptidase (PTP-A), although their role in asaccharolytic growth is unclear. The present study was carried out to elucidate the role of these dipeptidyl/tripeptidyl aminopeptidases on the asaccharolytic growth of P. gingivalis .
Material and Methods:  Knockout mutants for the DPPIV ( dpp ), dpp7 and/or PTP-A genes were constructed. Brain–heart infusion medium supplemented with sterile hemin and menadione (BHIHM) was used as a complex medium, and the minimal medium used was GA, in which the sole energy source was a mixture of immunoglobulin G and bovine serum albumin. Growth of P. gingivalis was monitored by measuring the optical density of the culture.
Results:  All knockout mutants for DPPIV , dpp7 and PTP-A grew as well as strain W83 in BHIHM. In GA, growth of single-knockout and double-knockout mutants was similar to that of W83, whereas growth of a triple-knockout mutant (83-47A) was reduced. We purified recombinant DPPIV and recombinant PTP-A from recombinant Escherichia coli overproducers, and purified DPP-7 from the triple-knockout mutant 83-4A. GA supplemented with the three purified dipeptidyl/tripeptidyl aminopeptidases supported the growth of 83-47A.
Conclusion:  DPPIV, DPP-7 and PTP-A contribute to the normal growth of P. gingivalis by cleaving substrate peptides into short-chain polypeptides that are efficient energy sources for P. gingivalis.     

Effects of dipeptide bestatin on Porphyromonas gingivalis and epithelial cells.

BACKGROUND: Dipeptide bestatin has been previously reported to selectively inhibit the growth of Porphyromonas gingivalis. The aims of this study were to investigate the mechanism of action of bestatin and to evaluate its effect on epithelial cells. METHODS: The inhibitory effect of bestatin on P. gingivalis was tested in vitro (culture medium) and in vivo (guinea pig model). Radiolabeled compounds were used to investigate the effect of bestatin on the uptake of amino acids and peptides. The cytotoxic effect of bestatin was evaluated using a keratinocyte cell line. RESULTS: The growth inhibition of P. gingivalis by bestatin was concentration-dependent. Even at high concentrations, compounds possessing a chemical structure or an aminopeptidase inhibitor activity related to bestatin had no effect on growth of P. gingivalis. When injected in the presence of P. gingivalis, bestatin was able to prevent the development of a necrotic abscess in a guinea pig model. Data were obtained suggesting that bestatin does not act on proteinases of P. gingivalis. Rather, bestatin was found to inhibit the intracellular uptake of radioactivity from 14C-labeled amino acids or heat-denatured type I collagen. This was not observed with a spontaneous mutant of P. gingivalis, whose growth was not affected by bestatin. In the second part of the study, bestatin was found to have no effect on epithelial cell viability in culture at concentrations effective on P. gingivalis. In addition, bestatin did not show effects on epithelial cell migration or production of gelatinases. CONCLUSIONS: This study suggests that bestatin selectively inhibits growth of P. gingivalis by affecting the intracellular uptake of amino acids and peptides, which serve as energy and nitrogen sources for this bacterial species. Bestatin has no cytotoxicity and may represent a therapeutic molecule for local treatment of P. gingivalis-associated periodontitis.     

Inhibitory effect of green tea catechins on cysteine proteinases in Porphyromonas gingivalis

The purpose of this study was to examine the effects of catechins and their derivatives on the activities of Arg-gingipain (Rgp) and Lys-gingipain (Kgp) in Porphyromonas gingivalis. Catechin derivatives, which included (-)-epigallocatechin gallate, (-)-epicatechin gallate, (-)-gallocatechin gallate, and (-)-catechin gallate, significantly inhibited the Rgp activity. The 50% inhibitory concentrations (IC50s) of these catechin derivatives for Rgp ranged from 3 to 5 microm. While (-)-epigallocatechin and (-)-gallocatechin moderately inhibited Rgp activity (IC50s, 20 microm), (-) -epicatechin, (+)-catechin, and gallic acid were not effective, with IC50s greater than 300 microm. Further, some of the catechin derivatives tested also inhibited the Kgp activity, though to a lesser extent than inhibition of the Rgp activity. These findings suggest that green tea catechins may have the potential to reduce periodontal breakdown resulting from the potent proteinase activity of P. gingivalis.     

Effects of Porphyromonas gingivalis on human gingival fibroblasts from healthy and inflamed tissues

Background and Objective:  This study compared the ability of human gingival fibroblasts (HGFs) isolated from healthy and inflamed gingival tissues to degrade collagen in the presence and absence of Porphyromonas gingivalis supernatant.
Material and Methods:  Human gingival fibroblasts were cultured from explants of 21 healthy and 21 inflamed periodontal tissues. The HGFs that grew out of the explants were seeded in the center of six-well plates coated with collagen in the presence and absence of 10% P. gingivalis supernatant. An inflamed and a healthy cell line were also evaluated with Arg-gingipain. After 6 days, Coomassie Blue was used to visualize the collagen cleavage.
Results:  The collagen was totally cleaved in 12 (aggressive) of the 21 cell lines isolated from the inflamed tissues in the presence of P. gingivalis . The remaining nine cell lines (non-aggressive) cleaved only the collagen underneath the cell colonies in the presence of P. gingivalis . Of the healthy tissues, five (aggressive) of the 21 cell lines cleaved all the collagen and 16 cell lines (non-aggressive) only cleaved the collagen underneath the cell colonies in the presence of P. gingivalis . All the collagen was cleaved by an aggressive cell line and only the collagen underneath the cell colonies was cleaved by a non-aggressive cell line in the presence of Arg-gingipain.
Conclusion:  The collagen in the wells was more readily cleaved by the inflamed than by the healthy cell lines, and the difference was statistically significant ( p  = 0.0278). Arg-gingipain gave identical results to the P. gingivalis supernatant.     

Survey of a receptor protein in human erythrocytes for hemagglutinin of Porphyromonas gingivalis

The purpose of this study is to survey a receptor protein in human erythrocyte membrane for the hemagglutinin (HA) of Porphyromonas gingivalis. Human erythrocytes were modified by either chymotrypsin or P. gingivalis HA along with the disappearance of their hemagglutinating ability and the removal of the band 3 protein. By preparative electrophoresis, this protein was isolated and purified from human erythrocytes. The purified protein showed strong inhibitory activity for hemagglutination and the binding to P. gingivalis cells, whose binding sites were calculated to be approximately 9000, suggesting its binding to the active site of HA. Hemagglutinin purified from P. gingivalis by affinity absorption to sheep erythrocyte ghosts possessed strong trypsin-like activity, and both the HA and the enzyme activities were inhibited by arginine. Specific modification of arginyl residues in human erythrocytes by phenylglyoxal diminished the hemagglutinating ability. From the similarity of the inhibition profile and possible active sites between HA and the trypsin-like protease, it is suggested that hemagglutination may occur as a result of the primary reaction of the enzyme (protease) and the substrate. These results suggest that band 3 may be a key protein in human erythrocyte membrane for HA from P. gingivalis and its binding sites may be arginyl residues of the protein.     

Arg-gingipain A DNA vaccine prevents alveolar bone loss in mice

One major pathogenic factor of Porphyromonas gingivalis is Arg-gingipain (Rgp), an arginine-specific cysteine proteinase. To clarify the effect of rgpA DNA vaccine, we immunized BALB/c mice via the abdomen with a Gene Gun or via the nasal cavity weekly for 6 weeks. After immunization, the mice were challenged orally with P. gingivalis. Immunization elicited IgG responses against P. gingivalis in both groups. Nasal immunization also induced sIgA against P. gingivalis, although Gene Gun immunization did not. Reduction of alveolar bone loss was observed in both groups at 42 days following initial infection. This effect was more pronounced in the intranasal immunization group than in the Gene Gun group. The results of this study suggest that immunization with rgpA DNA vaccine via the nasal cavity is an effective method for preventing alveolar bone loss incurred by infection with P. gingivalis.     

Participation of an arginyl residue of insulin chain B in the inhibition of hemagglutination by Porphyromonas gingivalis

Insulin chain B, containing each one arginyl and lysyl residue in its peptide chain, inhibited hemagglutination by Porphyromonas gingivalis. To determine the further inhibitory profile, chain B was digested into 4 fragments by protease, which was contained in the preparation of hemagglutinin from P. gingivalis. Identification of each fragment by the amino acid analysis revealed that the chain was cleaved at the carboxyl site of arginyl and/or lysyl residues, but one fragment contained citrulline instead of arginine at its carboxyl terminal. This citrulline might have originated from arginine by an arginine deaminase-like enzyme of P. gingivalis. Only one fragment that contained the arginyl residue exhibited inhibitory activity on hemagglutination, but it was considerably weakened compared with that of the intact chain B. The difference in the inhibitory activity seemed to depend on the position of an arginyl residue in the peptide; this was also confirmed using several derivatives of bradykinin. The present result suggests that the internal arginyl residue in a peptide chain may be critical for the inhibition of the hemagglutination by P. gingivalis.     

The prevalence and pathogenic differences of Porphyromonas gingivalis fimA genotypes in patients with aggressive periodontitis

BACKGROUND: The fimA gene, which encodes fimbrillin (FimA), is found in Porphyromonas gingivalis and has been classified into six genotypes based on nucleotide sequence. P. gingivalis that possesses the type II fimA gene is prevalent in adult periodontitis. OBJECTIVES: The aim of this study was to investigate the prevalence of P. gingivalis fimA genotypes in Japanese aggressive periodontitis patients, and to examine their virulence. METHODS: Subgingival plaque samples were obtained from 223 sites in 18 aggressive periodontitis patients and 95 sites in 22 periodontally healthy young adults. Actinobacillus actinomycetemcomitans, P. gingivalis and Tannerella forsythensis detection, determination of the fimA genotype in P. gingivalis, and the quantification of P. gingivalis were analyzed by polymerase chain reaction (PCR) methods. The proteolytic activities of the P. gingivalis fimA type I and fimA type II were also examined. RESULTS: In aggressive periodontitis patients, the most prevalent fimA genotype was the type II (46.7%), followed by the type Ib and type I, whereas in healthy subjects, the type I fimA was the only genotype detected. The number of P. gingivalis pathogens was the greatest in the type I fimA positive sites, and the frequency of coexisting A. actinomycetemcomitans and T. forsythensis was highest in the type II fimA positive sites in the aggressive periodontitis patients. Both the arginine-specific cysteine proteinase (Arg-gingipain) and lysine-specific cysteine proteinase (Lys-gingipain) activity of the P. gingivalis fimA type I strain were significantly higher than those of the fimA type II strains. CONCLUSIONS: These results suggest that differences in virulence exist among different fimA genotypes. Coadherence with other pathogens in P. gingivalis fimA type II-associated aggressive periodontitis and quantitative increases in P. gingivalis in fimA type I-associated aggressive periodontitis are related to this virulence.     

Correlation of haemagglutination activity with trypsin-like protease activity of Porphyromonas gingivalis.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with various forms of periodontal disease. Several characteristics of P. gingivalis are thought to contribute to its pathogenicity; these include haemagglutination and trypsin-like protease activity. Previous studies suggest an association between haemagglutination and trypsin-like protease activity of P. gingivalis. To investigate this, two complementary quantitative experimental approaches were taken. Five independent mutants of P. gingivalis deficient in trypsin-like protease activity were shown to exhibit reduced haemagglutination activity. In addition, enhancers (cysteine and dithiothreitol) and inhibitors (N-ethylmaleimide, N-p-tosyl-L-lysine-chloromethyl ketone, and phenylmethylsulphonyl fluoride) of trypsin-like protease activity were shown, respectively, to significantly enhance and inhibit haemagglutination activity of washed, wild-type P. gingivalis cells (p less than 0.05, paired t-test). Statistical analysis indicated a strong correlation between haemagglutination and trypsin-like protease activity (r = 0.85, p less than 0.001, Spearman rank correlation). The effect of the protease enhancers and inhibitors on haemagglutination activity was specific for P. gingivalis, as they did not significantly change the haemagglutination activity of Fusobacterium nucleatum. These results suggest that the proteolytic site of the trypsin-like protease participates in haemagglutination activity of P. gingivalis.     

Isolation and characterization of protease from culture supernatant of Bacteroides gingivalis

Caseinolytic protease was isolated and purified, with high yield, from culture supernatant of Bacteroides gingivalis 381 by procedures including acetone fractionation, gel filtration on Sepharose CL-6B, solubilization with 0.8% 1-O-N-octyl-β-D-glucopyranoside and affinity chromatography on arginine-Sepharose 4B. By the affinity chromatography, the protease was separated into three isoenzymes, one of which was unadsorbed on the arginine-Sepharose 4B, but the other two of which were adsorbed and eluted with the buffer at a different concentration of arginine. Caseinolytic activities of the latter two were highly dependent on dithiothreitol and were inhibited by both thiol-blocking reagents and some microbial protease inhibitors; but such inhibition was not found in the former isoenzyme. The higher the affinity of the enzyme to arginine-Sepharose, the stronger the thiol dependency and the inhibition by these inhibitors. Phosphoramidone and ethylenediamine tetraacetate had no effect on the activity of any of the three peaks, indicating that the enzyme is not a metaloprotease. Besides the enzymehydrolyzed synthetic substrates of trypsin, such as BA p NA, TAME, substrates of chymotrypsin and kallikrein were also hydrolyzed. The salivary and egg-white lysozymes were also degraded by this enzyme. These findings suggest that the protease produced by B. gingivalis may play a role as a periodontopathogen not only in directly destroying periodontal tissues but also in weakening the oral antibacterial mechanism.     

Serum IgG1 and IgG2 antibody responses to Porphyromonas gingivalis in patients with periodontitis

BACKGROUND/AIMS: Protein and carbohydrate antigens of Porphyromonas gingivalis interact with the host to produce antibody of different subclasses. IgG1 and IgG2 antibodies frequently account for approximately 90% of the total serum IgG. This work aimed to investigate serum IgG1 and IgG2 antibody responses of periodontitis patients to protein and carbohydrate-rich antigens of P. gingivalis. METHODS: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blots of P. gingivalis antigens and proteinase K digested antigens rich in carbohydrates were used to investigate the molecular weight of antigen recognised by serum IgG1 and IgG2. Enzyme-linked immunosorbent assay was used to measure levels of IgG1 and IgG2 antibody to P. gingivalis and radial immunodiffusion was used to estimate the total concentration of IgG1 and IgG2 in serum. RESULTS: Serum IgG antibodies bound to antigens of molecular weights 47, 39 and 32 kDa. Antigen most frequently recognised by both IgG1 and IgG2 antibody had a molecular weight of 47 kDa. Serum IgG2 antibody bound to carbohydrate antigen with a molecular weight of 32 kDa but there was no recognition of carbohydrate antigens by IgG1 antibodies. There was no correlation between the titre of anti-P. gingivalis IgG1 or IgG2 antibody and the total concentration of serum IgG1 or IgG2 antibodies of all specificities. CONCLUSION: Both IgG1 and IgG2 antibodies recognised a dominant antigen of 47 kDa, probably Arg-gingipain. Much of the response to carbohydrate antigen is of the IgG2 subclass. Neither the level of IgG1 nor the IgG2 antibody specific to P. gingivalis was related to the total serum concentration of that antibody.     

Identification of amino acid residues involved in hemin binding in Porphyromonas gingivalis hemagglutinin 2

Porphyromonas gingivalis (P. gingivalis) is a major etiological agent in the development and progression of chronic periodontitis. It produces cysteine proteases (gingipains), including a lysine‐specific gingipain and two arginine‐specific gingipains. Heme binding and uptake are fundamental to the growth and virulence of P. gingivalis. The recombinant hemagglutinin 2 domain (rHA2) of gingipain binds hemin with high affinity. The aim of the present work was to identify the key residues involved in its hemin‐binding activity. A functional rHA2 was expressed and bound to hemin‐agarose, and then digested with endopeptidases. The peptides bound to hemin‐agarose were identified by mass spectrometry and the amino acids were assessed by mutation and peptide binding inhibition analysis. The DHYAVMISK sequence was identified in peptides derived from both Asp‐N and Lys‐C endopeptidase digestions of rHA2. A monoclonal antibody, mAb QB, was produced and its epitope was associated with the DGFPGDHYAVMISK peptide within the HA2 domain. Hemin was shown to competitively inhibit the immunoreactivity of rHA2 or the peptide to mAb QB. The peptide DHYAVMISK inhibited hemin‐binding activity; although, this inhibition was not seen when the peptide contained the H1001E mutation (DEYAVMISK). Based on these results, we propose that residue His1001 is involved in the hemin‐binding mechanism of the P. gingivalis rHA2 and the peptide containing this residue, DHYAVMISK, may be an inhibitor of hemin binding.     

Effect of pH on the growth and proteolytic activity of Porphyromonas gingivalis and Bacteroides intermedius

The effect of pH on the growth and proteolytic activity of the type strain and fresh isolates of Porphyromonas gingivalis and Bacteroides intermedius was investigated. B. intermedius strains grew with and without glucose at a pH as low as 5.0. These bacteria grew almost as well as Streptococcus mutans at pH 5.0 and better than Actinomyces viscosus at pH 5.5 and 5.0. Some B. intermedius strains raised the culture pH when grown at a low pH without glucose. In contrast, P. gingivalis strains grew only at pH 6.5 to 7.0. The P. gingivalis strains had proteolytic activities against azocoll, azocasein, and azoalbumin, while the B. intermedius strains degraded azocasein and azoalbumin, but not azocoll. B. intermedius showed maximum proteolytic activity at pH 7.0, and high activity over a wide pH range. In contrast, the optimum pH of proteolytic activity in P. gingivalis was pH 7.5 to 8.0. The P. gingivalis activities were more sensitive than those of B. intermedius to low pH. The capacity of B. intermedius to degrade proteins to more readily metabolizable substrates at low pH might explain the growth of this bacterium in an acidic environment. These differences between B. intermedius and P. gingivalis could explain their capacity to survive at different sites in the oral cavity and indicate how B. intermedius might positively influence the growth of P. gingivalis in subgingival plaque.     

Effect of hydroxamic acid-based matrix metalloproteinase inhibitors on human gingival cells and Porphyromonas gingivalis

BACKGROUND: Matrix metalloproteinases (MMPs) are considered to play key roles in tissue destruction during periodontitis. In this study, we evaluated the cytotoxicity of hydroxamic acid-based MMP inhibitors (ONO-4817, ONO-MI1-514, and ONO-MI1-570), and their inhibitory effects on MMP-2 and -9 activities and growth of Porphyromonas gingivalis. METHODS: Human gingival fibroblasts (HGF) and human gingival epithelial cells (HGE) were incubated with test inhibitors prior to investigating cell viability, cell proliferation, and mRNA expression for MMP-2 and -9. Gelatin zymography and a colorimetric MMP assay were performed to study the inhibitory effects on MMP-2 and -9 activities derived from HGF and HGE, respectively. The effect of MMP inhibitors on keratinocyte migration and P. gingivalis growth was also tested. RESULTS: Cell viability was not affected by any of the inhibitors at a final concentration of 50 microM, nor was cell proliferation at 20 microM. All inhibitors clearly inhibited MMP-2 produced by HGF and MMP-9 produced by HGE in a dose-dependent manner. No change was found in mRNA expression of MMPs by gingival cells treated with the inhibitors. ONO-4817 and ONO-MI1-514 inhibited keratinocyte migration. ONO-4817 showed a slightly inhibitory effect on the growth of P. gingivalis. CONCLUSION: Data obtained in this study support the potential use of the three MMP inhibitors for the prevention and treatment of periodontal disease.     

Inhibitory effects of macrocarpals on the biological activity of Porphyromonas gingivalis and other periodontopathic bacteria

BACKGROUND/AIMS: Macrocarpals, which are phloroglucinol derivatives contained in eucalyptus leaves, exhibit antimicrobial activity against a variety of bacteria including oral bacteria. This study examined effects of macrocarpals A, B, and C on periodontopathic bacteria, especially Porphyromonas gingivalis. METHODS: Macrocarpals A, B, and C were purified from a 60% ethanol-extract of Eucalyptus globules leaves. To investigate antibacterial activity, representative periodontopathic bacteria were cultured in media with or without various amounts of macrocarpals; subsequently, the optical density at 660 nm was measured. Macrocarpal inhibition of P. gingivalis Arg- and Lys-specific proteinases was assessed by spectrofluorophotometric assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The effect of macrocarpals on P. gingivalis binding to saliva-coated hydroxyapatite beads was examined with (3)H-labeled P. gingivalis. RESULTS: Growth of P. gingivalis was inhibited more strongly than growth of Prevotella intermedia or Prevotella nigrescens and Treponema denticola by macrocarpals, however, Actinobacillus actinomycetemcomitans and Fusobacterium nucleatum were much more resistant. Macrocarpals inhibited P. gingivalis Arg- and Lys-specific proteinases in a dose-dependent manner. The enzyme-inhibitory effect of macrocarpals was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in which hemoglobin degradation by P. gingivalis proteinase was inhibited by macrocarpals. P. gingivalis binding to saliva-coated hydroxyapatite beads was also strongly attenuated by macrocarpals. CONCLUSIONS: Macrocarpals A, B and C demonstrated antibacterial activity against periodontopathic bacteria. Among tested bacteria, P. gingivalis displayed the greatest sensitivity to macrocarpals; additionally, its trypsin-like proteinase activity and binding to saliva-coated hydroxyapatite beads were inhibited by macrocarpals. These results indicate that eucalyptus leaf extracts may be useful as a potent preventative of periodontal disease.     

Moderate effect of enamel matrix derivative (Emdogain Gel) on Porphyromonas gingivalis growth in vitro

OBJECTIVE: The aim of this study was to analyse the antibacterial effects of Emdogain Gel or its constituents on the growth of the suspected periodontopathogen Porphyromonas gingivalis. STUDY DESIGN: The effects of the proteins of enamel matrix derivative (EMD), the commercial product Emdogain Gel or its vehicle propylene glycol alginate (PGA) (Straumann, Switzerland) on P. gingivalis growth were determined by two methods: broth dilution assay (BDA) and agar diffusion assay (ADA). RESULTS: BDA-Emdogain Gel inhibited moderately the growth of P. gingivalis, whereas EMD showed no effect. The PGA vehicle inhibited the growth completely. ADA-Emdogain Gel resulted in some inhibition in growth but was not significantly different from control. EMD revealed no zone of inhibition. PGA demonstrated statistically significant zones of inhibition. CONCLUSION: Emdogain Gel shows moderate antibacterial activities against P. gingivalis. These properties seem to be due to the PGA component of the gel preparation.