牙龈卟啉单胞菌牙龈素在牙周炎症过程中的作用及其机制

牙周炎是口腔临床实践中最为常见的疾病之一。牙龈卟啉单胞菌与牙周炎密切相关,牙龈卟啉单胞菌表面的牙龈素(gingipains)、菌毛和脂多糖被认为是其最重要的毒力因子,是近年来牙周炎机制研究的热点,引起研究者的广泛关注。本文结合近年来的研究进展,就牙龈卟啉单胞菌gingipains的结构、引起牙周炎症的机制和所涉及的信号通路作一综述。     

Lipoprotein modifications by gingipains of Porphyromonas gingivalis

Background and Objective

Several studies have shown an association between periodontitis and cardiovascular disease (CVD). Atherosclerosis is the major cause of CVD, and a key event in the development of atherosclerosis is accumulation of lipoproteins within the arterial wall. Bacteria are the primary etiologic agents in periodontitis and Porphyromonas gingivalis is the major pathogen in the disease. Several studies support a role of modified low‐density lipoprotein (LDL) in atherogenesis; however, the pathogenic stimuli that induce the changes and the mechanisms by which this occur are unknown. This study aims to identify alterations in plasma lipoproteins induced by the periodontopathic species of bacterium, P. gingivalis, in vitro.

Material and Methods

Plasma lipoproteins were isolated from whole blood treated with wild‐type and gingipain‐mutant (lacking either the Rgp‐ or Kgp gingipains) P. gingivalis by density/gradient‐ultracentrifugation and were studied using 2‐dimensional gel electrophoresis followed by matrix‐assisted laser desorption/ionization mass spectrometry. Porphyromonas gingivalis‐induced lipid peroxidation and antioxidant levels were measured by thiobarbituric acid‐reactive substances and antioxidant assay kits, respectively, and lumiaggregometry was used for measurement of reactive oxygen species (ROS) and aggregation.

Results

Porphyromonas gingivalis exerted substantial proteolytic effects on the lipoproteins. The Rgp gingipains were responsible for producing 2 apoE fragments, as well as 2 apoB‐100 fragments, in LDL, and the Kgp gingipain produced an unidentified fragment in high‐density lipoproteins. Porphyromonas gingivalis and its different gingipain variants induced ROS and consumed antioxidants. Both the Rgp and Kgp gingipains were involved in inducing lipid peroxidation.

Conclusion

Porphyromonas gingivalis has the potential to change the expression of lipoproteins in blood, which may represent a crucial link between periodontitis and CVD.     

Mixed infection of Porphyromonas gingivalis and Bacteroides forsythus in a murine abscess model: involvement of gingipains in a synergistic effect

Several microorganisms including Porphyromonas gingivalis and Bacteroides forsythus have been implicated to be etiologically important agents of periodontal disease. In this study, we determined the ability of combinations of periodontopathogenic microorganisms to cause tissue destruction in a murine abscess model. Although all bacterial combinations used in this study produced larger abscesses than did monoinfection of each bacterium, the combination of P. gingivalis and B.forsythus showed a synergistic effect on abscess formation. Since these two bacteria have been frequently found together in lesions of periodontitis, these results suggest the significance of their co-infection in the progression of periodontitis. P. gingivalis produces extracellular and cell-associated cysteine proteinases (gingipains) which appear to be involved in its virulence. The rgpA rgpB double and kgp mutants induced significantly smaller abscesses than the wild type. Moreover, the rgpA rgpB kgp triple (gingipain-null) mutant hardly showed lesion formation at all with the experimental conditions used in this study, indicating that these genes encoding gingipains are important for virulence of P. gingivalis. Mixed infection of these P. gingivalis mutants with B. forsythus showed an additive effect on abscess formation, indicating that the gingipains of P. gingivalis may play an important role in the pathological synergism between P. gingivalis and B. forsythus.     

The host cytokine response to Porphyromonas gingivalis is modified by gingipains

Background/aims:  Clinical studies indicate that primary proinflammatory cytokines, such as interleukin-1β (IL-1β) are elevated in the gingival crevice around teeth with periodontitis but the secondary cytokines and chemokines, IL-6 and IL-8, are not. The human gingival epithelial cells (HGECs) lining the gingival sulcus respond to perturbation by microbes of dental plaque by releasing a wide range of cytokines. Porphyromonas gingivalis , a putative periodontal pathogen, possesses numerous virulence factors some of which directly impact on the host response. In the present study, we sought to determine how P. gingivalis influences the inflammatory cytokine responses.
Methods:  HGECs were challenged with P. gingivalis and other putative periodontal pathogens, and the resultant production of IL-1β, IL-6, and IL-8 was assayed by enzyme-linked immunosorbent assay (ELISA). Culture supernatants and recombinant human cytokines were challenged with live P. gingivalis wild-type and gingipain-deficient strains and the resultant cytokine profile was assessed by ELISA and Western blot.
Results:  We show here that primary HGECs challenged with live P. gingivalis result in high levels of IL-1β but not the related secondary cytokines IL-6 and IL-8. We further demonstrate that cytokine response differences are the result of the action of P. gingivalis proteases, with lysine gingipain being the most effective.
Conclusion:  We conclude that P. gingivalis , through lysine gingipain, can subvert the protective host proinflammatory response by direct cytokine degradation. Changes in the crevicular cytokine profile have consequences in periodontal disease pathogenesis that should be considered in the development of diagnostic and therapeutic modalities.     

Inflammatory response of uric acid produced by Porphyromonas gingivalis gingipains

Uric acid is a potential metabolite that serves as a danger‐associated molecular pattern (DAMP) and induces inflammatory responses in sterile environments. Porphyromonas gingivalis is a keystone periodontopathogen, and its gingipain proteases play a critical role in the pathogenesis of periodontitis. In this study, we demonstrate that P. gingivalis gingipains play a role in THP‐1 macrophage uric acid production by increasing the expression and activity of xanthine oxidoreductase (XOR). Uric acid sodium salt induces caspase‐1 activation, cell death, and the expression of proinflammatory cytokines, including IL‐1α, IL‐6, and IL‐8, in the human keratinocyte HOK‐16B cell line. Our results suggest that gingipain‐induced uric acid can mediate inflammation in periodontal tissue cells.     

Pancreatic secretory trypsin inhibitor acts as an effective inhibitor of cysteine proteinases gingipains from Porphyromonas gingivalis

Background and Objective: Porphyromonas gingivalis has been implicated as the major pathogen of periodontitis in adults. This organism produces an array of virulence factors, of which cysteine proteinases, referred to as gingipains K and R, are believed to play a crucial role in pathogenicity. The aim of this study was to investigate the susceptibility of gingipains K and R to inhibition by a pancreatic secretory trypsin inhibitor. Material and Methods: Enzyme activities were measured spectrophotometrically using chromogenic turnover substrates. To estimate the value of the association constant (K_a), constant amounts of enzyme were reacted with increasing amounts of inhibitor to reach equilibrium. The K_a was calculated by fitting the experimental data to the given equation. Results: In this study it was shown that gingipains are susceptible to pancreatic Kazal‐type trypsin inhibitors (pancreatic secretory trypsin inhibitors). Bovine pancreatic secretory trypsin inhibitor, having an Arg residue at the P₁ position of the reactive site, specifically inhibited the activity of the Arg‐specific cysteine proteinase gingipain R, whereas porcine inhibitor, possessing a Lys residue at the P₁ position, exhibited activity only against the Lys‐specific cysteine proteinase gingipain K. The K_a values for the inhibitor–proteinase interaction were 1.6 × 10⁶ m ^?1 and 2.0 × 10⁴ m ^?1 for gingipain R and gingipain K, respectively. Conclusion: This finding is the first demonstration of the inhibitory potency of the Kazal‐type specific trypsin inhibitors against cysteine proteinases. These discoveries open new possibilities for the use of naturally occurring inhibitors, displaying activity across enzyme families, as a model in designing new molecules of therapeutic significance.     

Porphyromonas gingivalis gingipains cause G(1) arrest in osteoblastic/stromal cells

INTRODUCTION: The program for mammalian cell growth and division consists of four successive phases; G(1), S, G(2), and M. Porphyromonas gingivalis may manipulate the host cell cycle to benefit bacterial virulence expression, which likely causes the cell and tissue tropism observed in chronic periodontal infections. We examined P. gingivalis for its effects on cell-cycle modulation in mouse ST2 osteoblastic/stromal cells. METHODS: Synchronized ST2 cells were infected with P. gingivalis ATCC33277 (wild-type, WT), gingipain-mutants [KDP136 (DeltargpADeltargpBDeltakgp), KDP129 (DeltargpADeltargpB), and KDP133 (Deltakgp)], and a fimbria-deficient mutant (KDP150) for 24 h, then the cell cycle was evaluated using flow cytometry. Cell-cycle-related molecule expression was examined with a microarray, as well as with quantitative real-time polymerase chain reaction and Western blotting assays. RESULTS: Both the WT and KDP150 strains significantly inhibited cellular proliferation and arrested the cell cycle in the G(0)/G(1) phase, while the expression levels of the cell-cycle regulatory molecules cyclin D and cyclin E were also decreased. In contrast, KDP136 did not show any effects. G(1) arrest was also clearly induced by KDP129 and KDP133, with KDP129 being more effective. CONCLUSION: The present findings suggest that P. gingivalis gingipains reduce cyclin expression and cause early G(1) arrest, leading to the inhibition of cellular proliferation.     

Activity of anti‐Porphyromonas gingivalis egg yolk antibody against gingipains in vitro

Introduction: We investigated the effect of anti‐Porphyromonas gingivalis egg yolk antibody against gingipains [immunoglobulin Y (IgY)‐GP] on gingipain activity in vitro. Methods: IgY‐GP was isolated from the yolks of White Leghorn hens immunized with purified gingipains. Control antibody (IgY) was isolated from the yolks of non‐immunized hens. Gingipain activity was assessed according to the rate of enzymatic substrate hydrolysis. Human epithelial cells were cultured with or without gingipains and with gingipains pretreated with either IgY‐GP or IgY. Results: Hydrolytic activity decreased in the presence of IgY‐GP. Cells incubated with gingipains showed a dose‐dependent loss of adhesion activity. Pretreatment of gingipains with IgY‐GP was associated with strong inhibition of cell detachment, whereas pretreatment with IgY was not. Conclusion: Our findings suggest that IgY‐GP may be an effective immunotherapeutic agent in the treatment of periodontitis.     

Antibody responses to Porphyromonas gingivalis infection in a murine abscess model--involvement of gingipains and responses to re-infection

BACKGROUND: Porphyromonas gingivalis is one of the most important periodontopathogens. It produces cysteine proteinases named gingipains. We previously examined the effect of gingipains on abscess formation in a murine model. The rgpA rgpB double and kgp mutants induced smaller abscesses than the wild type. Moreover, the rgpA rgpB kgp triple (gingipain-null) mutant hardly showed lesion formation at all under the experimental conditions used, indicating that genes encoding gingipains are important for P. gingivalis virulence. OBJECTIVES: Here, we further report the humoral immune responses induced by P. gingivalis strains. METHODS: After the lesions were apparently cured, sera were collected from the mice and immunoglobulin G (IgG) responses against the whole cell antigens of wild-type P. gingivalis were measured. RESULTS: Wild-type strain was found to induce a strong antibody reaction. On the other hand, the rgpA rgpB kgp triple and kgp mutants induced significantly lower antibody responses compared to the wild type. Western blotting analysis confirmed the differences in antibody production. Next, these mice were re-infected with wild-type strain. Mice that were first infected with wild-type strain showed significantly smaller lesion formation than control mice that were first infected with medium only. On the other hand, mice that were first infected with mutant strains devoid of gingipain activities did not show resistance to re-infection and immunoglobulins directed against gingipains may be protective. CONCLUSIONS: These results suggest that gingipains play an important role in abscess formation in mice, and humoral immune responses seem to be partly responsible for the resistance to re-infection by P. gingivalis.     

Effect of chlorhexidine on the adherence properties of Porphyromonas gingivalis

Abstract Chlorhexidine is a bisbiguanide compound that possesses substantive and antibacterial properties. The aim of the present investigation was to evaluate the effect of chlorhexidine on the adherence of Porphyromonas gingivalis to epithelial cells and erythrocytes. Both pretreatment of bacterial cells with chlorhexidine (>20 μg/ml) and incorporation of chlorhexidine in an adherence assay markedly affected the ability of P. gingivalis to adhere to epithelial cells. Chlorhexidine also strongly inhibited hemagglotination by P. gingivalis. It is suggested that chlorhexidine binds to cells, altering the structural conformation of the outer membrane and reducing adherence. This ability of chlorhexidine to prevent bacterial adherence may represent an additional mechanism by which this antimicrobial agent exerts its beneficial clinical effect when used as an adjunct to periodontal therapy.     

The role of Porphyromonas gingivalis gingipains in platelet activation and innate immune modulation

Platelets are considered to have important functions in inflammatory processes and as actors in the innate immunity. Several studies have shown associations between cardiovascular disease and periodontitis, where the oral anaerobic pathogen Porphyromonas gingivalis has a prominent role in modulating the immune response. Porphyromonas gingivalis has been found in atherosclerotic plaques, indicating spreading of the pathogen via the circulation, with an ability to interact with and activate platelets via e.g. Toll‐like receptors (TLR) and protease‐activated receptors. We aimed to evaluate how the cysteine proteases, gingipains, of P. gingivalis affect platelets in terms of activation and chemokine secretion, and to further investigate the mechanisms of platelet–bacteria interaction. This study shows that primary features of platelet activation, i.e. changes in intracellular free calcium and aggregation, are affected by P. gingivalis and that arg‐gingipains are of great importance for the ability of the bacterium to activate platelets. The P. gingivalis induced a release of the chemokine RANTES, however, to a much lower extent compared with the TLR2/1‐agonist Pam₃CSK₄, which evoked a time‐dependent release of the chemokine. Interestingly, the TLR2/1‐evoked response was abolished by a following addition of viable P. gingivalis wild‐types and gingipain mutants, showing that both Rgp and Kgp cleave the secreted chemokine. We also demonstrate that Pam₃CSK₄‐stimulated platelets release migration inhibitory factor and plasminogen activator inhibitor‐1, and that also these responses were antagonized by P. gingivalis. These results supports immune‐modulatory activities of P. gingivalis and further clarify platelets as active players in innate immunity and in sensing bacterial infections, and as target cells in inflammatory reactions induced by P. gingivalis infection.     

壳聚糖和氯己定协同抗菌斑作用的实验研究

目的：评估壳聚糖和氯己定协同控制菌斑的作用效果。方法：用伴放线放线杆菌（Actinobacillus actinomycetemcomitans Aa）诱导Wistar大鼠牙周炎模型,24只大鼠模型随机分两组,实验组用0.1%氯己定和0.2%壳聚糖混合制剂口腔冲洗,对照组用0.1%氯己定单独冲洗,1周后对大鼠牙菌斑和颊黏膜处Aa取样菌落计数分析,并进行菌斑指数和牙龈指数评分。结果：实验组和对照组颊黏膜Aa菌落计数（×104cfu/ml）分别为0.00±0.12和0.02±0.09,无显著性差异（P〉0.05）,牙菌斑Aa菌落计数（×10^4cfu/ml）分别为0.19±0.08和0.47±0.14,实验组少于对照组,有显著性差异（P〈0.05）;两组牙菌斑Aa菌落计数均大于颊黏膜（P〈0.05）。实验组菌斑指数小于对照组（P〈0.05）,而牙龈指数无显著性差异（P〉0.05）。结论：壳聚糖和氯己定具有协同抗菌斑作用,联合使用比单独使用氯己定能取得更好的菌斑控制效果。     

Stimulation of growth of Porphyromonas gingivalis by cell extracts from Tannerella forsythia

OBJECTIVE: In order to examine if Tannerella forsythia stimulates the growth of Porphyromonas gingivalis, an in vitro study was performed. Background: P. gingivalis and T. forsythia are often isolated simultaneously from active periodontitis sites, indicating that these bacteria somewhat interact in the periodontal environment. We reported previously that mixed infection of P. gingivalis and T. forsythia synergistically induced lesion formation in a murine abscess model, and gingipains of P. gingivalis played an important role in this synergism. One of the possible mechanisms of this synergism is growth promotion by coinfection of the two bacteria. METHODS: Cell extracts of T. forsythia were added to the nutrition-decreased medium and the promotion of growth of P. gingivalis was examined. RESULTS: Sonicated extract of T. forsythia stimulated growth of P. gingivalis in nutrition-decreased medium in a dose-dependent manner. Proteins appeared to be the nature of growth-promoting factor, and the cell extract of T. forsythia had no stimulating effect on the growth of P. gingivalis strain devoid of gingipain activities. CONCLUSION: A product or a component of T. forsythia seemed to stimulate growth of P. gingivalis under nutrition-limited conditions. Gingipains are considered to play an important role in digestion or uptake of this growth-promoting factor. The interaction between T. forsythia and P. gingivalis in growth may be in part related with the synergistic virulence in a murine model.     

Serum antibodies against the hemoglobin-binding domain (HA2) of Porphyromonas gingivalis

BACKGROUND: The hemoglobin-binding domain (HA2) of the Porphyromonasgingivalis gingipains and hemagglutinins strongly binds hemoglobin and hemin and is thought to play a key role in acquisition of this essential metabolite by the microorganism. METHODS: In this report, we partially characterized human anti-HA2 humoral antibodies and their relationship to periodontal disease in an analysis of titer and function. RESULTS: Overall, serum anti-HA2 antibodies were relatively low and dominated by the immunoglobulin M (IgM) isotype. Pre-therapy titers had a direct association with periodontal health. Levels of P. gingivalis in the plaque were directly related to pre-therapy anti-HA2 IgG levels, and were an important covariant in a significant direct relationship between pre- and post-therapy anti-HA2 titers. Post-therapy anti-HA2 IgG antibody titers were directly related to the capacity of serum IgG fractions to neutralize hemoglobin binding by Lys-gingipain (Kgp). Further, lower levels of neutralizing activity post-therapy were directly related to severe periodontitis within the patient cohort. CONCLUSIONS: These data suggest that anti-HA2 IgG antibodies correspond directly with periodontal health, possibly through their ability to neutralize P. gingivalis hemoglobin capture. The data also suggest that inadvertent or therapeutic inoculation of P. gingivalis in the plaque may contribute to generation of neutralizing anti-HA2 IgG and improvement of periodontal prognosis.     

Inhibition of plaque formation and plaque acidogenicity by zinc and chlorhexidine combinations

Abstract – Zinc ions and chlorhexidine (CH) were found to exhibit a synergistic inhibitory effect on in vitro growth of S. sobrinus OMZ 176 and of S. sanguis 10556. A clinical mouthrinsing experiment was performed in a group of 10 volunteers to assess the plaque-inhibiting capacity of this combination. Sucrose enhanced plaque accumulations were assessed (Plaque Index, Silness & Löe) after 4 days of twice daily mouthrinses with 10 ml aqueous solutions of either 10.0 mM zinc or 0.55 mM CH, or with a combination of zinc ions and CH, during which period no mechanical toothcleaning was performed. The Zn–CH combination showed improved inhibition properties compared to the individual agents. The effects on plaque acidogenicity of 8.0 mM zinc, 0.44 mM CH, and of zinc and CH in combination were also assessed in a test panel of five volunteers. The Zn–CH combination inhibited acid production by dental plaque significantly (P< 0.05) more than the individual agents 1 h 30 min after a single rinse.     

Inhibition of orally produced volatile sulfur compounds by zinc,chlorhexidine or cetylpyridinium chloride--effect of concentration

Zinc ions, chlorhexidine (CHX) and cetylpyridinium chloride (CPC) are all known to inhibit production of volatile sulfur compounds (VSCs). The objective was to examine the anti-VSC dose-response effects of each of the above agents. Oral malodor was induced in 13 test subjects using the cysteine challenge method. The oral VSC response to rinses with 6 mm l-cysteine (pH 7.2) before and 1, 2 and 3 h after rinsing with zinc ions (Zn2+: 0.1, 0.3 and 1.0%), CHX and CPC (0.025 and 0.2%) was measured. Mouth air was analysed for VSC by gas chromatography (GC) according to current methodology. Zinc had a marked dose- and time-dependent anti-VSC effect. Zinc at 1% concentration had a somewhat unpleasant taste, whereas the lowest concentration was found acceptable. Chlorhexidine maintained a moderate anti-VSC effect over time. At 3 h, 0.2% CHX was the most effective agent but tasted relatively unpleasant. Cetylpyridinium at a concentration of 0.2% was only marginally more effective than 0.025% CHX over the 3 h, while 0.025% CPC had no better anti-VSC effect than water at both 2 h and 3 h. It was concluded that the three test agents demonstrated different anti-VSC kinetics. Although Zn had the best anti-VSC effect at 1 h, 0.2% CHX was at least as effective as 1% Zn at 3 h, most likely as a result of its unique substantivity.     

Suppression of inflammatory responses of human gingival fibroblasts by gingipains from Porphyromonas gingivalis

The interaction between human gingival fibroblasts (HGFs) and Porphyromonas gingivalis plays an important role in the development and progression of periodontitis. Porphyromonas gingivalis possesses several virulence factors, including cysteine proteases, the arginine‐specific (Rgp) and lysine‐specific (Kgp) gingipains. Studying the mechanisms that P. gingivalis, and its derived virulence, use to propagate and interact with host cells will increase the understanding of the development and progression of periodontitis. In this study, we aimed to elucidate how P. gingivalis influences the inflammatory events in HGFs regarding transforming growth factor‐β₁ (TGF‐β₁), CXCL8, secretory leucocyte protease inhibitor (SLPI), c‐Jun and indoleamine 2,3‐dioxygenase (IDO). HGFs were inoculated for 6 and 24 h with the wild‐type strains ATCC 33277 and W50, two gingipain‐mutants of W50 and heat‐killed ATCC 33277. The P. gingivalis regulated CXCL8 and TGF‐β₁ in HGFs, and the kgp mutant gave significantly higher immune response with increased CXCL8 (P < 0.001) and low levels of TGF‐β₁. We show that HGFs express and secrete SLPI, which was significantly suppressed by P. gingivalis (P < 0.05). This suggests that by antagonizing SLPI, P. gingivalis contributes to the tissue destruction associated with periodontitis. Furthermore, we found that P. gingivalis inhibits the expression of the antimicrobial IDO, as well as upregulating c‐Jun (P < 0.05). In conclusion, P. gingivalis both triggers and suppresses the immune response in HGFs. Consequently, we suggest that the pathogenic effects of P. gingivalis, and especially the activity of the gingipains on the inflammatory and immune response of HGFs, are crucial in 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.     

The effect of triclosan, stannous fluoride and chlorhexidine products on: (II) Salivary bacterial counts

A previous study demonstrated that triclosan and stannous fluoride containing oral hygiene products reduced plaque regrowth compared to saline but were not more effective than a conventional commercial fluoride/anionic detergent toothpaste. To further understand these results, this study measured the persistance of antimicrobial activity of the same products by recording the duration of salivary bacterial count reductions following a single exposure to each product. Comparison was also made with a chlorhexidine rinse as the positive control. From a panel of 16 volunteers, in an 8-cell randomised cross-over designed study, salivary bacterial counts were recorded at baseline and to 420 min. All test and control products were significantly more effective than saline and significantly less effective than chlorhexidine at suppressing bacterial counts. Unlike chlorhexidine, evidence of bacterial recovery was apparent after the 30-min sampling time. There were essentially no significant differences between the test and control products, although the stannous fluoride toothpaste performed marginally better than other products. The findings are consistent with the plaque regrowth results previously obtained and again demonstrate to date that it is difficult to surpass the antimicrobial and plaque inhibitory properties of conventional commercially available toothpastes by the addition of antimicrobial agents such as triclosan and metal salts.