RESUMEN
The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded ß-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.
Asunto(s)
Aminoaciltransferasas/química , Periodontitis/microbiología , Porphyromonas gingivalis/enzimología , Prevotella intermedia/enzimología , Aminoaciltransferasas/antagonistas & inhibidores , Aminoaciltransferasas/genética , Aminoaciltransferasas/ultraestructura , Dominio Catalítico/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Periodontitis/tratamiento farmacológico , Periodontitis/genética , Porphyromonas gingivalis/patogenicidad , Prevotella intermedia/patogenicidad , Estructura Terciaria de Proteína/efectos de los fármacos , Ácido Pirrolidona Carboxílico/química , Ácido Pirrolidona Carboxílico/metabolismo , Tannerella forsythia/enzimología , Tannerella forsythia/patogenicidadRESUMEN
BACKGROUND: Coffee is a major dietary source of polyphenols. Previous research found that coffee had a protective effect on periodontal disease. In this study, we aimed to investigate whether coffee extract and its primary phenolic acid, chlorogenic acid, affect the growth and protease activity of a periodontopathogen Porphyromonas gingivalis (P. gingivalis). METHODS: Coffee extract and chlorogenic acid were prepared by a two-fold serial dilution. The turbid metric test and plate count method were used to examine the inhibitory effects of chlorogenic acid on P. gingivalis. The time-kill assay was used to measure changes in the viability of P. gingivalis after exposure to chlorogenic acid for 0-24 h. The protease activity of P. gingivalis was analyzed using the optical density of a chromogenic substrate. RESULTS: As a result, the minimum inhibitory concentration (MIC) of chlorogenic acid was 4 mg/mL, and the minimum bactericidal concentration was 16 mg/mL. Chlorogenic acid at concentrations above MIC resulted in a longer-lasting inhibitory effect on P. gingivalis viability and significantly reduced associated protease activity. The coffee extract showed antibacterial activity as observed by the disk diffusion test, whereas these inhibitory effects were not affected by different roast degrees of coffee. CONCLUSIONS: Collectively, our novel findings indicate that chlorogenic acid not only has antimicrobial activity but also reduced the protease activity of P. gingivalis. In addition, coffee extract inhibits the proliferation of P. gingivalis, which may partly be attributed to the effect of chlorogenic acid.
Asunto(s)
Antibacterianos/farmacología , Infecciones por Bacteroidaceae/prevención & control , Ácido Clorogénico/farmacología , Coffea/química , Periodontitis/tratamiento farmacológico , Extractos Vegetales/farmacología , Porphyromonas gingivalis/efectos de los fármacos , Antibacterianos/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Infecciones por Bacteroidaceae/microbiología , Ácido Clorogénico/aislamiento & purificación , Pruebas Antimicrobianas de Difusión por Disco , Viabilidad Microbiana/efectos de los fármacos , Péptido Hidrolasas/metabolismo , Periodontitis/microbiología , Extractos Vegetales/aislamiento & purificación , Porphyromonas gingivalis/enzimología , Porphyromonas gingivalis/patogenicidad , Semillas/química , Factores de Tiempo , Factores de Virulencia/metabolismoRESUMEN
Antimicrobial resistance is a global public threat and raises the need for development of new antibiotics with a novel mode of action. The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to a new class of serine peptidases, family S46. Because S46 peptidases are not found in mammals, these enzymes are attractive targets for novel antibiotics. However, potent and selective inhibitors of these peptidases have not been developed to date. In this study, a high-resolution crystal structure analysis of PgDPP11 using a space-grown crystal enabled us to identify the binding of citrate ion, which could be regarded as a lead fragment mimicking the binding of a substrate peptide with acidic amino acids, in the S1 subsite. The citrate-based pharmacophore was utilized for in silico inhibitor screening. The screening resulted in an active compound SH-5, the first nonpeptidyl inhibitor of S46 peptidases. SH-5 and a lipophilic analog of SH-5 showed a dose-dependent inhibitory effect against the growth of P. gingivalis. The binding mode of SH-5 was confirmed by crystal structure analysis. Thus, these compounds could be lead structures for the development of selective inhibitors of PgDPP11.
Asunto(s)
Benzoatos/farmacología , Ácido Cítrico/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/química , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Porphyromonas gingivalis/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Benzoatos/química , Sitios de Unión , Dominio Catalítico , Simulación por Computador , Cristalografía por Rayos X , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , Fosfatos de Inositol , Modelos Moleculares , Conformación ProteicaRESUMEN
The gingival epithelium, a stratified squamous tissue that acts as an interface between the external environment and the underlying connective tissue, plays an active role in maintaining periodontal health. The aim of the present study was to investigate the ability of green tea catechins to enhance gingival epithelial barrier function and protect against the disruption of epithelial integrity induced by Porphyromonas gingivalis. Both the green tea extract and epigallocatechin-3-gallate (EGCG) dose- and time-dependently increased the transepithelial electrical resistance (TER) of a gingival keratinocyte model and decreased the permeability of the cell monolayer to fluorescein isothyocyanate-conjugated 4.4-kDa dextran. This was associated with the increased expression of zonula occludens-1 (ZO-1) and occludin, two tight junction proteins. Treating the gingival keratinocyte monolayer with P. gingivalis caused a reduction in TER and affected the distribution of ZO-1 and occludin, allowing P. gingivalis to translocate through the cell monolayer. These deleterious effects mediated by P. gingivalis were abolished by the green tea extract and EGCG. This protection may be in part related to the ability of tea catechins to inhibit the protease activities of P. gingivalis. Given the above properties, green tea catechins may represent promising preventive and therapeutic molecules against periodontal disease.
Asunto(s)
Antibacterianos/farmacología , Catequina/análogos & derivados , Queratinocitos/efectos de los fármacos , Porphyromonas gingivalis/efectos de los fármacos , Té/química , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Antibacterianos/aislamiento & purificación , Traslocación Bacteriana , Catequina/aislamiento & purificación , Catequina/farmacología , Línea Celular Transformada , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Dextranos/metabolismo , Impedancia Eléctrica , Pruebas de Enzimas , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Cisteína-Endopeptidasas Gingipaínas , Encía/efectos de los fármacos , Encía/metabolismo , Encía/microbiología , Humanos , Queratinocitos/metabolismo , Queratinocitos/microbiología , Modelos Biológicos , Ocludina/genética , Ocludina/metabolismo , Porphyromonas gingivalis/enzimología , Porphyromonas gingivalis/crecimiento & desarrollo , Porphyromonas gingivalis/patogenicidad , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
BACKGROUND: Currently, in the field of rheumatology, there is much attention given towards the possible causality between periodontitis and rheumatoid arthritis (RA), specifically regarding the role of Porphyromonas gingivalis (Pg). This bacterium is unique, having a citrullinating enzyme. Antibodies against citrullinated proteins are rather specific for RA. METHODS: Because causality is ultimately tested in longitudinal cohort studies which currently do not exist for periodontitis and RA, this commentary applied Bradford Hill criteria on the existing literature to assess causality as the most likely interpretation of this association. CONCLUSIONS: From an epidemiologic point of view, patients with RA have a higher incidence of periodontal disease than individuals without RA. In addition, there is a dose-response pattern in the association between the severity of periodontitis and RA disease activity. There are indications that periodontitis precedes RA, but there is no evidence yet available to show that Pg plays a direct role in this temporal relationship. The role of the unique characteristic of citrullination by Pg remains unexplained. However, in animal models, citrullination by Pg plays a distinct role in the development and aggravation of experimental arthritis. Although the role of Pg in RA remains speculative, a causative role for periodontitis as a chronic inflammatory disease caused by infectious agents in RA seems biologically plausible.
Asunto(s)
Artritis Reumatoide/microbiología , Periodontitis/microbiología , Porphyromonas gingivalis/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Autoanticuerpos/inmunología , Citrulina/inmunología , Humanos , Hidrolasas/inmunología , Inmunidad Celular/inmunología , Neutrófilos/inmunología , Periodontitis/inmunología , Porphyromonas gingivalis/enzimología , Desiminasas de la Arginina Proteica , Ligando RANK/inmunología , Factor Reumatoide/inmunología , Factores de Riesgo , Especificidad de la Especie , Células Th17/inmunologíaRESUMEN
BACKGROUND: In the present study, we explored the effect of the ethanol extract of Osmanthus fragrans (EOF) on the growth and collagenase activity of Porphyromonas gingivalis (P. gingivalis). We also investigated the capacity of EOF to attenuate P. gingivalis lipopolysaccharide (LPS)-induced inflammatory responses and the possible signalling pathway. METHODS: EOF was obtained by soaking the O. fragrans powder in the ethanol and concentrating the extracts under reduced pressure. Microplate dilution assays were used to determine the effect of EOF on P. gingivalis growth. Collagenase inhibition was detected using fluorometric and colorimetric assays. The effects of EOF on the production of the cytokines interleukin-6 (IL-6) and IL-8 were assessed using enzyme-linked immunosorbent assays (ELISAs). The oxidative stress biomarkers were assayed using commercial kits. The effects of EOF on the expression of cytoprotective enzymes and nucleoprotein nuclear factor erythroid 2-related factor (Nrf2) were tested by Western blot analysis. RESULTS: EOF significantly inhibited the growth of P. gingivalis, especially in the iron-limited culture medium. The inhibitory effect of EOF on P. gingivalis collagenase activity was time- and concentration-dependent. The P. gingivalis LPS-stimulated production of IL-6 and IL-8 was attenuated by EOF. LPS significantly induced the production of nitric oxide (NO) and malondialdehyde (MDA), and decreased the expression of superoxide dismutase (SOD) while pretreatment with EOF alleviated these effects. The presence of EOF markedly upregulated the expression levels of the cytoprotective enzymes and nucleoprotein Nrf2. CONCLUSION: This study suggests that the potent Nrf2 activation capacity of O. fragrans may be useful in the adjunctive treatment of periodontal disease.
Asunto(s)
Lipopolisacáridos/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Oleaceae/química , Extractos Vegetales/farmacología , Porphyromonas gingivalis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Adolescente , Adulto , Western Blotting , Colagenasas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Malondialdehído/metabolismo , Óxido Nítrico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Porphyromonas gingivalis/enzimología , Superóxido Dismutasa/metabolismoRESUMEN
Porphyromonas gingivalis (P. gingivalis) expres-ses the enzyme peptidylarginine deiminase (PPAD), which has a strong preference for C-terminal arginines. Due to the combined activity of PPAD and Arg-specific gingipains, P. gingivalis on the cell surface is highly citrullinated. To investigate the contribution of PPAD to the interaction of P. gingivalis with primary human gingival fibroblasts (PHGF) and P. gingivalis-induced synthesis of prostaglandin E2 (PGE2 ), PHGF were infected with wild-type P. gingivalis ATCC 33277, an isogenic PPAD-knockout strain (∆ppad) or a mutated strain (C351A) expressing an inactive enzyme in which the catalytic cysteine has been mutated to alanine (PPAD(C351A) ). Cells were infected in medium containing the mutants alone or in medium supplemented with purified, active PPAD. PHGF infection was assessed by colony-forming assay, microscopic analysis and flow cytometry. Expression of cyclo-oxygenase 2 (COX-2) and microsomal PGE synthase-1 (mPGES-1), key factors in the prostaglandin synthesis pathway, was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), while PGE2 synthesis was evaluated by enzyme immunoassay. PHGF were infected more efficiently by wild-type P. gingivalis than by the ∆ppad strain, which correlated with strong induction of COX-2 and mPGES-1 expression by wild-type P. gingivalis, but not by the PPAD activity-null mutant strains (Δppad and C351A). The impaired ability of the Δppad strain to adhere to and/or invade PHGF and both Δppad and C351A to stimulate the PGE2 -synthesis pathway was fully restored by the addition of purified PPAD. The latter effect was strongly inhibited by aspirin. Collectively, our results implicate PPAD activity, but not PPAD itself, as an important factor for gingival fibroblast infection and activation of PGE2 synthesis, the latter of which may strongly contribute to bone resorption and eventual tooth loss.
Asunto(s)
Dinoprostona/biosíntesis , Fibroblastos/microbiología , Encía/microbiología , Hidrolasas/metabolismo , Porphyromonas gingivalis/patogenicidad , Adhesinas Bacterianas/metabolismo , Alanina/genética , Aspirina/farmacología , Adhesión Bacteriana , Células Cultivadas , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Cisteína/genética , Cisteína Endopeptidasas/metabolismo , Dinoprostona/metabolismo , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Cisteína-Endopeptidasas Gingipaínas , Encía/citología , Humanos , Inmunoensayo , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Mutación , Porphyromonas gingivalis/enzimología , Porphyromonas gingivalis/genética , Prostaglandina-E Sintasas , Desiminasas de la Arginina Proteica , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de SeñalRESUMEN
The periodontal pathogen Porphyromonas gingivalis produces a unique class of cysteine proteinases termed gingipains that comprises Arg-gingipain (Rgp) and Lys-gingipain (Kgp). Growing evidence indicates that these 2 types of gingipains synergistically contribute to the entire virulence of the organism and increase the risk of periodontal disease (PD) by disrupting the host immune system and degrading the host tissue and plasma proteins. Therefore, a dual inhibitor of both gingipains would have attractive clinical potential for PD therapy. In this study, a novel, potent, dual inhibitor of Rgp and Kgp was developed through structure-based drug design, and its biological potency was evaluated in vitro and in vivo. This inhibitor had low nanomolar inhibitory potency (Ki=40 nM for Rgp, Ki=0.27 nM for Kgp) and good selectivity for host proteases and exhibited potent antibacterial activity against P. gingivalis by abrogating its manifold pathophysiological functions. The therapeutic potential of this inhibitor in vivo was also verified by suppressing the vascular permeability that was enhanced in guinea pigs by the organism and the gingival inflammation in beagle dog PD models. These findings suggest that a dual inhibitor of Rgp and Kgp would exhibit noteworthy anti-inflammatory activity in the treatment of PD.
Asunto(s)
Adhesinas Bacterianas/efectos de los fármacos , Cisteína Endopeptidasas/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/uso terapéutico , Oligopéptidos/uso terapéutico , Periodontitis/tratamiento farmacológico , Porphyromonas gingivalis/enzimología , Animales , Permeabilidad Capilar/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Medios de Cultivo Condicionados/toxicidad , Inhibidores de Cisteína Proteinasa/farmacología , Citocinas/metabolismo , Progresión de la Enfermedad , Perros , Evaluación Preclínica de Medicamentos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Cisteína-Endopeptidasas Gingipaínas , Cobayas , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Periodontitis/microbiología , Porphyromonas gingivalis/patogenicidad , Proteolisis , Especificidad por Sustrato , VirulenciaRESUMEN
Rheumatoid arthritis and periodontitis are two prevalent chronic inflammatory diseases in humans and are associated with each other both clinically and epidemiologically. Recent findings suggest a causative link between periodontal infection and rheumatoid arthritis via bacteria-dependent induction of a pathogenic autoimmune response to citrullinated epitopes. Here we showed that infection with viable periodontal pathogen Porphyromonas gingivalis strain W83 exacerbated collagen-induced arthritis (CIA) in a mouse model, as manifested by earlier onset, accelerated progression and enhanced severity of the disease, including significantly increased bone and cartilage destruction. The ability of P. gingivalis to augment CIA was dependent on the expression of a unique P. gingivalis peptidylarginine deiminase (PPAD), which converts arginine residues in proteins to citrulline. Infection with wild type P. gingivalis was responsible for significantly increased levels of autoantibodies to collagen type II and citrullinated epitopes as a PPAD-null mutant did not elicit similar host response. High level of citrullinated proteins was also detected at the site of infection with wild-type P. gingivalis. Together, these results suggest bacterial PAD as the mechanistic link between P. gingivalis periodontal infection and rheumatoid arthritis.
Asunto(s)
Artritis/microbiología , Proteínas Bacterianas/metabolismo , Infecciones por Bacteroidaceae/microbiología , Modelos Animales de Enfermedad , Hidrolasas/metabolismo , Periodontitis/microbiología , Porphyromonas gingivalis/enzimología , Animales , Artritis/inmunología , Artritis/patología , Artritis/fisiopatología , Autoanticuerpos/análisis , Proteínas Bacterianas/genética , Infecciones por Bacteroidaceae/inmunología , Infecciones por Bacteroidaceae/patología , Infecciones por Bacteroidaceae/fisiopatología , Resorción Ósea/etiología , Citrulina/metabolismo , Progresión de la Enfermedad , Eliminación de Gen , Hidrolasas/genética , Articulaciones/inmunología , Articulaciones/metabolismo , Articulaciones/microbiología , Articulaciones/patología , Masculino , Ratones Endogámicos DBA , Infiltración Neutrófila , Periodontitis/inmunología , Periodontitis/metabolismo , Periodontitis/patología , Porphyromonas gingivalis/inmunología , Porphyromonas gingivalis/aislamiento & purificación , Prevotella intermedia/enzimología , Prevotella intermedia/inmunología , Prevotella intermedia/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Desiminasas de la Arginina Proteica , Índice de Severidad de la EnfermedadRESUMEN
Porphyromonas gingivalis is a key etiologic agent of chronic periodontitis. This Gram-negative anaerobic bacterium produces several virulence factors and can induce a host inflammatory response that contributes to periodontal disease. In the present study, we investigated green tea, white tea, oolong tea, and black tea extracts with a high polyphenol content for their effects on (i) the growth and adherence of P. gingivalis, (ii) the activity of host and bacterial proteases, and (iii) cytokine secretion by oral epithelial cells. All the tea extracts inhibited the growth of P. gingivalis (minimal inhibitory concentrations ranging from 200 to 500 µg/mL; minimal bactericidal concentrations=500 µg/mL). In addition, they dose dependently reduced the adherence of P. gingivalis to oral epithelial cells. Tea extracts also inhibited the catalytic activity of matrix metalloproteinase (MMP)-9, neutrophil elastase, and P. gingivalis collagenase. Lastly, the tea extracts dose dependently inhibited the secretion of interleukin (IL)-6, IL-8, and chemokine (C-C motif) ligand 5 (CCL-5) by P. gingivalis-stimulated oral epithelial cells. No marked differences in the various effects were observed among the four tea extracts. Extracts from green tea, white tea, oolong tea, and black tea show promise for controlling periodontal disease by their capacity to interfere with P. gingivalis growth and virulence properties, host destructive enzymes, and inflammatory mediator secretion. Such extracts may be incorporated to oral hygiene products or locally delivered into diseased periodontal sites.
Asunto(s)
Antibacterianos/administración & dosificación , Antiinflamatorios/administración & dosificación , Adhesión Bacteriana/efectos de los fármacos , Camellia sinensis/química , Enfermedades Periodontales/tratamiento farmacológico , Extractos Vegetales/administración & dosificación , Porphyromonas gingivalis/efectos de los fármacos , Inhibidores de Proteasas/administración & dosificación , Antibacterianos/análisis , Antiinflamatorios/análisis , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Línea Celular , Citocinas/genética , Citocinas/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Humanos , Péptido Hidrolasas/metabolismo , Enfermedades Periodontales/enzimología , Enfermedades Periodontales/metabolismo , Enfermedades Periodontales/microbiología , Extractos Vegetales/análisis , Porphyromonas gingivalis/enzimología , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/fisiología , Inhibidores de Proteasas/análisisRESUMEN
Arg-specific gingipain (Rgp) is a major pathogenic determinant of Porphyromonas gingivalis which is a major pathogen in periodontal disease. We prepared protein extracts with Rgp-inhibitory activity from polished rice (Oryza sativa) and evaluated the effects of these extracts on the growth and pathogenicity of P. gingivalis. The extracts inhibited the proteolytic degradation of human proteins by P. gingivalis proteinases, and repressed the growth and homotypic biofilm formation of P. gingivalis. The disruption of adhesion of epithelial cells by P. gingivalis was also restricted by the rice protein extracts. Our results suggested that the rice protein extracts suppressed the pathogenicity and growth of P. gingivalis by inhibiting the bacterial proteinase activities, implying that the Rgp-inhibitory proteins prepared from rice may be potentially valuable as nutraceutical agents for preventing periodontal diseases.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Oryza/química , Extractos Vegetales/farmacología , Proteínas de Plantas/farmacología , Porphyromonas gingivalis/efectos de los fármacos , Infecciones por Bacteroidaceae/prevención & control , Biopelículas/crecimiento & desarrollo , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidores de Cisteína Proteinasa/aislamiento & purificación , Pruebas de Enzimas , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Cisteína-Endopeptidasas Gingipaínas , Humanos , Extractos Vegetales/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Porphyromonas gingivalis/enzimología , Porphyromonas gingivalis/crecimiento & desarrollo , Proteolisis/efectos de los fármacosRESUMEN
BACKGROUND AND OBJECTIVES: Host- and bacteria-derived proteinases are considered to play critical roles in periodontitis progression. This study investigated the ability of a blackcurrant extract and its major anthocyanins (cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside and delphinidin-3-O-rutinoside) to inhibit the activity of matrix metalloproteinases (MMPs), neutrophil elastase and periodontopathogen (Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola) proteinases. MATERIAL AND METHODS: Enzyme inhibition was detected using fluorometric and colorimetric assays after incubating blackcurrant extract and its major anthocyanins (at concentrations of 6.25, 12.5, 25 and 50 µg/mL) with MMPs, elastase or bacterial proteinases, along with their specific substrates. Substrate degradation was recorded every hour for up to 4 h. RESULTS: The blackcurrant extract (50 µg/mL) inhibited all proteinases tested. MMP-1 and MMP-9 were significantly inhibited by pure anthocyanins at concentrations ranging from 6.25 to 50 µg/mL. Elastase activity was inhibited by cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside in the range of 6.25-50 µg/mL and by delphinidin-3-O-rutinoside at 50 µg/mL. P. gingivalis, T. forsythia and T. denticola proteinases were also significantly inhibited by pure anthocyanins. In all cases, enzyme inhibition was time-dependent. CONCLUSION: Our study showed that a blackcurrant extract and its major anthocyanins were able to inhibit the activity of host- and bacteria-derived proteinases. This suggests that such natural compounds may represent promising agents for use in adjunctive treatments for periodontitis.
Asunto(s)
Antocianinas/farmacología , Extractos Vegetales/farmacología , Inhibidores de Proteasas/farmacología , Ribes/química , Proteínas Bacterianas/antagonistas & inhibidores , Bacteroides/enzimología , Glucósidos/farmacología , Humanos , Elastasa de Leucocito/efectos adversos , Metaloproteinasa 1 de la Matriz/efectos adversos , Inhibidores de la Metaloproteinasa de la Matriz , Porphyromonas gingivalis/enzimología , Proteolisis , Treponema denticola/enzimologíaRESUMEN
Periodontitis is a common chronic inflammatory disorder of bacterial origin, which affects the tooth-supporting tissues. A wide range of evidences suggests that Porphyromonas gingivalis plays a key role in the initiation and progression of chronic periodontitis. This Gram-negative anaerobic bacterium produces several types of proteolytic enzymes, including gingipains, collagenases, and a dipeptidyl aminopeptidase IV. Although these enzymes have physiological functions for P. gingivalis, they have been suggested to play multiple roles in the pathogenic process of periodontitis. Indeed, P. gingivalis proteases hydrolyze a variety of serum and tissue proteins thus contributing to neutralize the immune defense system and to cause tissue destruction. Considering the key roles that P. gingivalis proteases may play in the pathogenesis of periodontitis, inhibitors of these enzymes are considered potentially new therapeutics agents. In recent years, several groups have identified natural plant-derived inhibitors effective on P. gingivalis proteases. More specifically, polyphenols isolated from cranberry and green tea were found to inhibit several proteases produced by P. gingivalis. This paper will discuss the pathological roles of P. gingivalis proteases and review the scientific literature for bioactive plant-derived compounds endowed with a capacity to inhibit these enzymes.
Asunto(s)
Péptido Hidrolasas/metabolismo , Porphyromonas gingivalis/enzimología , Inhibidores de Proteasas/farmacología , Factores de Virulencia/antagonistas & inhibidores , Factores de Virulencia/metabolismo , Animales , Humanos , Terapia Molecular Dirigida , Extractos Vegetales/química , Extractos Vegetales/farmacología , Porphyromonas gingivalis/patogenicidad , Inhibidores de Proteasas/uso terapéutico , VirulenciaRESUMEN
Host and bacterial proteases play a vital role in periodontitis. Inhibitors of these proteases are necessary for control of this disease. The purpose of this study was to evaluate the effect of lanthanides on proteins from Porphyromonas gingivalis, a major pathogen in periodontitis. Benzoyl-L-Arg-p-nitroanilide (BAPNA); H-Gly-Pro-pNA x HCl and gelatin were used to evaluate the activity of P. gingivalis proteins in the presence of lanthanides. Proteins extracted from cell surfaces and culture media of P. gingivalis were assessed for activity in the presence of different lanthanides by BAPNA assay. Only gadolinium chloride was used for H-Gly-Pro-pNA x HCl assay and gelatin-zymography. Concentration-dependent reduction of absorbance was observed in the presence of lanthanides with BAPNA and a similar observation was made with gadolinium chloride using H-Gly-Pro-pNa. Collagenolytic activity in cell surface extracts and culture media-precipitated proteins was absent in the presence of gadolinium chloride. These results suggest that the lanthanide gadolinium can be a potential inhibitor of P. gingivalis proteases.
Asunto(s)
Proteínas Bacterianas/efectos de los fármacos , Gadolinio/farmacología , Péptido Hidrolasas/efectos de los fármacos , Porphyromonas gingivalis/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Proteínas Bacterianas/metabolismo , Colagenasas/efectos de los fármacos , Colagenasas/metabolismo , Medios de Cultivo Condicionados/farmacología , Evaluación Preclínica de Medicamentos , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Péptido Hidrolasas/metabolismo , Porphyromonas gingivalis/enzimología , Serina Endopeptidasas/efectos de los fármacos , Serina Endopeptidasas/metabolismoRESUMEN
Porphyromonas gingivalis is known to be a major etiologic agent in the onset and progression of chronic periodontitis. Among various virulence factors that this bacterium produces, Arg- and Lys-specific cysteine proteinases (gingipains) are believed to be major determinants of the pathogenicity of P. gingivalis. Here, we report on our finding that there are inhibitors of these cysteine proteinases in a rice protein fraction. Comprehensive affinity chromatography and MS analyses resulted in the identification of 17 Arg-gingipain (Rgp)-interacting proteins in the rice endosperm. Of these, four proteins (i.e., a 26 kDa globulin, a plant lipid transfer/trypsin-alpha amylase inhibitor, the RA17 seed allergen, and an alpha amylase/trypsin inhibitor) were estimated to account for 90% of the Rgp inhibitory activity in the rice protein fraction, using a two-dimensional gel system of double-layer reverse zymography. In addition, a synthetic peptide derived from an Rgp-interacting protein, cyanate hydratase, could inhibit the growth of P. gingivalis and showed inhibitory activity against both the Arg- and Lys-gingipains. These results suggest that these rice proteins may be useful as nutraceutical ingredients for the prevention and management of periodontal diseases.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Inhibidores Enzimáticos/metabolismo , Oryza/química , Proteínas de Plantas/metabolismo , Porphyromonas gingivalis/enzimología , Proteómica/métodos , Adhesinas Bacterianas/genética , Secuencia de Aminoácidos , Cisteína Endopeptidasas/genética , Suplementos Dietéticos , Electroforesis en Gel Bidimensional , Inhibidores Enzimáticos/química , Cisteína-Endopeptidasas Gingipaínas , Humanos , Focalización Isoeléctrica , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Enfermedades Periodontales/microbiología , Proteínas de Plantas/química , Proteínas de Plantas/genética , Porphyromonas gingivalis/patogenicidadRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Medios de Cultivo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Endopeptidasas/metabolismo , Porphyromonas gingivalis/crecimiento & desarrollo , Adhesinas Bacterianas/metabolismo , Aminopeptidasas , Animales , Proteínas Bacterianas/genética , Bovinos , Cisteína Endopeptidasas/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Endopeptidasas/genética , Técnicas de Inactivación de Genes , Cisteína-Endopeptidasas Gingipaínas , Hemina , Inmunoglobulina G , Mutación , Péptidos/metabolismo , Porphyromonas gingivalis/enzimología , Proteínas Recombinantes/metabolismo , Albúmina Sérica Bovina , Vitamina K 3RESUMEN
BACKGROUND/AIMS: Gingipains, proteolytic enzymes produced by the periodontal pathogen Porphyromonas gingivalis, are regarded as virulence factors in the pathogenesis of periodontitis. Inhibition of gingipain activity therefore may have therapeutic potential, and it has been suggested that chlorhexidine may inhibit the activities of these enzymes. The purposes of the present study were to examine systematically the inhibitory effects of chlorhexidine on three purified gingipains and to determine the effect of Zn(II) on chlorhexidine inhibition. METHODS: The activities of lys-gingipain (Kgp) and two forms of arg-gingipain (RgpB and HRgpA) were measured in the presence of varying concentrations of chlorhexidine and with chlorhexidine supplemented with Zn(II). Inhibition constants (K(i)'s) were determined for chlorhexidine alone and in the presence of Zn(II). Fractional inhibitory constant indices were calculated to assess the synergy of the chlorhexidine-Zn(II) inhibition. RESULTS: RgpB, HRgpA, and Kgp were all inhibited by chlorhexidine with K(i)'s in the micromolar range. For RgpB and HRgpA, the inhibitory effects of chlorhexidine were enhanced 3-30-fold by Zn(II). The chlorhexidine-Zn(II) interaction was synergistic for inhibition of HRgpA and RgpB. For Kgp, the effect of Zn(II) on chlorhexidine inhibition was antagonistic. CONCLUSIONS: Chlorhexidine is an effective inhibitor of gingipains, and the inhibition of R-gingipains is enhanced by Zn(II). A mixture of chlorhexidine and Zn(II) may be useful as an adjunct in the treatment of periodontitis and in the post-treatment maintenance of periodontitis patients.
Asunto(s)
Adhesinas Bacterianas/efectos de los fármacos , Antiinfecciosos Locales/farmacología , Clorhexidina/farmacología , Cisteína Endopeptidasas/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Porphyromonas gingivalis/enzimología , Zinc/farmacología , Antiinfecciosos Locales/administración & dosificación , Clorhexidina/administración & dosificación , Clorhexidina/análogos & derivados , Inhibidores de Cisteína Proteinasa/administración & dosificación , Antagonismo de Drogas , Sinergismo Farmacológico , Cisteína-Endopeptidasas Gingipaínas , Humanos , Zinc/administración & dosificación , Acetato de Zinc/administración & dosificación , Acetato de Zinc/farmacologíaRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Eucalyptus , Extractos Vegetales/farmacología , Porphyromonas gingivalis/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Adhesión Bacteriana/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Endopeptidasas/metabolismo , Hojas de la Planta , Porphyromonas gingivalis/enzimología , Porphyromonas gingivalis/patogenicidad , Sesquiterpenos/farmacología , Virulencia/efectos de los fármacosRESUMEN
Garlic (Allium sativum) has long been known to have antibacterial, antifungal and antiviral properties but there are few data on its effects against oral bacterial species particularly putative periodontal pathogens or their enzymes. Filter sterilised, aqueous extract of garlic was tested for ability to inhibit the growth of a range of oral species and to inhibit the trypsin-like and total protease activity Porphyromonas gingivalis. The garlic extract (57.1% (w/v), containing 220 microg/ml allicin) inhibited the growth and killed most of the organisms tested. In general, the minimal inhibitory and minimum bactericidal concentrations for the Gram-negative strains (garlic MIC range 35.7-1.1 mg/ml; allicin mean MIC 4.1 microg/ml; mean MBC 7.9 microg/ml) were lower than those for the Gram-positive strains tested (garlic MIC range 142.7-35.7 mg/ml; allicin mean MIC 27.5 microg/ml; mean MBC 91.9 microg/ml). Also, of the organisms tested, the putative periodontal pathogens had among the lowest MICs (17.8-1.1 mg/ml garlic) and MBCs (35.7-1.1 mg/ml garlic). Time-kill curves for Streptococcus mutans and P. ginigvalis, showed that killing of the latter started almost immediately, whereas there was a delay before S. mutans was killed. The garlic extract also inhibited the trypsin-like and total protease activity of P. gingivalis by 92.7% and 94.88%, respectively. These data indicate that garlic extract inhibits the growth of oral pathogens and certain proteases and so may have therapeutic value, particularly for periodontitis.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Ajo , Boca/microbiología , Fitoterapia , Bacterias/crecimiento & desarrollo , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Péptido Hidrolasas/metabolismo , Extractos Vegetales/farmacología , Porphyromonas gingivalis/efectos de los fármacos , Porphyromonas gingivalis/enzimología , Porphyromonas gingivalis/crecimiento & desarrollo , Inhibidores de Proteasas/farmacologíaRESUMEN
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.