RESUMEN
Bacteria-derived glucosyltransferases (Gtf) (EC 2.4.1.5), through synthesizing glucan polymers from sucrose and starch hydrolysates, play an essential role in the etiology and pathogenesis of caries. We attempted to correlate the levels of Gtf in whole saliva with the prevalence of carious lesions in young children. We examined saliva from children who were either free of overt carious lesions, or had severe early childhood caries (mean dmfs = 18.72 +/- 9.0 SD), for Gtf by direct enzyme assay. The levels of GtfB, GtfC and GtfD from Streptococcus mutans in the saliva using monoclonal/specific antibodies in an enzyme-linked immunosorbent assay were determined. Multiple logistic regression analyses with model selection showed that GtfB levels correlated with dmfs values of the subjects (p = 0.006). There was no correlation between total Gtf activity as measured by direct enzyme assay and dmfs values. There was a strong correlation between mutans streptococci populations in saliva and caries activity. Collectively, these data show that GtfB levels in saliva correlate strongly with presence of clinical caries and with number of carious lesions in young children. It is also possible to measure different Gtfs, separately, in whole saliva. These observations may have important clinical implications, may lead to development of a chair side caries activity test and support the importance of GtfB in the pathogenesis of dental caries.
Asunto(s)
Pruebas de Actividad de Caries Dental/métodos , Caries Dental/enzimología , Glucosiltransferasas/análisis , Saliva/química , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Preescolar , Caries Dental/epidemiología , Caries Dental/microbiología , Ensayo de Inmunoadsorción Enzimática/métodos , Métodos Epidemiológicos , Femenino , Glucosiltransferasas/metabolismo , Humanos , Masculino , Conejos , Streptococcus mutans/aislamiento & purificaciónRESUMEN
Several active enzymes have been identified as components of acquired enamel pellicle. In the present study, the interactions of Streptococcus mutans glucosyltransferase B (GtfB) with lysozyme in solution and on the surface of hydroxyapatite (HA) beads were studied. Experiments were also performed to investigate whether structural differences exist between glucans formed by GtfB enzyme in the presence or absence of lysozyme in solution and on the surface of HA. Hen egg-white lysozyme (HEWL) and saliva were used as the sources of lysozyme; lysozyme-depleted saliva was used as control. Lysozyme activity was significantly reduced when adsorbed onto HA beads compared with that in solution. The GtfB enzyme did not affect the activity of lysozyme in solution or that of adsorbed lysozyme onto HA. The presence of HEWL increased GtfB activity; bovine serum albumin had an even greater enhancing effect. Depletion of lysozyme from whole saliva increased GtfB activity in solution, but not on the surface of saliva-coated HA. The presence of lysozyme affected the amount of glucan formation by GtfB, but not the structure of glucans formed in solution and on the surface. Therefore, the interaction of lysozyme and GtfB enzymes on HA surface may modulate the formation of glucan and dental plaque.
Asunto(s)
Durapatita , Glucanos/metabolismo , Glucosiltransferasas/metabolismo , Muramidasa/metabolismo , Streptococcus mutans/enzimología , Animales , Bovinos , Embrión de Pollo , Glucanos/química , Humanos , Saliva/enzimología , SolucionesRESUMEN
OBJECTIVES: Apigenin is a potent inhibitor of glucosyltransferases and tt-farnesol affects the membrane integrity of Streptococcus mutans. We investigated the influence of apigenin and tt-farnesol, alone and in combination, on the accumulation, polysaccharide composition and viability of S. mutans UA159 biofilms. METHODS: Initially, biofilms were grown for 54 h; then, the early-formed biofilms were treated for 1 min twice daily with one of the following: (i). 1.33 mM tt-farnesol; (ii). 1.33 mM apigenin; (iii). apigenin + tt-farnesol (1.33 mM each); (iv). vehicle control (20% ethanol with 0.75% dimethyl sulphoxide); (v). 0.12% chlorhexidine (1.33 mM); or (vi). physiological saline (145 mM NaCl). The procedure was repeated at biofilm ages of 78 and 102 h, and biofilms were harvested at 126 h. The dry weight, protein concentration, number of cfu, and polysaccharide composition per biofilm were determined. RESULTS: The dry weights of the biofilms treated with the test agents were significantly less (30-50%) than those treated with vehicle control (P < 0.05). Biofilms treated with the test agents also resulted in lower amounts of extracellular alkali-soluble glucans, intracellular iodophilic polysaccharides and, to a lesser extent, fructans. The fructosyltransferase activity was affected only by apigenin and apigenin + tt-farnesol. The recoverable viable counts of S. mutans were slightly lower (0.5 to 1 log10 decrease in cfu/biofilm) after apigenin and tt-farnesol treatments compared with the vehicle control. Chlorhexidine displayed potent bactericidal activity, and virtually halted the further accumulation of early-formed (54 h old) biofilms. CONCLUSIONS: Apigenin and tt-farnesol affected the accumulation and polysaccharide content of S. mutans biofilms without major impact on the bacterial viability.
Asunto(s)
Biopelículas/efectos de los fármacos , Farnesol/farmacología , Flavonoides/farmacología , Polisacáridos Bacterianos/biosíntesis , Streptococcus mutans/efectos de los fármacos , Antiinfecciosos Locales/farmacología , Apigenina , Biopelículas/crecimiento & desarrollo , Clorhexidina/farmacología , Recuento de Colonia Microbiana , Glucosiltransferasas/metabolismo , Humanos , Streptococcus mutans/crecimiento & desarrolloRESUMEN
The aim of our study was to determine whether the structure of glucans formed by glucosyltransferase from Streptococcus sanguinis (GtfSs) on a surface differ from those formed in solution and to explore the effects of antiserum to Gtfs, control normal rabbit serum, starch hydrolysates (STH) and dextran on S. sanguinis (GtfSs) glucan. Linkage analyses showed that solution-formed glucans are predominantly alpha-1,6-linked and have a small amount of alpha-1,3-linked glucose. Surface-formed glucans have enhanced susceptibility to mutanase. Solution- and surface-formed glucans made in the presence or absence of sera, STH, and dextran contain linkages which differ in both amount and type from control glucans. The GtfSs enzyme in solution exposed to antiserum behaves as if it is adsorbed to a surface. Binding of Streptococcus mutans GS-5 and Actinomyces viscosus OMZ105E (Ny1) to S. sanguinis glucan differs if the glucan is formed in the presence of antiserum. The information could help to define the role of glucans in the formation of pellicle, colonization of tooth surfaces and the accumulation of dental plaque.
Asunto(s)
Glucanos/biosíntesis , Streptococcus sanguis/enzimología , Análisis de Varianza , Adhesión Bacteriana , Cromatografía por Intercambio Iónico/métodos , Película Dental , Glucanos/análisis , Glucanos/química , Glucosiltransferasas/metabolismo , Streptococcus sanguis/patogenicidad , Relación Estructura-ActividadRESUMEN
Propolis, a resinous hive product collected by Apis mellifera bees, has been used for thousands of years in folk medicine. Ethanolic extracts of propolis (EEP) have been shown to inhibit the activity of a mixture of crude glucosyltransferase (Gtf) enzymes in solution. These enzymes synthesize glucans from sucrose, which are important for the formation of pathogenic dental plaque. In the present study, the effects of propolis from two different regions of Brazil on the activity of separate, purified Gtf enzymes in solution and on the surface of saliva-coated hydroxyapatite (sHA) beads were evaluated. The EEP from Minas Gerais (MG; Southeastern Brazil) and Rio Grande do Sul (RS; Southern Brazil) were tested for their ability to inhibit the enzymes GtfB (synthesis of insoluble glucan), GtfC (insoluble/soluble glucan) and GtfD (soluble glucan). The effects of propolis on Gtf from Streptococcus sanguis (soluble glucan synthesis) was also explored. The EEP from both regions effectively inhibited the activity of all Gtfs in solution (75-95%) and on the surface of sHA beads (45-95%) at concentrations between 0.75 and 3.0 mg of propolis/ml. However, the two samples of propolis showed different levels of inhibition on each of the enzymes tested. In general, EEP RS demonstrated a significantly higher inhibitory activity on GtfB and C activities (both solution and surface assays) than EEP MG at concentrations between 0.047 and 0.187 mg/ml (p<0.05). EEP MG, on the other hand, exhibited a greater inhibitory effect on the activities of surface GtfD (at 0.375, 0.75 and 1.5 mg/ml) and S. sanguis Gtf (at 1.5 and 3.0 mg/ml; p<0.05). These data indicate that EEP is a potent inhibitor of Gtf enzymes in solution and adsorbed on an experimental pellicle; however, its effect on Gtf activity is variable depending on the geographical origin of the propolis samples. There is a need to identify the active compounds of propolis.
Asunto(s)
Glicosiltransferasas/antagonistas & inhibidores , Própolis/farmacología , Streptococcus sanguis/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Brasil , Película Dental , Durapatita , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Própolis/química , Saliva/enzimología , Especificidad de la EspecieRESUMEN
We have shown in previous studies that the glucosyltransferase (Gtf) enzymes of Streptococcus mutans have distinct properties when adsorbed to a surface. In the present study, we compared the activity of Gtf from Streptococcus sanguis, designated GtfSs, in solution and on the surface of saliva-coated hydroxyapatite (sHA) beads, and determined the ability of its product glucan to support the adherence of oral microorganisms. Gtf from S. sanguis 804 NCTC 10904 was purified from culture supernatant fluids by means of hydroxyapatite chromatography. Enzyme and the substrate were prepared in buffers at pH values from 3.5 to 7.5. Maximum activity of GtfSs occurred between pH 5.5 and pH 6.5, whether in solution or adsorbed onto a surface. The solubilized and insolubilized enzymes showed highest activity at 40 degrees C; activity was reduced by 50(+/-2)% at 20 and 30 degrees C. The enzyme did not form glucans in either phase at 10 or 60 degrees C. The K(m), determined from Lineweaver-Burk plots, for the enzyme in solution was 4.3(+/-0.4) mmol/l sucrose, and the K(m) for the enzyme on sHA beads was 5.0(+/-1.0) mmol/l sucrose. The ability of the GtfSs glucan synthesized on the surface of sHA beads to support the adherence of oral bacteria was investigated. (3)H-thymidine-labeled bacteria (S. mutans GS-5, S. sobrinus 6715, S. sobrinus 6716, S. sanguis 10904, Actinomyces viscosus OMZ105E, A. viscosus 2085, and A. viscosus 2086) were incubated with sHA beads coated with GtfSs glucan. S. mutans GS-5 displayed the highest level of binding numerically. These results show that the GtfSs of S. sanguis is active on sHA beads, that the pH optimum for activity on a surface differs slightly from that in solution, and that its product glucan can support the adherence of oral microorganisms.
Asunto(s)
Glucosiltransferasas/fisiología , Streptococcus sanguis/enzimología , Actinomyces viscosus/fisiología , Adsorción , Adulto , Adhesión Bacteriana/fisiología , Durapatita/química , Femenino , Glucanos/metabolismo , Glucosiltransferasas/metabolismo , Humanos , Polisacáridos Bacterianos/metabolismo , Radiofármacos , Saliva/fisiología , Soluciones , Streptococcus mutans/fisiología , Streptococcus sanguis/metabolismo , Streptococcus sobrinus/fisiología , Sacarosa/metabolismo , Propiedades de Superficie , Temperatura , TritioRESUMEN
The formation of acquired enamel pellicle on hydroxyapatite (HA) discs of known surface area carried in the mouth was studied; discs were carried in the mouth for 30 s, 1, 5, 10 and 20 min. Similar amounts of protein were found on the discs at each time-point, as determined by ninhydrin analyses. The amounts of amylase and lysozyme detected remained stable after 5 min of exposure of the discs to the mouth. Assay of the discs for fructosyl- and glucosyltransferase activities revealed that fructosyltransferase activity increased up to 1 min of exposure to the mouth and decreased when kept in the mouth for longer periods; glucosyltransferase activity, in contrast, increased the longer the discs were kept in the mouth. This in situ model provides insight into the activities of various enzymes during the first 20 min of pellicle formation. The effects of rinsing with sucrose and sugar alcohols on pellicle formation on the discs were also explored. The discs were placed in the mouth for 30 s, 1, 5, 10 and 20 min, preceded by rinsing with either distilled deionized water, sucrose, sorbitol, xylitol or phosphate-buffered saline. Western blot analyses of disc eluates with antiserum/antibody preparations to various salivary components revealed distinct patterns of deposition of bacterial and salivary components depending on the composition of the rinse. These studies confirm that salivary molecules and bacteria are deposited on apatitic surfaces in a selective manner and reveal that pellicle formation may be influenced by composition of diet. It is apparent that this in situ model could be used in screening potential antiplaque agents.
Asunto(s)
Depósitos Dentarios/química , Durapatita/química , Adulto , Amilasas/análisis , Anticuerpos Monoclonales , Adhesión Bacteriana , Western Blotting , Depósitos Dentarios/metabolismo , Depósitos Dentarios/microbiología , Película Dental , Sacarosa en la Dieta/farmacología , Femenino , Glucosiltransferasas/análisis , Hexosiltransferasas/análisis , Humanos , Indicadores y Reactivos , Muramidasa/análisis , Ninhidrina , Proteínas/análisis , Proteínas y Péptidos Salivales/química , Proteínas y Péptidos Salivales/inmunología , Sorbitol/farmacología , Streptococcus mutans/metabolismo , Streptococcus mutans/fisiología , Propiedades de Superficie , Edulcorantes/farmacología , Factores de Tiempo , Xilitol/farmacologíaRESUMEN
The effects of milk and kappa-casein rinses on the salivary pellicle formed on hydroxyapatite discs carried in the mouth were studied. SDS-PAGE analyses revealed an increase in the number of proteins deposited onto the discs carried after the water and milk rinses only. Scanning electron microscopy studies revealed the deposition of an amorphous material, small, micelle-like structures, cocci and rods on discs carried in the mouth after the water rinse. Large, micelle-like structures were seen on discs carried in the mouth after the milk and kappa-casein rinses; bacteria were not seen. Glucosyltransferase (Gtf) activity on discs carried in the mouth after the milk and kappa-casein rinses were 45+/-5 and 67+/-2% lower than the activity of Gtf on discs carried in the mouth after a water rinse, respectively. These data suggest that milk and kappa-casein may influence pellicle formation in vivo.
Asunto(s)
Caseínas , Depósitos Dentarios/química , Leche , Animales , Adhesión Bacteriana/efectos de los fármacos , Proteínas Bacterianas/análisis , Proteínas Bacterianas/metabolismo , Unión Competitiva , Biopelículas/efectos de los fármacos , Caseínas/metabolismo , Caseínas/farmacología , Depósitos Dentarios/enzimología , Película Dental , Durapatita , Electroforesis en Gel de Poliacrilamida , Femenino , Glucosiltransferasas/metabolismo , Hexosiltransferasas/metabolismo , Humanos , Micelas , Leche/metabolismo , Proteínas de la Leche/análisis , Proteínas de la Leche/metabolismo , Peso Molecular , Unión Proteica , Saliva/química , Saliva/enzimología , Saliva/metabolismo , Proteínas y Péptidos Salivales/análisis , Proteínas y Péptidos Salivales/metabolismoRESUMEN
The binding specificities of Streptococcus glucosyltransferase (Gtf) B, C and D for hydroxyapatite (HA), saliva-coated hydroxyapatite (SHA), and bacterial surfaces were examined. For HA beads the following values were obtained: (K = affinity; N = number of binding sites) GtfB, K = 46 x 10(5) ml/mumol, N = 0.65 x 10(-6) mumol/m2; GtfC, K = 86 x 10(5) ml/mumol, N = 4.42 x 10(-6) mumol/m2.; GtfD, K = 100 x 10(5) ml/mumol, N = 0.83 x 10(-6) mumol/m2. For SHA beads, the following values were obtained: GtfB, K = 14.7 x 10(5) ml/mumol, N = 1.03 x 10(-6) mumol/m2; GtfC, K = 21.3 x 10(5) ml/mumol, N = 3.66 x 10(-6) mumol/m2; GtfD, K = 1.73 x 10(5) ml/mumol, N = 8.88 x 10(-6) mumol/m2. The binding of GtfB to SHA beads was reduced in the presence of parotid saliva, but the binding of GtfC and D was unaffected. The binding of GtfB to SHA in the presence of parotid saliva supplemented with GtfC and D was reduced when compared with its binding to SHA in the presence of parotid saliva alone. In contrast, te binding of GtfC and SHA was unaffected when parotid saliva was supplemented with the other Gtf enzymes. GtfB bound to several bacterial strains (Strep, mutans GS-5, Actinomyces viscosus OMZ105E and Lactobacillus casei 4646) in an active form, while GtfC and D did not bind to bacterial surfaces. It is concluded that of the three Gtf enzymes, GtfC has the highest affinity for HA and SHA surfaces and can adsorb on the the SHA surface in the presence of the other two enzymes. GtfD also binds to SHA in the presence of the other enzymes but has a very low affinity for the surface. GtfB does not bind to SHA in the presence of the other Gtf enzymes but binds avidly to bacterial surfaces in an active form. Therefore, GtfC most probably binds to apatitic surfaces, while GtfB binds to bacterial surfaces.
Asunto(s)
Actinomyces viscosus/fisiología , Adhesión Bacteriana/fisiología , Durapatita/química , Glucosiltransferasas/química , Lacticaseibacillus casei/fisiología , Saliva/fisiología , Streptococcus mutans/fisiología , Streptococcus/enzimología , Adsorción , Antígenos Bacterianos/química , Antígenos Bacterianos/metabolismo , Sitios de Unión , Glucosiltransferasas/metabolismo , Humanos , Glándula Parótida/metabolismo , Streptococcus/fisiologíaRESUMEN
Streptococcus mutans glucosyltransferases (GtfB, -C, and -D) and their products formed from sucrose, glucans, play an essential role in the pathogenesis of dental caries. Enzymatically active Gtf is found in whole human saliva (solution), and incorporated into the salivary pellicle that is formed on teeth in vivo (surface). GtfB glucans are predominantly 1,3-linked; however, surface-formed glucans from GtfB contain greater amounts of 3-linked glucose than glucans formed in solution. In contrast, the major linkage of glucans formed on the surface by GtfB in the presence of sucrose and starch hydrolysates in 4-linked glucose. GtfC-derived glucans in solution have a major linkage of 6-linked glucose, while surface-formed glucans from the same enzyme have 3-linked glucose as the major linkage. GtfD glucans formed either in solution or on the surface are predominantly 1,6-linked; however, surface-formed glucans contain more 6-linked glucose than solution-formed glucans. Digestion with the glucanohydrolases mutanase and dextranase shows differences in susceptibility among glucans formed either in solution or on the surface by each of the Gtf enzymes, and differences are also seen in the soluble end products from these digestions. Our results show that the same Gtf enzyme can form structurally distinct glucans in solution and on a surface. These observations are important in the study of naturally occurring microbial films.
Asunto(s)
Durapatita/química , Glucanos/química , Conformación de Carbohidratos , Película Dental , Dextranasa/metabolismo , Glucanos/metabolismo , Glicósido Hidrolasas/metabolismo , Soluciones , Streptococcus/enzimologíaRESUMEN
The salivary pellicle consists of various proteins and glycoproteins which may interact with one another. Experiments were performed to elucidate the interactions of streptococcal glucosyltransferase (Gtf) enzymes with human salivary alpha-amylase in solution and on the surface of saliva-coated hydroxyapatite (SHA) beads. The Gtf enzymes -B, -C and -D, when immobilized on to SHA beads, reduced the activity of adsorbed amylase; GtfD showed the highest inhibition of salivary amylase activity. The presence of glucan produced by immobilized GtfD did not further reduce amylase activity. The amount of amylase adsorbed on to hydroxyapatite beads was reduced when salivary amylase was added simultaneously with any of the Gtf enzymes, suggesting that amylase and Gtfs may compete with each other for binding sites on hydroxyapatite. Starch hydrolysates produced by SHA-surface-bound salivary amylase were tested for their effect on glucan production from sucrose by Gtf enzymes in solution and on SHA beads; glucan production by SHA-immobilized GtfB was stimulated in the presence of starch hydrolysates. Glucan synthesized by SHA-immobilized GtfB in the presence of starch hydrolysates was less susceptible to hydrolysis by the fungal enzyme mutanase than was glucan made by SHA-immobilized GtfB in the absence of starch hydrolysates. Glucan production by GtfB associated with streptococci immobilized on to SHA was also enhanced in the presence of starch hydrolysates. The adhesion of oral micro-organisms to SHA coated with glucan made in the presence and absence of starch hydrolysates was investigated, and some bacteria displayed higher adhesion activities for the glucan made in the presence of the hydrolysates. Therefore, the interaction of amylase and Gtf enzymes on a SHA surface may modulate the formation of glucan and the adherence of oral micro-organisms.
Asunto(s)
Amilasas/metabolismo , Adhesión Bacteriana/fisiología , Placa Dental/metabolismo , Glucosiltransferasas/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Streptococcus/enzimología , Adsorción , Adulto , Proteínas Bacterianas/metabolismo , Unión Competitiva , Película Dental , Dextranasa/metabolismo , Durapatita/metabolismo , Femenino , Proteínas Fúngicas/metabolismo , Glucanos/biosíntesis , Glicósido Hidrolasas/metabolismo , Humanos , Hidrólisis , Saliva/enzimología , Almidón/metabolismoRESUMEN
Activities of glucosyltransferases (Gtf)-B, -C and -D adsorbed on the surface of saliva-coated hydroxyapatite were compared with those of Gtfs of donor whole saliva. Antiserum raised against a mixture of the three Gtfs reduced the activity of GtfB only, and had no effect on donor Gtf activities. GtfB, and not the Gtfs of the donors, was stimulated in the presence of starch hydrosylates. GtfD and GtfC activities were enhanced on the surfaces, as were the Gtf activities of donor salivas. The activities of GtfD and GtfB, but not GtfC, were stimulated by dextran. The donor Gtf activities were unaffected by dextran. Therefore, Gtf activity in pellicles has properties similar to those of GtfC.
Asunto(s)
Depósitos Dentarios/enzimología , Glicosiltransferasas/química , Proteínas y Péptidos Salivales/química , Streptococcus mutans/enzimología , Adsorción , Proteínas Bacterianas/química , Unión Competitiva , Depósitos Dentarios/química , Durapatita/química , Glucanos/biosíntesis , Glicosiltransferasas/metabolismo , Humanos , Saliva/enzimologíaRESUMEN
GlucosyltransferaseB, GtfC, and GtfD were purified by hydroxyapatite column chromatography, followed by ultrafiltration from the culture supernatant fluids of three Streptococcus milleri constructs (gift from Dr. H.K. Kuramitsu) which harbored individual gtf genes of Streptococcus mutans GS5. GtfB, GtfC, and GtfD were enzymatically active both in solution and in an experimental pellicle (HA-CWS-Gtf) formed by adsorbing Gtf onto the surface of clarified human whole saliva (CWS)-coated hydroxyapatite (HA). The Km values for sucrose for all three enzymes were lower when the enzyme was adsorbed to a surface, compared with when it was in solution. In solution phase assays, and in the absence of primer dextran, glucan production was enhanced 75% when both GtfB and GtfD were present in the reaction mixture, compared with the sum of the individual enzyme activities (p < 0.005). This enhancement did not occur when GtfC was additionally present, or when the GtfB+GtfD enzyme pair was adsorbed onto HA-CWS. In additional experiments, glucan formed by GtfB or GtfC, but not by GtfD, on a HA-CWS-Gtf surface increased adherence of Streptococcus mutans GS5 and Streptococcus sobrinus 6715 by seven- to nine-fold compared with adherence when no glucan was present on the pellicle surface (p < 0.001). Further, treatment of the HA-CWS-GtfB-glucan or HA-CWS-GtfC-glucan pellicle with alpha-1,6 dextranase significantly reduced adherence of both streptococcal strains (p < 0.001). These results show that GtfB, GtfC, and GtfD are enzymatically active in an adsorbed state and that the nature of their product glucan can influence the adherence of cariogenic oral streptococci to an experimental pellicle.
Asunto(s)
Durapatita/química , Glucosiltransferasas/metabolismo , Adsorción , Análisis de Varianza , Adhesión Bacteriana , Cromatografía/instrumentación , Cromatografía/métodos , Película Dental , Interacciones Farmacológicas , Electroforesis en Gel de Poliacrilamida , Genes Bacterianos , Glucanos/biosíntesis , Glucosiltransferasas/análisis , Glucosiltransferasas/aislamiento & purificación , Humanos , Microscopía Electrónica de Rastreo , Saliva , Soluciones , Streptococcus/enzimología , Streptococcus/genética , Streptococcus mutans/enzimología , Streptococcus mutans/patogenicidad , Streptococcus sobrinus/enzimología , Streptococcus sobrinus/patogenicidad , Propiedades de SuperficieRESUMEN
Many dietary components such as carbohydrates, lipids and proteins may be incorporated into the salivary pellicle and thus may affect glucosyltransferase (GTF) activity on pellicle surfaces. The effect of milk on streptococcal GTF activity was determined. Milk, when coated onto buffer-coated hydroxyapatite or saliva-coated hydroxyapatite (sHA), reduced the subsequent adsorption of GTF onto the surfaces. Milk also reduced the expression of enzymatic activity of GTF adsorbed onto sHA. kappa-Casein, when present on the surface of sHA, reduced the adsorption of GTF activity onto sHA, resulting in reduced glucan formation. alpha-Casein had no effect on the adsorption of GTF onto sHA or on subsequent glucan formation. Both milk and kappa-casein reduced activity of the enzyme in solution. The presence of milk and kappa-casein fractions on the surface of sHA and in solution with GTF can clearly modulate glucan formation in vitro.
Asunto(s)
Cariostáticos/farmacología , Caseínas/farmacología , Depósitos Dentarios/metabolismo , Glicosiltransferasas/metabolismo , Leche , Adulto , Animales , Proteínas Bacterianas/metabolismo , Unión Competitiva , Película Dental , Durapatita/metabolismo , Femenino , Glucanos/biosíntesis , Humanos , Saliva/fisiología , Streptococcus mutans/enzimología , Propiedades de Superficie/efectos de los fármacos , Adherencias TisularesRESUMEN
Experiments sought to determine the nature of the binding of milk proteins to hydroxyapatite (HA) and to saliva-coated hydroxyapatite (sHA), and to determine the effect of milk and casein on the adherence of Streptococcus mutans GS-5 to sHA. The binding of radiolabelled alpha-casein to HA was reduced when incubated simultaneously with parotid saliva, and enhanced in the presence of milk. The binding of beta- and kappa-casein to HA was unaffected by the presence of parotid saliva and enhanced by the presence of milk. The in vitro bacterial adherence of Strep. mutans GS-5 to sHA beads was reduced when beads were coated with milk instead of buffer, or when bacteria were added to sHA in the presence of milk instead of buffer. Casein proteins (alpha, beta, kappa) added to sHA simultaneously with bacteria inhibited the adherence of Strep. mutans GS-5 to sHA. kappa-Casein, when bound to sHA, inhibited streptococcal adherence to sHA; alpha- and beta-casein, when bound to sHA, had no effect on streptococcal adherence. Fractionation of kappa-casein by anion-exchange chromatography revealed the anti-adherence activity of kappa-casein was mediated primarily by a 40,000 mol. wt. glycoprotein-containing fraction. These data show that milk, particularly kappa-casein fractions, can modulate the adherence of Strep. mutans GS-5 to SHA surfaces in vitro.
Asunto(s)
Adhesión Bacteriana/efectos de los fármacos , Caseínas/farmacología , Leche , Streptococcus mutans/fisiología , Adulto , Animales , Caseínas/química , Caseínas/metabolismo , Cromatografía por Intercambio Iónico , Durapatita/metabolismo , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Unión Proteica , Saliva/fisiología , Propiedades de Superficie , Factores de TiempoRESUMEN
Human umbilical vein endothelial cells (HUVEC) were used as an experimental host model to investigate the mechanism(s) of streptococcal adhesion in infective endocarditis. Adhesion activity of Streptococcus gordonii was maximal during the logarithmic phase of growth and was greatly reduced or eliminated by pretreatment of bacteria with heat, formaldehyde, or trypsin. At saturating numbers of streptococci, an average of 81 bacteria were bound per HUVEC. Streptococcal adhesion was inhibited by low-molecular-weight dextran and heparin but not by sucrose, fibronectin, or laminin. Adhesion was also prevented by pretreatment of HUVEC with proteins dissociated from the surface of S. gordonii with 10 mM EDTA or isolated from spent culture medium. Western blot (immunoblot) assays detected a single adhesion protein of 153 kDa (AP153) on HUVEC after incubation with unfractionated extracts of streptococci. The adhesin exhibited glucosyltransferase (GTF) activity when incubated with sucrose and Triton X-100 after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The AP153 was purified by affinity chromatography on dextran beads and show to have binding activity for HUVEC, GTF activity, an amino acid composition similar to that reported for GTF of S. gordonii, and the ability to inhibit S. gordonii adhesion. Incubation of the streptococci with antibodies to the adhesin inhibited bacterial attachment to HUVEC monolayers. These results indicate that surface-localized GTF mediates adhesion of S. gordonii to HUVEC in vitro and may serve as a mechanism for colonization of the endocardium in infective endocarditis.