Initial catabolism of sorbitol in Actinomyces naeslundii and Actinomyces viscosus

The initial steps of sorbitol catabolism were studied in 4 strains of Actinomyces naeslundii and Actinomyces viscosus that had been isolated from human dental plaque. Cell-free extracts were prepared from cells grown in the presence of either sorbitol, xylitol or glucose. The extracts from all strains grown on sorbitol had nicotinamide adenine dinucleotide-linked dehydrogenase activities for sorbitol and xylitol and reduced nicotinamide adenine dinucleotide-linked reductase activities for fructose and xylulose. Two of the strains also exhibited these activities when grown in the presence of xylitol, and all glucose-grown cells lacked them. The results indicate that sorbitol metabolism in oral actinomyces involve oxidation of sorbitol to fructose by an inducible enzyme, nicotinamide adenine dinucleotide-linked sorbitol dehydrogenase. This step is followed by the phosphorylation of fructose with guanosine triphosphate as a main phosphoryl donor. Thus, the initial catabolic pathway of sorbitol in A. naeslundii and A. viscosus is different from those described for other oral bacteria.     

The induction of oral tolerance to Actinomyces viscosus in mice

OBJECTIVES: To determine whether oral tolerance with the oral bacterium Actinomyces viscosus was inducible in mice. MATERIALS AND METHODS: Mice were intragastrically (i.g.) and then intraperitoneally (i.p.) immunized with heat-killed A. viscosus. A control group of mice received only saline. A delayed type hypersensitivity (DTH) response and the levels of isotype specific antibodies were assessed. Spleen cells from mice that were i.g. immunized with A. viscosus were transferred to A. viscosus-primed mice in vivo and in vitro. Furthermore, mice were i.g. immunized with saline or A. viscosus and then challenged i.p. with saline, A. viscosus, or Porphyromonas gingivalis. RESULTS: Intragastric immunization with A. viscosus suppressed both DTH and serum specific antibodies to A. viscosus. DTH suppression lasted until week 4, while serum immunoglobulin (Ig)A and both IgG and IgM specific antibody levels remained suppressed up to week 8 and 12 respectively. IgG specific antibody suppression was transferable. The DTH response and serum antibodies specific to A. viscosus were suppressed in mice after i.g. challenged with A. viscosus but not P. gingivalis. CONCLUSION: Mucosal presentation of A. viscosus in mice led to the suppression of immune response to this bacterium in an antigen-specific fashion. Tolerance of DTH response was short lived, while suppression of antigen-specific IgG antibodies in mucosally tolerized mice was long-lasting.     

Stimulatory effect of bicarbonate on the glycolysis of Actinomyces viscosus and its biochemical mechanism

The effects of bicarbonate on acid production by 4 human strains of Actinomyces viscosus were estimated under anaerobic conditions. The rate of acid production was accelerated by bicarbonate 3-4 times as much as that without bicarbonate. The analyses of intracellular glycolytic intermediates, NAD and NADH revealed a decrease in NADH:NAD ratio and an increase in the level of 3-phosphoglycerate in the cells when bicarbonate was present. Furthermore, when bicarbonate was available, malate dehydrogenase and fumarate reductase in the succinate pathway were expected to function as NADH-oxidizing enzymes in addition to lactate dehydrogenase. These observations indicate the efficient regeneration of NAD in the presence of bicarbonate. Thus, the stimulation of A. viscosus glycolysis by bicarbonate was thought to stem from the activation of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) by the decrease in the level of NADH, because NADH was a strong inhibitor of G3PDH in this microorganism.     

Catabolic pathway for aerobic degradation of lactate by Actinomyces naeslundii

The aerobic metabolism of lactate by oral Actinomyces was studied. Six of 7 strains of Actinomyces naeslundii increased their growth in the presence of lactate under aerobic conditions. Washed cells grown on lactate aerobically degraded lactate and pyruvate to acetate with a concomitant consumption of oxygen. In the presence of catalase, the molar ratios of oxygen consumed to acetate produced were 1 for lactate degradation and 0.5 for pyruvate degradation. The enzymatic activities found in cell extracts revealed that lactate could be converted to pyruvate by NAD-independent lactate dehydrogenase (iLDH) and further to acetyl CoA by pyruvate dehydrogenase (PDH). The acetyl CoA formed could be metabolized into acetate by phosphotrans-acetylase (PTA) and acetate kinase (AK) with the formation of ATP. These results indicate that A. naeslundii metabolizes lactate into acetate by the sequential enzymatic reactions iLDH, PDH, PTA and AK and that hydrogens produced by iLDH and PDH are transferred to oxygen. The activity of lactate degradation and oxygen consumption may modify the environmental conditions of dental plaque.     

Actinomyces viscosus and Actinomyces naeslundii agglutinins in human saliva

Abstract – The objectives were to determine the degree of Actinomyces agglutinating activity in human saliva and to begin characterizing the agglutination mechanism. Agglutination titres of whole saliva collected from adults and 6-yr-old children were compared. Titres for A. naeslundii were always higher than for A. viscosus. The mean A. naeslundii titre for the adults’ and children's samples were equivalent. The children had a slightly lower mean titre than the adults for A. viscosus. No correlation was found between IgA concentration and agglutination titre. Agglutinating activity was partially impaired by incubation with anti-IgA serum. Activity in submandibular/sublingual saliva was resistant to heat at 56°C but sensitive to boiling. Boiling the bacteria had no effect. In sugar inhibition tests, only galactosides (β-Gal) and glucosamine (for A. viscosus) affected Actinomyces agglutination but impairment was only temporary. Agglutinating activity was diminished by incubating saliva with hydroxyapatite. Thus, Actinomyces agglutinins 1) are probably distinct from IgA but may complex with it; 2) may include both β-Gal and higher affinity sites; and 3) may contribute to salivary pellicle.     

老年人根面菌斑内氏放线菌临床分离株基因型多样性分析

目的分析老年人根面菌斑中内氏放线菌临床株的基因型多样性,探讨内氏放线菌基因型与根面龋的关系。方法选择老年根面龋患者20例设为根面龋组,无根面龋老年人20例设为无龋组。根面龋组以暴露的无龋根面和根面龋损部位为取样位点,无龋组以暴露的根面为取样位点,刮取菌斑进行临床株的分离鉴定,并利用基因外重复回文序列聚合酶链反应（REP-PCR）分析内氏放线菌基因型的多样性。结果2组共分离出内氏放线菌299株,选择156株进行REP-PCR分析,分离出61个不同的基因型。根面龋组无龋根面分离的57株内氏放线菌有25个基因型,根面龋损部位分离的34株有25个基因型,无龋组分离的65株有26个基因型：内氏放线菌基因型存在多样性。单个取样位点的基因型数目存在差异（P<0.05）。结论多种基因型的内氏放线菌参与了根面龋的发生。     

尿素水解对内氏放线菌增殖及耐酸力的影响

目的通过研究尿素水解对内氏放线菌的生长和耐酸能力的影响,了解尿素水解对内氏放线菌的生理作用。方法比较内氏放线菌利用尿素或其它物质作为氮源的生长浊度;采用耐酸实验研究尿素水解与细菌耐酸能力的关系。结果与其他口腔中常见氮源物质相比,尿素可以促进内氏放线菌生长,获得更高的A值;在临床牙菌斑能够检测到的pH4.0~7.0范围内,尿素水解可以提高内氏放线菌的耐酸性,在pH3.0,尿素水解对细菌没有保护作用。结论尿素水解可以促进内氏放线菌生长,提高酸性环境中细菌的耐酸能力,增强细菌的生存竞争力。     

Induction of suppressor cells following mucosal presentation of Actinomyces viscosus in mice

Mucosal presentation of Actinomyces viscosus results in antigen-specific systemic immune suppression, known as oral tolerance. The aim of the present study was to determine the mechanism by which this oral tolerance is induced. DBA/2 mice were gastrically immunized with A. viscosus. Serum, Peyer's patch (PP) and spleen cells were transferred to syngeneic recipients which were then systemically challenged with the sameiA. viscosus strain. To determine antigen-specificity of cells from gastrically immunized mice, recipients which received immune spleen cells were also challenged with Porphyromonas gingivalis. One week after the last systemic challenge, the delayed type hypersensitivity (DTH) response was determined by footpad swelling and the level of serum IgG, IgA and IgM antibodies to A. viscosus or P. gingivalis measured by an ELISA. No suppression of DTH response or of specific serum antibodies was found in recipients which received serum from gastrically immunized mice. Systemic immune suppression to A. viscosus was observed in recipients which had been transferred with PP cells obtained 2 days but not 4 and 6 days after gastric immunization with A. viscosus. Conversely, suppressed immune response could be seen in recipients transferred with spleen cells obtained 6 days after gastric immunization. The immune response to P. gingivalis remained unaltered in mice transferred with A. viscosus-gastrically immunized cells. The results of the present study suggest that oral tolerance induced by A. viscosus may be mediated by antigen-specific suppressor cells which originate in the PP and then migrate to the spleen.     

Binding of Actinomyces viscosus to collagen: association with the type 1 fimbrial adhesin

Treatment of hydroxyapatite (HA) with human type I or type III collagen strongly promoted adhesion of Actinomyces viscosus LY7 cells. Treatment with human type V collagen was somewhat less effective while treatment with human type IV or rat type I collagen was significantly less effective. Electron microscopic observations revealed that A. viscosus cells also attached to fibrils prepared from human type I collagen. The alpha 1 (1) polypeptide chain derived from type I collagen was ineffective in promoting binding and the alpha 2 (1) polypeptide chain exhibited moderate activity. Heat- or urea-denatured type I collagens were also ineffective in promoting binding. Mutants of A. viscosus that possess type 1 fimbriae, but not type 2 fimbriae or no fimbriae, also bound to collagen-treated HA; this suggests that the adhesin responsible was associated with type 1 fimbriae. Strains of Actinomyces israelii and Actinomyces odontolyticus also exhibited strong binding to collagen-treated HA, while Actinomyces naeslundii ATCC 12104 did not. The avidity of Actinomyces species for collagen would seem to be at least partially responsible for the high proportions of these organisms found on cemental and root tooth surfaces.     

Effect of acetate on sorbitol fermentation by oral lactobacilli

The rate of acid production and end-products from sorbitol were measured under anaerobic conditions in washed-cell suspensions of oral strains of Lactobacillus casei subsp, casei and Lactobacillus casei subsp, rhamnosus . The enzymatic activities were assayed in cell extracts of these strains. The cells fermented sorbitol to lactate, formate, ethanol and acetate under anaerobic conditions. Exposure of the cells to air (oxygen) led to inactivation of pyruvate formatelyase and inhibition of anaerobic sorbitol fermentation. In the presence of acetate, air-exposed cells fermented sorbitol with a concomitant consumption of acetate and production of ethanol and lactate. Acetate also enhanced acid production from sorbitol in cells kept under anaerobic conditions and resulted in formation of lactate and ethanol. Cell extracts of all the strains had NADH-coupled acetate-reducing activity, which consisted of sequential reactions of acetate kinase, phosphotrans-acetylase, acylating aldehyde dehydrogenase and alcohol dehydrogenase. These findings indicate that oral lactobacilli can utilize acetate as an electron acceptor for maintaining their intracellular redox balance during anaerobic sorbitol fermentation in the absence of pyruvate formate-lyase activity.     

木糖醇对黏性放线菌生长及产酸影响的体外研究

目的对比不同质量浓度下木糖醇对黏性放线菌生长及产酸的影响。方法分别用含不同质量浓度（128、64、32、16、8、4 g·L^-1）木糖醇的脑心浸液（BHI）液体培养基在厌氧条件下培养黏性放线菌,测定其最小抑菌质量浓度（MIC）;然后测量对照组以及1/2、1/4、1/8 MIC和MIC质量浓度时培养1.5、3、6、12、24、48 h液体培养基的pH值,计算ΔpH值,同时测量2、4、6、8、10、12 h液体培养基的光密度（OD550）值;最后测定抑制50%及 90%黏性放线菌生物膜形成的最低木糖醇质量浓度（即MBIC50和MBIC90）。运用SPSS 19.0进行数据分析。结果木糖醇能抑制黏性放线菌的生长,MIC为64 g·L^-1。培养12 h后,各组之间的ΔpH值差异均有统计学意义（P<0.05）,且ΔpH值随MIC质量浓度的增大而减小;此时,除1/2 MIC以及MIC组之外,其余各组的OD550差异均无统计学意义（P>0.05）,OD550值随MIC质量浓度的增加而减小。提示黏性放线菌在含1/2 MIC及MIC的木糖醇培养基里生长及产酸能力随木糖醇质量浓度的升高而下降。木糖醇的MIBC50为64 g·L^-1,MIBC90为128 g·L^-1,说明木糖醇培养基能抑制黏性放线菌生物膜的形成。结论木糖醇能有效地抑制黏性放线菌的生长、黏附以及产酸,有一定的抑制致龋菌和防治龋病的效果。     

镍铬合金析出的镍离子对内氏放线菌糖代谢和尿素代谢的影响

目的研究镍铬合金析出的镍离子对内氏放线菌糖代谢和尿素代谢的影响。方法分别用含0.260mg/L和0.625mg/L镍离子的BHI培养基厌氧培养内氏放线菌WVU45型菌株,计算pH的变化值,检测乳酸脱氢酶(LDH)含量,测定尿素酶活性。结果镍离子对内氏放线菌产酸和产乳酸脱氢酶的作用不明显(P>0.05),对尿素酶活性有促进作用(P<0.05)。结论镍铬合金析出的镍离子可以增加内氏放线菌尿素酶活性,促进尿素代谢。     

Preparation and characterization of an Actinomyces naeslundii aggregation factor that mediates coaggregation with Porphyromonas gingivalis

Intergeneric coaggregation is responsible for the complexity of the microbiota in human dental plaque and is believed to be important in the initial bacterial colonization of the human oral cavity. Actinomyces naeslundii, an early colonizer of the tooth surface, may enhance subsequent colonization by Porphyromonas gingivalis which is associated with adult periodontitis. The purpose of this study was to isolate and characterize the A. naeslundii aggregation factor (AnAF) that mediates coaggregation with P. gingivalis. AnAF was isolated from A. naeslundii sonic extract (SE) by gel nitration on a Sephacryl S-400HR, by hydrophobic interaction chromatography on a HiTrap Octyl Sepharose 4FF, and by ion exchange chromatography on a HiTrap Q. The specific activity increased 12-fold with a yield of 2.5%. SDS-PAGE analysis of AnAF revealed a protein band of high molecular weight in excess of 200 kDa. Carbohydrate was detected as the only material coinciding with the protein band, indicating that the AnAF was a glycoprotein. Immunoblotting analysis indicated that AnAF directly bound to P. gingivalis cells. AnAF was sensitive to sodium metaperiodate treatment but not to heat or protease treatments. These results suggest that the AnAF carbohydrate component mediated coaggregation with P. gingivalis cells. AnAF also inhibited coaggregation with other periodontal disease-associated bacteria such as Prevotella intermedia, Fusobacterium nucleatum, Capnocytophaga ochracea, but not streptococci.     

Reduction in adherence of Actinomyces viscosus after exposure to low-frequency acoustic energy

The ability of low-frequency (200 Hz) acoustic energy to reduce the adherence of Actinomyces viscosus T14V to saliva-treated hydroxyapatite (SHA) disks was studied. An acoustic pressure range between 0 and 65 kPa and exposure durations between 0 and 8 min were used to study the levels necessary to significantly alter adherence. The effects of acoustic exposure on both bacteria in liquid and bacteria already adhering to SHA disks were studied. A modified enzyme-linked immunosorbent assay was used to assess bacterial adherence. For bacterial suspensions exposed prior to addition to SHA disks, it was found that reductions in adherence were greater for lower bacterial concentrations. Exposure of bacteria already adhering to SHA disks resulted in a decrease in adherence that was independent of the bacterial concentration and linearly related to the logarithm of the exposure duration. In addition to affecting adherence, acoustic energy also dispersed bacterial aggregates. Our results support the concept that low-frequency sonic energy applied orally may be of therapeutic value in reducing adherence and colonization of teeth by plaque bacteria.     

外源性右旋糖酐酶和氟化钠对粘性放线菌生物膜的影响

目的：探讨外源性右旋糖苷酶（dextranase ,Dex）和氟化钠（NaF）对粘性放线菌生物膜的影响。方法用结晶紫（crystal violet,CV）染色法测量不同浓度外源性Dex和NaF作用下粘性放线菌生物膜量;用扫描电镜观察不同实验组生物膜的形态和结构。结果外源性Dex和NaF的浓度升高,对粘性放线菌生物膜形成的抑制作用增强;在实验条件下,0.25 U/mL Dex与40/80μg/mL NaF联用对生物膜形成的抑制作用明显强于二者单独作用。结论外源性Dex和NaF能够抑制粘性放线菌生物膜的形成,且在一定条件下,它们对粘性放线菌生物膜的形成表现出协同抑制作用。     

两种材料修复体的冠边缘粘性放线菌的检测

目的 研究老年患者中,两种材料修复体冠修复前后不同时期冠边缘龈下菌斑中粘性放线菌的定植变化。 方法 收集门诊就诊老年患者中下颌第一磨牙要求行冠修复的患者共60例,随机编入二氧化锆组与钯银合金烤瓷冠组,采用自身前后对照法,将牙体预备前的患牙设为对照组,冠修复后6个月、12个月的患牙设为试验组,并分别在这3个时期收集冠边缘龈下菌斑,采用荧光定量聚合酶链式反应技术对临床样本进行粘性放线菌的相对定量分析。 结果 与修复前比较,二氧化锆全瓷冠组修复后6个月、12个月的粘性放线菌相对定量值差异无统计学意义（P＞0.05）;钯银合金烤瓷冠组修复后6个月、12个月的粘性放线菌相对定量值差异有统计学意义（P＜0.05）。 结论 二氧化锆全瓷冠可能可以更好地降低冠修复后患继发龋的风险。     

Inhibition of Actinomyces viscosus – Porphyromonas gingivalis coadhesion by trypsin and other proteins

Protease activity is associated with the coadhesion of Actinomyces viscosus and Porphyromonas gingivalis. To try to distinguish whether the recognition/adhesion or degradative functions of proteases are more crucial for coadhesion, we determined the effect of trypsin and other purchased proteases and proteins on coadhesion when they were incorporated in the coadhesion assay buffer or when A. viscosus cells were pretreated with trypsin. Coadhesion was measured by the decrease in turbidity caused by the absorption of A. viscosus cells from aqueous suspension by P. gingivalis-coated hexadecane droplets. Pretreatment of A. viscosus with trypsin had no obvious effect on the kinetics of coadhesion. Likewise, trypsinization of A. viscosus failed to aid or enhance coaggregation by chemically induced, trypsin activity-deficient mutants of B. gingivalis. In contrast, incorporating trypsin in the buffer during the coadhesion assay yielded a concentration-dependent inhibition of coadhesion greater than the inhibition found with the same concentration of other proteases. Coadhesion was also impaired to a greater extent by similar wt/vol concentrations of nonproteolytic proteins (bovine serum albumin (BSA), defatted BSA, gelatin, and casein), by antisera against whole P. gingivalis cells and fimbriae, by preimmune serum, and by the amino acid arginine but not lysine. These findings suggest that the role of proteases in coadhesion is not solely to enzymatically "prime" A. viscosus for more avid coadhesion and that their role as potential protein or peptide seeking adhesins should be considered.     

Inhibition of Actinomyces viscosus – Porphyromonas gingivalis coadhesion by trypsin and other proteins

Protease activity is associated with the coadhesion of Actinomyces viscosus and Porphyromonas gingivalis. To try to distinguish whether the recognition/adhesion or degradative functions of proteases are more crucial for coadhesion, we determined the effect of trypsin and other purchased proteases and proteins on coadhesion when they were incorporated in the coadhesion assay buffer or when A. viscosus cells were pretreated with trypsin. Coadhesion was measured by the decrease in turbidity caused by the absorption of A. viscosus cells from aqueous suspension by P. gingivalis-coated hexadecane droplets. Pretreatment of A. viscosus with trypsin had no obvious effect on the kinetics of coadhesion. Likewise, trypsinization of A. viscosus failed to aid or enhance coaggregation by chemically induced, trypsin activity-deficient mutants of B. gingivalis. In contrast, incorporating trypsin in the buffer during the coadhesion assay yielded a concentration-dependent inhibition of coadhesion greater than the inhibition found with the same concentration of other proteases. Coadhesion was also impaired to a greater extent by similar wt/vol concentrations of nonproleolytic proteins (bovine serum albumin (BSA), defatted BSA, gelatin, and casein), by antisera against whole P. gingivalis cells and fimbriae, by preimmune serum, and by the amino acid arginine but not lysine. These findings suggest that the role of proteases in coadhesion is not solely to enzymatically “prime” A. viscosus for more avid coadhesion and that their role as potential protein or peptide seeking adhesins should be considered.