变异链球菌葡萄糖基转移酶转录调控因子的研究进展

葡萄糖基转移酶是变异链球菌自身合成的一种重要的固有酶,是变异链球菌重要的致龋因子,它以蔗糖作为代谢底物,合成生物膜重要的胞外多糖葡聚糖,葡聚糖一方面介导细菌蔗糖依赖的生物膜形成,另一方面当外源性糖源缺乏时,被降解供细菌继续代谢。因此可以通过控制葡萄糖基转移酶而控制龋病的发生发展。本文就变异链球菌葡萄糖基转移酶基因转录的调控因子作一综述。     

口腔链球菌果糖基转移酶的分子结构和调控机制

果糖基转移酶是口腔细菌细胞外多糖合成酶之一,它以蔗糖为底物催化合成果聚糖.口腔链球菌果糖基转移酶是口腔重要的致龋蛋白之一.笔者下面就果糖基转移酶的分子结构、反应机制和编码基因及其调节作一综述.     

果聚糖代谢及其致龋作用

变形链球菌能分泌果糖基转移酶,利用蔗糖合成果聚糖,它由ftf基因编码。同时还能分泌果聚糖水解酶,由fru A基因编码。当碳源消耗后,fru A活性被激活,水解果聚糖为单一的果糖产物。以上两种酶和聚合多糖有助于细菌和牙釉质膜的作用,加速致龋菌在牙面的集聚和增强产酸能力,与龋病的发生发展密切相关。     

果聚糖代谢及其致龋作用

变形链球菌能分泌果糖基转移酶，利用蔗糖合成聚糖，它由ｆｔｆ基因编码。同时还能分泌果聚糖水解酶，由ｆｒｕＡ基因编码。当碳源消耗后，ｆｒｕＡ活性被激，水解果聚糖为单一果糖产物。以上两种酶和聚合多糖有助于细菌和牙釉质膜的作用，加速致龋菌在牙面的集聚和增产酸能力，与龋病的发生发展密切相关。     

糖类型对混合致龋菌产酸影响的研究

本文分析五种致龋菌在混合培养时利用蔗糖,葡萄糖和果糖产酸,了解糖类型对细菌产酸的影响。结果表明混合致龋菌在利用蔗糖、葡萄糖和果糖时产生的酸的类型是相同的。蔗糖和葡萄糖产生的总酸量相同,果糖产生的总酸量明显低于前二者。各种糖产生的乳酸量无明显差异。     

菌斑细菌糖酵解前后胞内乳酸脱氢酶活性的研究

目的 研究细菌胞内乳酸脱氢酶(lactate dehydrogenase,LDH)在糖酵解过程中的表达特性,以了解菌斑细菌代谢的产酸机制.方法 取3种临床分离菌株(发酵乳杆菌、变形链球菌及寡发酵链球菌)和临床混合牙菌斑,在给糖前后经超声破壁后采用连续动力学法检测细菌胞内LDH活性.结果 单菌和集合菌斑细菌糖酵解前后LDH活性均无变化.但不同细菌LDH活性不同,发酵乳杆菌LDH活性[(52.91 ±8.97) U/mg]显著高于寡发酵链球菌[(0.61 ±0.05) U/mg]和变形链球菌[(0.32 ±0.07) U/mg],P＜0.01;集合菌斑LDH活性为(16.48±3.83) U/mg,加入1,6-二磷酸果糖(fructose 1,6-diphosphate,FDP)后活性显著增高[(26.37 ±8.26) U/mg] (P＜0.01).结论 不同菌种胞内LDH的活性不同,牙菌斑中存在FDP依赖型的细菌.     

两种糖对变异链球菌产酸能力影响的比较研究

目的:通过测量变异链菌代谢高果糖玉米糖浆(high-fructose corn syrup,HFCS)和蔗糖后△pH值的变化,比较两种糖对变异链球菌产酸能力的影响。方法:配制质量浓度为0.25%、0.5%、1%、3%和5%的高果糖玉米糖浆和蔗糖培养基,将变异链球菌UA159于上述各培养基37℃微需氧培养,分别于培养1、4、8、24、48 h各个时间点,用酸度计测量培养前后的pH值,计算ΔpH值,代表变异链球菌的产酸能力。结果:在本研究设定的糖浓度范围和培养时间段内,高果糖玉米糖浆(HFCS)和蔗糖培养基中的ΔpH值均随时间的延长而增大,在培养4～8 h内,HFCS培养基内pH值下降速度快于蔗糖培养基。在培养4、8、24 h时,5种浓度的HFCS培养基中ΔpH值均明显大于蔗糖培养基,差异均有统计学意义(P<0.05),而在培养1 h和48 h时,两种糖培养基中ΔpH值的变化无统计学意义(P>0.05)。结论:在培养4～8 h内,HFCS利于变异链球菌代谢产酸,而在24～48 h内,蔗糖则显示出较强的持续产酸能力。     

高果糖玉米糖浆对变形链球菌粘附能力的影响

目的：以目前食品加工业中广泛使用的高果糖玉米糖浆（HFCS）为研究对象,通过与蔗糖、葡萄糖和果糖的比较,研究高果糖玉米糖浆对变形链球菌粘附能力的影响。方法：将变形链球菌uA159分别接种于0．25％、0．5％、1％、3％、5％五组不同浓度TSB糖培养基培养18h后,用磷酸盐缓冲液洗脱2次并采用紫外分光光度计540nm检测各试管中的A值,计算粘附比,行方差分析,比较粘附力。结果：4种饮食糖培养基的粘附比存在统计学差异（P〈0．01）,HFCS组的变形链球菌粘附比显著低于蔗糖组,与葡萄糖和果糖组之间无显著差异;5组浓度糖培养基的变形链球菌粘附比之间的差异也具有统计学意义（P〈0．01）。结论：HFCS对变形链球菌粘附力的促进弱于蔗糖,与葡萄糖和果糖无明显差异。     

口腔细菌代谢产碱及其分子生物学研究进展

口腔细菌代谢产碱对牙菌斑生物膜菌群平衡和酸碱平衡极其重要,口腔中唾液和细菌的代谢产碱水平与龋病的发生呈负相关。口腔中代谢产碱是由多细菌多途径组成的复杂体系,受诸多环境因素的密切调控。本文就生物膜中细菌代谢产碱的主要途径、生物膜中细菌代谢产碱与龋病的关系、口腔细菌代谢产碱的分子生物学研究进展等作一综述。     

生物膜中细菌的代谢特点及影响因素

在口腔中的牙齿表面,细菌是以生物膜的方式生长,由于特定的生长方式,细菌将启动一套完全不同的基因系统,其生物学特征明显不同于游离状态的细菌。本文将结合生物膜的研究进展着重对牙菌斑中细菌的代谢特点及其影响因素作一综述。     

Effects of various antiplaque agents on fructosyltransferase activity in solution and immobilized onto hydroxyapatite

Fructosyltransferases (FTFs) are extracellular enzymes which synthesize fructans from sucrose. Cell free FTFs are found in the dental plaque biofilm as well as in saliva. Fructans play an important role in the progression of dental caries, mainly by serving as an extracellular nutrition reservoir for bacteria. The objective of the present study was to compare the effects of several antiplaque agents on the synthesis of fructans by FTF immobilized on hydroxyapatite (HA) or in solution. The effect of chlorhexidine, cetylpyridinium chloride, sodium lauryl sulfate and Tween on FTF activity was tested using radioactive assays. Their effect on fructan structure was tested using circular dichrosim-optical rotative dispersion (CD-ORD) analysis and Fourier transform infrared (FT-IR) spectroscopy. Our results show that the antiplaque agents tested had an inhibitory effect on FTF activity both in the immobilized phase and in solution, although the inhibitory effect was more pronounced in solution. Structural changes in fructans, due to the presence of the antiplque agents, were recorded as additional C-H or O-H bands demonstrated in FT-IR analysis. However, non-significant changes in peak location were detected in CD-ORD spectrum between fructans synthesized in solution and on HA surfaces, and after treatment with the different antiplaque agents. Our study shows that several antiplaque agents may affect FTF activity and the synthesis of fructans by FTF, immobilized on hydroxyapatite or in solution.     

The influence of sucralose on bacterial metabolism

Sucralose (1',4',6' trideoxy-trichloro-galactosucrose) is a nontoxic, intensely sweet sucrose derivative that has been shown to be non-cariogenic in experimental animals. The purpose of this study was to determine whether certain oral bacteria could utilize sucralose. Sucralose, as a sole carbon source, was unable to support growth of ten strains of oral bacteria and dental plaque. When sucrolose was incorporated into a liquid medium containing glucose or sucrose, all organisms tested displayed similar pH falls, compared with controls. The incorporation of 126 mmol/L sucralose into glucose agar medium caused total inhibition of growth of Streptococcus sobrinus 6715-17, Streptococcus sanguis 10904, Streptococcus sanguis Challis, Streptococcus salivarius, and Actinomyces viscosus WVU627. Sucralose had no effect on IPS production. Sucralose was not bound to, nor taken up by, cells. Sucralose inhibited the formation of glucan and fructan polymers in proportion to the sucralose-to-enzyme ratio, but independent of the sucrose concentration in the assay mixture. No radioactive polymer was formed from 14C-U-sucralose added to mixtures containing glucosyltransferase (GTF) or fructosyl-transferase (FTF). Inhibition of GTF and FTF by sucralose was removed following dialysis of the enzyme/sucralose mixture. These results show that sucralose was not utilized by the oral bacteria tested and that the inhibitory effect of sucralose on GTF and FTF was non-competitive and reversible. The results further support the concept that sucralose is non-cariogenic.     

An evaluation of remote communication versus face-to-face in clinical dental education

Distance learning and internet-based delivery of educational content are becoming very popular as an alternative to real face-to-face delivery. Clinical-based discussions still remain greatly face-to-face despite the advancement of remote communication and internet sharing technology. In this study we have compared three communication modalities between a learner and educator: audio and video using voice over internet protocol (VoIP) alone [AV]; audio and video VoIP with the addition of a three dimensional virtual artefact [AV3D] and physical face-to-face [FTF]. Clinical case discussions based on fictitious patients were held between a 'learner' and an 'expert' using the three communication modalities. The learner presented a clinical scenario to the experts, with the aid of a prop (partially dentate cast, digitised for AV3D), to obtain advice on the management of the clinical case. Each communication modality was tested in timed exercises in a random order among one of three experts (senior clinical restorative staff) and a learner (from a cohort of 15 senior clinical undergraduate students) all from the School of Clinical Dentistry, University of Sheffield. All learners and experts used each communication modality in turn with no prior training. Video recording and structured analysis were used to ascertain learner behaviour and levels of interactivity. Evaluation questionnaires were completed by experts and learners immediately after the experiment to ascertain effectiveness of information exchange and barriers/facilitators to communication. The video recordings showed that students were more relaxed with AV and AV3D than FTF (p = 0.01). The evaluation questionnaires showed that students felt they could provide (p = 0.03) and obtain (p = 0.003) more information using the FTF modality, followed by AV and then AV3D. Experts also ranked FTF better than AV and AV3D for providing (p = 0.012) and obtaining (p = 0) information to/from the expert. Physical face-to-face learning is a more effective communication modality for clinical case-based discussions between a learner and an expert. Remote, internet-based discussions enable a more relaxed discussion environment. The effectiveness of 3D supported internet-based communication is dependent upon a robust and simple to use interface, along with some prior training.     

Effect of biofilm formation on virulence factor secretion via the general secretory pathway in Streptococcus mutans

Objectives

To investigate the role of SecA in protein secretion, and to evaluate the effect of biofilm formation on protein secretion in Streptococcus mutans.

Design

S. mutans strains UA159 and GS-5 were used in this study. Cells grown in biofilm and planktonic conditions were observed using immunogold electron microscopy. The mRNA levels of ftf, gtfB, gtfC, gtfD, Pac and secA were analysed in different growth conditions using real-time quantitative polymerase chain reaction. The levels of wall proteins and whole-cell protein extracts were examined using Western blot analysis.

Results

A microdomain colocalised with SecA and virulence factors such as Pac (AgI/II) and glucosyltransferase (GTF) was observed. The mRNA level of secA was upregulated in the biofilm condition. The level of protein expression of SecA and wall protein levels of GTF, fructosyltransferase (FTF) and Pac (AgI/II) in the biofilm condition were significantly higher than in the planktonic condition.

Conclusions

These data suggest that S. mutans utilises the Sec pathway to secrete virulence factor proteins such as Pac (AgI/II), GTF and FTF, and protein secretion occurred at a distinct microdomain. The level of SecA, the key factor in the Sec pathway, was influenced significantly by biofilm formation in S. mutans.     

On the transformation of sulfur-containing amino acids and peptides to volatile sulfur compounds (VSC) in the human mouth

Halitosis is most often caused by oral conditions. Volatile sulfur compounds (VSC), constituting the major components of oral malodor, are produced by anaerobic, gram-negative bacteria retained mainly in periodontal pockets or on the tongue dorsum. Sulfur-containing amino acids serve as substrate for these bacteria. VSC have also been found to have unfavorable effect on the tissue. The aim of this study was to examine whether normal, healthy individuals with no history of halitosis were able to produce VSC from cysteine, when applied as a mouthrinse. A further aim of the study was to investigate and compare the potential of other sulfur-containing amino acids and peptides as substrates for oral VSC production and to localize the odor-production sites. A portable sulfide monitor was used for VSC registration. Results showed that all test subjects produced high oral concentrations of VSC upon rinses with cysteine, which thus seems to be a major substrate for VSC production. The other sulfur-containing substrates had much less effect. It was found that the tongue was the major site for VSC production, and that saliva per se caused low VSC production.     

Influence of irrigation regimens on the adherence of Enterococcus faecalis to root canal dentin

Enterococcus faecalis is frequently associated with post-treatment endodontic infections. Because adherence of bacteria to a substrate is the earliest stage in biofilm formation, eliciting the factors that links adherence of this bacterium to dentin would help in understanding its association with treatment-failed root canals. This investigation aimed to study the effects of endodontic irrigants on the adherence of E. faecalis to dentin. The bacteria adherence assay was conducted by using fluorescence microscopy, and the adhesion force was measured by using atomic force microscopy. There were significant increases in adherence and adhesion force after irrigation of dentin with ethylenediaminetetraacetic acid (EDTA), whereas sodium hypochlorite (NaOCl) reduced it. With the use of chlorhexidine (CHX), the force of adhesion increased, but the adherence assay showed a reduction in the number of adhering bacteria. The irrigation regimen of EDTA, NaOCl, and CHX resulted in the least number of adhering E. faecalis cells. This study highlighted that chemicals that alter the physicochemical properties of dentin will influence the nature of adherence, adhesion force, and subsequent biofilm formation of E. faecalis to dentin.     

Degradation of immunoglobulin G by periodontal bacteria

Several subgingival microorganisms were tested for their ability to utilize human immunoglobulin G (IgG) as a substrate for growth. This was done using a protein-free chemically defined medium, supplemented with IgG. Stimulation of growth was observed for Capnocytophaga ochracea, Porphyromonas asaccharolytica, Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, Prevotella oralis, Lactobacillus catenaforme and Streptococcus intermedius. Immunoelectrophoresis, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and a protein assay demonstrated that P. intermedia and P. endodontalis completely degraded the protein chains of IgG. Partial breakdown of IgG was observed for P. asaccharolytica and C. ochracea , whereas P. oralis cleaved the IgG heavy chain, yielding Fc and Fab fragments. All these bacteria utilized IgG as a substrate for growth. Binding studies using an enzyme-linked immunosorbent assay, revealed complete loss of in vitro antigen-antibody binding capacity after incubation of specific IgG with P. endodontalis and partial loss of binding with P. intermedia , P. gingivalis, C. ochracea or Fusobacterium nucleatum. Degradation or inactivation of IgG by oral bacteria is thought to be important in the causation of polymicrobial infections.     

五倍子对牙周可疑致病菌蛋白酶和糖苷酶活性的抑制作用

目的　体外观察五倍子水提取物对牙周可疑致病菌胞外酶 (蛋白水解酶和糖苷酶 )活性的影响。方法　应用荧光分光光度计检测荧光底物的降解来测定酶活力。结果　除中间普菌的L -arginine -arylamidase活性不受五倍子的影响外 ,浓度0 .0 5 %～ 0 .10 %的五倍子可以抑制所测其他细菌≥ 5 0 %胞外酶底物的降解。结论　五倍子水提取物可有效抑制牙周可疑致病菌蛋白水解酶和糖苷酶活性 ,其抑制作用可能会阻碍细菌生长 ,降低细菌毒力 ,减缓牙菌斑的形成     

In silico search of inhibitors of Streptococcus mutans for the control of dental plaque

Biofilm is an extremely complex microbial community arranged in a matrix of polysaccharides and attached to a substrate. Its development is crucial in the pathophysiology of oral infections like dental caries, as well as in periodontal, pulp, and periapical diseases. Streptococcus mutans is one of the most effective microorganisms in lactic acid production of the dental biofilm. Identifying essential Streptococcus mutans proteins using bioinformatics methods helps to search for alternative therapies. To this end, the bacterial genomes of several Streptococcus mutans strains and representative strains of other cariogenic and non-cariogenic bacteria were analysed by identifying pathogenicity islands and alignments with other bacteria, and by detecting the exclusive genes of cariogenic species in comparison to the non-pathogenic ones. This study used tools for orthology prediction such as BLAST and OrthoMCL, as well as the server IslandViewer for the detection of pathogenicity islands. In addition, the potential interactome of Streptococcus mutans was rebuilt by comparing it to interologues of other species phylogenetically close to or associated with cariogenicity. This protocol yielded a final list of 20 proteins related to potentially virulent factors that can be used as therapeutic targets in future analyses. The EIIA and EIIC enzymatic subunits of the phosphotransferase system (PTS) were prioritized, as well as the pyruvate kinase enzyme, which are directly involved in the metabolism of carbohydrates and in obtaining the necessary energy for the microorganism’s survival. These results will guide a subsequent experimental trial to develop new, safe, and effective molecules in the treatment of dental caries.     

Glycosaminoglycan-depolymerizing enzymes produced by anaerobic bacteria isolated from the human mouth

A number of obligately anaerobic bacteria, some implicated in periodontal disease, were screened for their ability to produce enzymes capable of degrading hyaluronic acid and chondroitin-4-sulphate. Two screening methods were used following anaerobic incubation at 37 degrees C for 7 days. One involved incorporating the respective substrates and bovine-serum albumin into agar plates and, after incubation, flooding the plates with 2 M acetic acid. Clear zones were produced around colonies which produced enzymes capable of depolymerizing the substrates. The second was a sensitive spectrophotometric procedure based on the ability of certain bacteria to produce eliminase enzymes, which degrade the substrates to unsaturated products having a characteristic u.v. absorption at 232 nm. Strains of Bacteroides gingivalis and Bacteroides melaninogenicus degraded both substrates whereas Bacteroides asaccharolyticus degraded neither substrate by either method. Some bacteria gave negative results with the plate method whereas the more sensitive spectrophotometric assay proved positive. The number of anaerobic bacteria capable of degrading hyaluronic acid and chondroitin-4-sulphate in vitro may therefore have been underestimated in previous studies.