葡聚糖结合蛋白A抗体对变形链球菌黏附影响的研究

目的：了解葡聚糖结合蛋白(CbP)A抗体对变形链球菌黏附的影响情况。方法：利用核素闪烁计数法，测定不同浓度的葡聚糖结合蛋白抗体影响两种变形链球菌对羧基磷灰石(HA)的黏附率。结果：葡聚糖结合蛋白抗体可明显抑制两种变形链球菌对羧基磷灰石的黏附率，并且随着葡聚糖结合蛋白抗体浓度的提高，抑制作用增强。结论：葡聚糖结合蛋白抗体可抑制变形链球菌对羧基磷灰石的黏附，在免疫防龋方面具有一定的意义。     

变形链球菌gbpA的葡聚糖结合区GBD肽段的纯化及葡聚糖结合功能实验

目的：纯化变形链球菌gbpA的葡聚糖结合区GBD肽段。方法：蛋白质技术：金属螯合亲和层析，葡萄糖结合实验。结果：通过蛋白质技术及金属螯合亲和层析，纯化出可融性融合蛋白GBD。此蛋白可与α－1，6键葡聚糖结合。结论：通过基因重组技术成功纯化出变形链球菌gbpA的葡聚糖结合区GBD肽段并证实此重组蛋白具有葡聚糖结合功能。     

变形链球菌葡聚糖结合蛋自的研究进展

本文介绍了一类对牙菌斑的发育、结构和致龋力具有重要意义的变形链球菌表面蛋白-葡聚糖结合蛋白,阐述了其作用、种类及分子结构等。     

变形链球菌葡聚糖结合蛋白的研究进展

本文介绍了一类对牙菌斑的发育，结构和致龋力具有重要意义的变形链球菌表面蛋白－葡聚糖结合蛋白，阐述了其作用，种类及分子结构等。     

变异链球菌葡聚糖结合蛋白B的生物学特性研究进展

变异链球菌是人类龋病的主要致病菌,葡聚糖结合蛋白是其重要的毒力因子之一.葡聚糖结合蛋白B在变异链球菌的致龋、维持细菌形态、发挥细胞壁生理功能等方面有重要作用,同时其N端具有免疫原性,可应用于免疫防龋.下面就葡聚糖结合蛋白B的生物学特性研究进展作一综述.     

变形链球菌葡聚糖结合蛋白B的纯化

目的：从变形链球菌S.mutans Ingbritt(serotype c)培养液上清中纯化葡聚糖结合蛋白B。方法：Sephadx G-200凝胶（α-1,6键葡聚糖）亲和层析和Q－SephroseFF离子交换层析。结果：纯化出约10μg具有葡聚糖结合特性的GbpB蛋白。结论：通过Sephadx G-200凝胶（α-1,6键葡聚糖）亲和层析和Q－Sephrose FF离子交换层析纯化出变链菌葡聚糖结合蛋白B。     

葡聚糖结合蛋白的生物学特性

葡聚糖（Ｇｌｕｃａｎ）是由变形链球菌产生的葡萄糖基转移酶（Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ,ＧＴＦ）利用蔗糖合成的由α－１,６和α－１,３糖苷键连结的细胞外多糖,它通过与位于细菌表面的特异性蛋白结合,介导细菌在牙面上粘附和集聚,调节牙面微生态系统,促进致病性牙菌斑微生态系统的成熟,起着十分重要的作用［１,２］。这类位于细菌表面能与葡聚糖结合的蛋白质,称为葡聚糖结合蛋白（Ｇｌｕｃａｎ－ｂｉｎｄｐｒｏｔｅｉｎ,ＧＢＰ）,通过与葡聚糖α－１,６糖苷键结合而凝集细菌的ＧＢＰ,称之为葡聚糖结合凝集素…     

变形链球菌葡聚糖结合蛋白D基因的分子克隆及其原核表达和初步纯化

目的:克隆变形链球菌葡聚糖结合蛋白D(gbpD)基因,在大肠杆菌中表达并对重组蛋白进行初步纯化,为进一步的蛋白功能研究奠定基础。方法:体外培养变形链球菌UA159菌株并以其基因组为模板,对gbpD基因编码区进行PCR扩增,连接pMD-18T载体并测序。随后将该片段克隆入原核表达载体pPROEX HTb中,构建表达质粒pPROEX/gbpD,转化大肠杆菌DH5α并用IPTG诱导表达。表达产物经镍-次氮基三乙酸(Ni-NTA)柱进行亲和层析纯化。结果:成功克隆变形链球菌gbpD基因并在大肠杆菌中得到可溶性表达,经Ni-NTA柱纯化后获得纯度大于95%、相对分子量(Mr)为76×103的变链菌GbpD蛋白。结论:获得Mr为76×103的变形链球菌GbpD蛋白。对进一步确定GbpD的葡聚糖结合能力,探讨其在变链菌致龋过程中的作用,以及在龋病预防领域的应用前景都具有重要的意义。     

口腔微生物与免疫学

变形链球菌葡聚糖结合蛋白B基因防龋疫苗的构建及其在哺乳动物细胞中的表达，变形链球菌耐氟菌株、远缘链球菌耐氟菌株与亲代菌株基因组AP-PCR的比较，变形链球菌体外耐酸能力的实验研究，测定牙菌斑氟浓度的超微型F-ISFET电极的研制，变形链球菌变链素粗提物活性的初步研究     

变形链球菌遗传Ⅰ型和Ⅲ型利用不同糖类合成葡聚糖的研究

对变形链球菌Ⅰ型ＪＢＰ和Ⅲ型６７１５利用蔗糖、麦芽糖、葡萄糖、果糖和甜菊糖合成葡聚糖的能力进行研究，并以红外光谱和薄层层析鉴定葡聚糖样品。结果显示两种变形链球菌只能利用蔗糖合成葡聚糖，且合成葡聚糖总体能力相似。     

十肽对变异链球菌作用的差异蛋白质组学研究

目的 利用差异蛋白质组学技术检测十肽对变异链球菌蛋白的作用反应,初步鉴定并验证变异链球菌致龋过程中的部分重要蛋白质。方法 人工合成十肽（氨基酸序列：KKVVFKVKFK-NH2）,利用双向电泳技术分离十肽作用前后变异链球菌的总蛋白,采用质谱分析联合生物信息学技术鉴定十肽作用前后发生差异性改变的变异链球菌蛋白质,并在蛋白质水平验证关键差异蛋白质烯醇酶的表达情况。结果 十肽作用前后变异链球菌蛋白质发生明显变化,其中发生差异性改变的蛋白质功能主要包括降解碳水化合物（经糖酵解途径）,蛋白质折叠、结合、运输及蛋白质翻译等。Western blot验证结果显示,十肽处理后烯醇酶的表达量明显降低。结论 人工合成抗菌肽十肽处理前后变异链球菌的蛋白质表达发生明显变化,推测其可能通过改变烯醇酶等标志性蛋白的表达而达到防龋作用。     

Effects of deuterium oxide on Streptococcus mutans and Pseudomonas aeruginosa

A complex aggregation of microorganisms growing on a solid substrate is termed a biofilm and is considered to be an etiological agents. Pseudomonas aeruginosa and Streptococcus mutans are representative bacteria in such biofilms. It is well known that deuterium oxide (D?O) causes toxic effects on a number of biological systems. We investigated the effects of D?O on growth and biofilm formation of P. aeruginosa and S. mutans. These bacteria were incubated in medium containing D?O (100%, 75% or 0%) at 37°C for 24hr, 48 hr or 72 hr. Growth of P. aeruginosa was inhibited by D?O within the first 48 hr. However, after 72 hr, growth rate was seen to increase in the D?O-containing medium compared with in medium without D?O. In contrast, the growth of S. mutans in the D?O medium was inhibited within 72 hr. The biofilm formation of P. aeruginosa was increased in the D?O medium. Biofilm formation of S. mutans in the D?O medium increased compared with in the medium without D?O, but this increase was only temporary in the case of P. aeruginosa. Compared to biofilm formation in 0% D?O medium marked as 100%, the biofilm formation rate of S. mutans in 75% D?O medium was 143% at 24 hr, 146% at 48 hr and 130% at 72 hr. In other D?O concentration media biofilm formation was lower. In 100% D?O medium, biofilm formation rate decreased from 114% at 24 hr to 56% at 72 hr. The biofilm formation rate of P. aeruginosa in 100% D?O medium was 172% at 24 hr, but decreased to 88% at 72 hr. Biofilm formation of P. aeruginosa in 75% and 0% D?O media showed no significant difference. We consider that these results were due to stress or alteration in bacterial metabolisms.     

Effects of human milk on adhesion of Streptococcus mutans to saliva-coated hydroxyapatite in vitro

Adhesion of bacteria to pellicle-coated tooth surfaces is the first step in biofilm formation on teeth. The aim of this study was to explore whether human milk prevents or promotes adhesion of cariogenic Streptococcus mutans to saliva-coated hydroxyapatite (HA) using an in vitro model system. S. mutans binding to HA coated with human parotid saliva (s-HA) or human milk was studied, in addition to binding inhibition to s-HA by human milk. S. mutans did not bind to HA coated with milk. S. mutans binding to s-HA was inhibited by milk from 15 (71 %) of 21 women, whereas milk from the remaining 6 mothers enhanced binding of S. mutans to s-HA. Inhibition of S. mutans binding correlated with bacterial binding to s-HA (r = 0.76). Human milk does not mediate adhesion of S. mutans to HA in vitro, but affects adhesion in an individually varying fashion. Phenotypic variations in milk and saliva glycosylation may explain the inhibitory capacity and possibly affect susceptibility to colonization by S. mutans in childhood.     

The oligopeptide (opp) gene cluster of Streptococcus mutans: identification, prevalence, and characterization

INTRODUCTION: The Opp system is an ATP-binding cassette-type transporter formed by membrane-associated proteins required for the uptake of oligopeptides in bacteria. In gram-positive bacteria, the Opp system, and particularly the oligopeptide-binding protein (OppA), has been shown to be involved in different aspects of cell physiology, including intercellular communication and binding to host proteins. METHODS: In the present study we began to investigate the Opp system of Streptococcus mutans, the main etiological agent of dental caries. RESULTS: Five opp genes (oppABCDF) organized in a single operon were identified in the genome of the S. mutans UA159 strain. Amino acid sequence analyses showed that the S. mutans OppA is closely related to an ortholog found in Streptococcus agalactiae. Incubation of S. mutans UA159 cells with an anti-OppA-specific serum did not inhibit biofilm formation on polystyrene plates. Moreover, S. mutans UA159 derivatives carrying deletions on the oppA or oppB genes did not show significant growth impairment, increased sensitivity to aminopterin, or defective capacity to form biofilms on polystyrene wells in the presence or not of saliva. Remarkably, only two out of three laboratory strains and one out of seven clinical strains recovered from tooth decay processes harbored a copy of the oppA gene and expressed the OppA protein. CONCLUSION: Collectively, these results indicate that, in contrast to other Streptococcus species, the S. mutans Opp system, and particularly the OppA protein, does not represent an important trait required for growth and colonization.     

Involvement of antigen I/II surface proteins in Streptococcus mutans and Streptococcus intermedius biofilm formation

BACKGROUND/AIM: Dental diseases are caused by microorganisms organized in biofilms. Streptococcus mutans and Streptococcus intermedius are commensals of the human oral cavity. S. mutans is associated with caries, whereas S. intermedius is associated with purulent infections. Oral streptococci including S. mutants and S. intermedius express a family of surface proteins termed antigen I/II (Ag I/II). Ag I/II is implicated in adhesion; however, its role in biofilm formation has not yet been investigated. METHODS: By using isogenic Ag I/II-deficient mutants of S. mutans and S. intermedius we studied the influence of Ag I/II on in vitro biofilm formation. Biofilm was quantified in polystyrene microtiter plates and visualized by scanning electron microscopy. Ag I/II expression in planktonic and biofilm cells, as well as in the presence or absence of saliva was investigated by immunoblotting. RESULTS: In the presence of saliva, the Ag I/II-deficient mutants formed 65% less biofilm than the wild-types. In the absence of saliva, no difference was observed in S. mutans, whereas the S. intermedius Ag I/II mutant formed 41% less biofilm. Ag I/II expression was reduced in the presence of saliva. No differences in expression were observed between biofilm and planktonic cells. CONCLUSION: The results indicated that Ag I/II may be important during biofilm formation particularly in the presence of saliva. These findings may provide useful information regarding the importance of Ag I/II in biofilm formation and in the search of new strategies to control biofilm-mediated infections.     

Role of sucrose in plaque formation

Results are presented which support the concept that the bacterial enzyme glucosyltransferase (GTF) plays a crucial role in sucrose induced plaque formation. GTF was shown to adhere strongly to anionic, hydrophobic and polysaccharide solid materials, and to be able to produce glucans in the adsorbed state. It appears conceivable that GTF adsorb to teeth and produce glucans. Glucan chains on (he surface of the bacteria and glucans on the tooth surfaces interact (pack) and form a strong binding mechanism. The rigid α1,3 linked glucans produced by Streptococcus mutans are particularly suited for interaction of this kind. This mechanism could account for sucrose-induced binding of bacteria to enamel, pellicle covered enamel and preformed plaque. S. mutans would adhere particularly strongly to tooth surfaces in the presence of sucrose, according to this model.     

Role of sucrose in plaque formation

Results are presented which support the concept that the bacterial enzyme glucosyltransferase (GTF) plays a crucial role in sucrose induced plaque formation. GTF was shown to adhere strongly to anionic, hydrophobic and polysaccharide solid materials, and to be able to produce glucans in the adsorbed state. It appears conceivable that GTF adsorb to teeth and produce glucans. Glucan chains on the surface of the bacteria and glucans on the tooth surfaces interact (pack) and form a strong binding mechanism. The rigid alpha 1,3 linked glucans produced by Streptococcus mutans are particularly suited for interaction of this kind. This mechanism could account for sucrose-induced binding of bacteria to enamel, pellicle covered enamel and preformed plaque. S. mutans would adhere particularly strongly to tooth surfaces in the presence of sucrose, according to this model.     

Real-time interaction of oral streptococci with human salivary components

Oral streptococci are present in large numbers in dental plaque and several types interact with the enamel salivary pellicle to form a biofilm on tooth surfaces. The respective affinity of individual streptococci for salivary components has an influence on the etiologic properties of oral biofilm in the development of dental caries. We studied real-time biospecific interactions between oral streptococci and salivary components utilizing biosensor technology to analyze surface plasmon resonance. Streptococcus sanguis and Streptococcus mutans showed significant responses for binding to salivary components, in comparison with other bacteria. Further, the association rates (4.1 x 10-11/bacterium) and dissociation rate (5.7 +/- 0.9 x 10-3 Second(s)-1) were higher for S. sanguis than for S. mutans (2.4 x 10-11 and 2.9 +/- 0.8 x 10-3) and Streptococcus mitis (1.3 x 10-11 and 3.5 +/- 1.3 x 10-3). However, the association equilibrium constants (8.2 S/bacterium) for S. mutans was 2 times higher in than S. mitis (3.8) and slightly higher than S. sanguis (7.2). These findings may provide useful information regarding the mechanism of early biofilm formation by streptococci on the tooth surface.     

Effect of sublethal CO2 laser irradiation on gene expression of streptococcus mutans immobilized in a biofilm

Streptococcus mutans colonizing on tooth surfaces is one of the major causative agents of human dental caries. Despite numerous studies conducted on lasers and oral tissue interactions, little is known about the effect of laser energy on S. mutans gene expression in a biofilm form. The aim of this study was to investigate the effect of sublethal energies of CO(2) laser on biofilm and gene expression of the oral bacteria S. mutans immobilized in biofilm. S. mutans biofilm was irradiated with CO(2) laser. Vitality and construction of the biofilm were observed by confocal laser scanning microscopy and scanning electron microscopy. The effect of laser irradiation on gene expression was evaluated by DNA microarray. CO(2) laser irradiation had a dose effect on the viability of S. mutans immobilized in biofilm. A nonsignificant lethal effect was observed at 31 J/cm(2) while at higher energy of 70 and 144 J/cm(2) an antibacterial effect was recorded. The mode of antibacterial action seems to be from the inner layers toward the outer layer of the biofilm, indicating the influence of the surface on the killing effect. At 31 J/cm(2), microarray analysis indicated a moderate effect on S. mutans gene expression due to CO(2) laser irradiation, mainly down-regulating genes related to bacterial stress response. In conclusion, laser irradiation at sublethal energy had an effect on gene expression of S. mutans.