GLUCAN BINDING PROTEINS OF ORAL STREPTOCOCCI

变形链球菌是龋病的病原菌，其具有多种葡聚糖结合蛋白(GBP)，有助于其在牙齿表面的黏附和定植。酶(GBP)是催化合成葡聚糖的葡糖基转移酶(GTF)。非酶(GBP)被认为有助于粘附性、聚集性和斑块的一致性，但它们的作用没有很好的文献记载。因此，尽管对变形链球菌的发病机制有了普遍的了解，但GBP的确切贡献和多样性的必要性仍有待确定。或许正因为如此，临床上缺乏专门干预这些变形链球菌毒力因子的措施。已经发现，编码非酶GBP的基因f gbpA的失活导致了大鼠模型毒力的变化，并导致了变异生物的积累，这些变异生物经历了相邻的gtfb和gtfc基因之间的重组，形成了杂交的gtfBC。与野生型相比，GTF重组体产生的葡聚糖量减少。有人认为，gbpA的失活会导致斑块结构的改变，进而导致GTF重组体的选择性保留。以下具体目的是在检验个体GBps对斑块基质形成的贡献的背景下检验这一假说。1)确定GTF重组子在GbpA突变体中的体内积累是由于重组频率的增加还是由于选择力的作用，并探讨了重组的分子机制。2)通过将多个拷贝的gbpA引入到生物体中，从基因上设计出一种过度表达GbpA的变形链球菌。3)在大鼠模型中检测基因工程GbpA++和GbpA+过表达基因对GTF重组体的定植、龋齿形成和蓄积的影响。4)制备含有变形链球菌GTf-I、GTf-SI、GTf-S葡聚糖结合区的融合蛋白，并测定其与葡聚糖、变构体的亲和力。将结果与GbpA获得的结果进行比较。5)通过检测突变链球菌LT11的插入突变体，确定gbpA上游的DNA是否编码影响葡萄糖基转移酶表达或分布的产物，该突变体具有增加的细胞表面GTF水平。总体而言，这些研究代表了一种新的方法，将GbpA作为一种降低葡萄糖基转移酶活性的机制，从而降低变形链球菌的毒力。

Streptococcus mutans, the etiologic agent of dental caries, possesses several glucan binding proteins (GBPs) which contribute to its ability to adhere to, and colonize, tooth surfaces. The enzymatic (GBPs) are the glucosyltransferases (GTFs) which catalyze the synthesis of the glucans. The nonenzymatic (GBPs) are thought to contribute to adherence, aggregation, and the coherence of plaque but their roles are not as well documented. Thus, despite the general understanding of S. mutans pathogenesis, the precise contributions and necessity of the multiplicity of GBPs have yet to be determined. Perhaps as a consequence, there are a lack of clinical measures to specifically interfere with these S. mutans virulence factors. It has been found that the inactivation f gbpA, a gene encoding a nonenzymatic GBP, results in a change in virulence in a rat model and the accumulation of variant organisms which have undergone a recombination between the contiguous gtfB and gtfC genes to form a hybrid gtfBC. The GTF recombinants produce decreased amounts of glucan relative to the wild type. It is proposed that the inactivation of gbpA leads to a change in plaque structure and this in turn brings about a selective retention of GTF recombinants. The following specific aims test this hypothesis within the context of examining the contribution of individual GBPs to the formation of the plaque matrix. 1) Determine whether the in vivo accumulation of GTF recombinants among the GbpA mutants is due to an increased frequency of recombination or is due to selection forces, and investigate the molecular mechanism of he recombination. 2) Genetically engineer a strain of S. mutans that overexpresses GbpA by introducing multiple copies of gbpA into the organism. 3) Test the genetically engineered GbpA++ and GbpA+++ overexpresser in a rat model to examine the effects on colonization, caries development, and accumulation of GTF recombinants. 4) Make fusion proteins containing the glucan binding domains of the S. mutans GTF-I, GTF-SI, and GTF-S and determine their affinities for dextran and mutan. Compare the results with those obtained for GbpA. 5) Determine if the DNA upstream of gbpA encodes products that affect the expression or distribution of the glucosyltransferases by examining an insertional mutant of S. mutans LT11 that possesses increased levels of cell surface GTFs. Overall these studies represent a novel approach which targets GbpA as a mechanism for reducing glucosyltransferase activity and thereby the virulence of S. mutans.