GLUCAN BINDING PROTEINS OF ORAL STREPTOCOCCI

变形链球菌是龋齿的病原，它具有几种葡聚糖结合蛋白（GBPs），这有助于它粘附和定植牙齿表面的能力。酶（GBPs）是催化葡聚糖合成的葡萄糖基转移酶（GTFs）。非酶（GBPs）被认为有助于斑块的粘附、聚集和一致性，但它们的作用尚未得到很好的证明。因此，尽管对变形链球菌的发病机制有了一般的了解，但GBPs多样性的确切贡献和必要性尚未确定。也许正因为如此，缺乏临床措施来专门干预这些变形链球菌的毒力因子。研究发现，编码非酶GBP的基因gbpA失活会导致大鼠模型中毒力的变化和变异生物的积累，这些变异生物在连续的gtfB和gtfC基因之间进行重组，形成杂交gtfBC。与野生型相比，GTF重组体产生的葡聚糖量减少。有人提出，gbpA的失活导致斑块结构的变化，而这反过来又导致GTF重组体的选择性保留。以下具体目的是在检查单个GBPs对斑块基质形成的贡献的背景下检验这一假设。1)确定GTF重组体在GbpA突变体中的体内积累是由于重组频率增加还是由于选择力，并研究重组的分子机制。2)通过向生物体中引入多个GbpA拷贝，对一株过度表达GbpA的变形链球菌进行基因工程改造。3)在大鼠模型中检测基因工程的gbpa++和gbpa++过表达子，研究GTF重组子对定植、龋发育和积累的影响。4)合成含有变形链球菌GTF-I、GTF-SI和GTF-S葡聚糖结合域的融合蛋白，确定其对葡聚糖和变形蛋白的亲和力。将结果与GbpA进行比较。5)通过检测具有增加细胞表面gtf水平的突变体S. mutans 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。总的来说，这些研究代表了一种新的方法，将GbpA作为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.