Mechanisms of Virulence Gene Regulation in Streptococcus mutans

描述（由申请人提供）：龋齿是人类最常见的细菌感染之一，在许多不发达国家仍未得到治疗。根据生物化学、流行病学和动物实验，变形链球菌被认为是龋齿的主要病原。代谢碳水化合物和在牙齿表面粘附并形成坚韧的生物膜的能力被认为与这种人类病原体的致龋性密切相关。变形链球菌通过三种葡萄糖转移酶（Gtf）从蔗糖合成葡聚糖，并在它们的配合下牢固地粘附在牙齿表面。它还产生葡聚糖结合蛋白（Gbps），在毒力中起主要作用。变形链球菌胞外多糖合成调控的具体机制尚未被发现。然而，最近发现一种孤儿反应调节因子GcrR可以调节至少一个Gtf和一个Gbp基因的表达。值得注意的是，gcrR的失活显著减少了大鼠生物膜的形成和龋齿的发生。因此，GcrR似乎对该生物的发病机制非常重要。此外，GcrR与致病性A组链球菌（GAS） CovR具有广泛的序列同源性（>80%），后者是一种应答调节因子，控制多达15%的GAS基因，包括许多重要的毒力因子。鉴于GcrR与CovR的高度相似性及其在生物膜和龋齿发生中的作用，人们预计GcrR可能是变形链球菌的全球调节剂。因此，我们选择关注GcrR的基因调控，具体目标如下：在目标1中，我们将通过DNA微阵列和纯化gcrR蛋白的体外DNA结合试验来鉴定gcrR调控子。我们将用蛋白质组学方法来证实我们的结果。在Aim 2中，我们将确定GcrR基因调控的机制。我们将在GcrR调控的基因（如gtfD， gbpC）的启动子上识别和表征GcrR结合基序。在Aim 3中，我们将研究gcrR的表达调控。由于GcrR的同源传感激酶在附近的GcrR位点缺失，我们将使用生化和遗传方法识别同源传感激酶以了解GcrR的调控。这项研究将促进我们对变形链球菌基因调控和信号转导的分子机制的理解，并促进旨在控制斑块生物膜形成和随后的龋齿发生的治疗方法的发展。

DESCRIPTION (provided by applicant): Dental caries is one of the most common bacterial infections in humans and remains untreated in many underdeveloped countries. Based on biochemical, epidemiological and animal experiments, Streptococcus mutans is considered to be the principal etiological agent of Dental caries. The ability to metabolize carbohydrates and to adhere to and form tenacious biofilms on the tooth surfaces are believed to be critically associated with the cariogenicity of this human pathogen. S. mutans synthesizes glucans from sucrose by three glucosyltransferases (Gtf), and adheres firmly to tooth surfaces with their cooperation. It also produces glucan-binding proteins (Gbps) which play major roles in virulence. The specific mechanisms governing regulation of exopolysaccharide synthesis in S. mutans have yet to be discovered. Nevertheless, recently it was found that an orphan response regulator, GcrR, modulates the expression of at least one Gtf and one Gbp gene. Remarkably, inactivation of gcrR drastically reduces biofilm formation and cariogenesis in rat. Therefore, GcrR appears to be very important for pathogenesis of this organism. In addition, GcrR shows extensive sequence homology (>80%) with the pathogenic group A streptococcus (GAS) CovR, a response regulator that controls as much as 15% of the GAS genes including many important virulence factors. Given the high degree of similarity with CovR and its effect in biofilm and cariogenesis, one would expect that GcrR may be a global regulator of S. mutans. Therefore, we have chosen to focus on gene regulation by GcrR with the following Specific Aims. In Aim 1, we will identify the gcrR regulon by DNA microarray and in vitro DNA binding assays using purified GcrR protein. We will confirm our results with proteomics approach. In Aim 2, we will determine the mechanisms of gene regulation by GcrR. We will identify and characterize the GcrR binding motif(s) on the promoters of genes (such as gtfD, gbpC) regulated by GcrR. In Aim 3, we will study the regulation of gcrR expression. Since the cognate sensor kinase of GcrR is absent in the nearby gcrR locus, using both biochemical and genetic approaches we will identify the cognate sensor kinase to understand gcrR regulation. This (investigation will promote our understanding of molecular mechanisms of gene regulation and signal transduction in S. mutans and facilitate the development of therapeutic approaches Aimed at controlling formation of plaque biofilm and subsequent cariogenesis.