GENE REGULATION IN S MUTANS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。变形链球菌被认为是人类龋病的主要病原菌，是口腔健康的全球性问题。变形链球菌也是感染性心内膜炎的主要原因。变形链球菌产生生物膜形成所需的一系列毒力因子，通常被称为牙菌斑。这些毒力因子受多个双组分系统(TCS)调控。典型的TCS由一个膜相关的传感器蛋白组成，它对特定的环境信号做出反应，并在保守的组氨酸残基上自动磷酸化。然后，这种磷酸化残基被转移到细胞质反应调节器(RR)进行激活。这种激活的RR调节某些靶基因的表达，例如那些形成生物膜所需的基因。在变形链球菌中，已鉴定出13个假定的TC。其中，COVR是一种重要的RR基因，在A组链球菌和B组链球菌中分别抑制15%和6%的基因表达。变形链球菌中COVR的失活会导致生物膜形成的改变，相应的突变体具有低龋性。利用DNA微阵列，我们实验室已经证明了变形链球菌中有大约100个基因受到COVR的调控。例如，COVR抑制编码葡聚糖结合蛋白的gbpc基因和编码葡萄糖转移酶的gtfb基因。与其他链球菌不同的是，变形链球菌中COVR的同源组氨酸激酶(HK)不在COVR附近。为了更好地了解牙菌斑形成所需的环境线索，本研究的重点是确定COVR的同源词HK(14个推定的HK)。为此，构建了14个质粒，通过单交叉整合使所有14个可能的HK基因失活。由于COVR调控PgbpC和PgtfB，因此也产生了转录报告融合，其中PgbpC-GusA或PgtfB-Gusa的单个拷贝被插入到异位染色体位置。本项目的目标是灭活PgbpC-Gusa或Pgtf-Gusa报告菌株中的所有14个HK基因，并研究这些启动子的GusA表达。我们期望找到一个或几个HK基因来调控这些启动子，因此可能是COVR的同源HK。一旦我们确定了同源的HK，我们未来的目标将是使用纯化的蛋白来研究COVR和HK的体外相互作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Streptococcus mutans is considered as the main etiological agent for human dental caries, a global problem in oral health. S. mutans is also a leading cause of infective endocarditis. S. mutans produces a wide range of virulence factors that are needed for the biofilm formation, commonly known as dental plaque. These virulence factors are regulated by several two-component systems (TCS). A typical TCS consists of a membrane- associated sensor kinase protein, which responds to specific environmental cues and is auto phosphorylated at a conserved histidine residue. This phosphorylated residue is then transferred to a cytoplasmic response regulator (RR) for activation. This activated RR modulates the expression of certain target genes, such as those needed for biofilm formation. In S. mutans, 13 putative TCS have been identified. Among them, CovR is an important RR that represses 15% of genes in Group A Streptococcus and 6% in Group B Streptococcus. The inactivation of the covR in S. mutans leads to to altered biofilm formation, and corresponding mutants are hypocariogenic. Using DNA microarray, our laboratory has shown that ~100 genes are regulated by CovR in S. mutans. For instance, CovR represses gbpC gene encoding a glucan-binding protein and gtfB gene encoding a glucosyl-transferase. Unlike other streptococcal species, the cognate histidine kinase (HK) for CovR in S. mutans is not located near the CovR locus. In order to better understand the environmental cues that are necessary for dental plaque formation, the focus of this research is to identify the cognate HK of CovR (out of 14 putative HK). Towards this end, 14 plasmids were constructed to inactivate all 14 putative HK genes by single-crossover integration. Since CovR regulates PgbpC and PgtfB, transcriptional reporter fusions were also generated in which a single copy of PgbpC-gusA or PgtfB-gusA was inserted in an ectopic chromosomal location. The goal of this project is to inactivate all 14 HK genes in the PgbpC-gusA or Pgtf- gusA reporter strains and study gusA expression from these promoters. We expect to find one or several HK genes will modulate these promoters and therefore may be the cognate HK for CovR. Once we identify the cognate HK, our future goal will be to study in vitro interaction of CovR and HK using purified proteins.