The competence regulon in Streptococcus mutans biofilms

变形链球菌存在于牙菌斑的生物膜中，它从饮食中产生酸。 碳水化合物引起龋齿。S.变形杆菌利用生物膜中的群体感应信号系统来激活 遗传能力、耐酸性和影响生物膜结构。该提案涉及 细胞-细胞和环境信号激活这种“生物膜表型”的机制。S.变异法定人数 传感系统由comCDE基因编码，comCDE基因编码感受态刺激肽（CSP）， 前体、组氨酸激酶和应答调节剂。我们期望回答1）哪些表型变化 CSP是否通过其各种假定的受体和调节剂调用，以及这些变化如何促进 在生物膜环境中优化生长/存活？2)这些途径是如何调节的？3)我们能抑制 这些途径减弱了S.变种人？DMA微阵列分析将用于 识别由CSP激活的基因和蛋白质。许多突变体在各种成分上都有缺陷 将用于阐明这些复杂的信号网络。基因编码产物， 将使用新的等位基因交换技术克隆和突变生物膜环境中改变的表达; 将测试突变体的遗传能力和生物膜形成能力。细胞分离和3D 突变生物膜的结构将通过SEM和共聚焦扫描激光显微镜进行评估 （CSLM）。在选定的基因中构建的报告基因融合体（gfp、lacZ和luc）将用于测量 使用荧光和发光分析以及CSLM， 深入了解CSP信号级联如何发挥作用以真实的时间影响生物膜表型。CSP 将测试类似物对信号转导过程的抑制。规管程序 将使用DNA-蛋白质结合测定、蛋白质-蛋白质结合测定和蛋白质-蛋白质结合测定来研究参与调节该网络的分子。 相互作用和磷酸化测定。这些实验将使我们深入了解 使用S。变形菌和潜在的其他生物膜形成病原体，以在生物膜中获得最佳存活， 希望能设计出控制它们的方法。

Streptococcus mutans resides in the biofilm of dental plaque where it produces acid from dietary carbohydrate to cause caries. S. mutans uses a quorum sensing signaling system in biofilms to activate genetic competence, acid tolerance, and influence biofilm architecture. This proposal addresses mechanisms by which cell-cell and environmental signals activate this 'biofilm phenotype'. S. mutans quorum sensing system is encoded by the comCDE genes that encode a competence-stimulating peptide (CSP) precursor, a histidine kinase and a response regulator. We expect to answer 1)Which phenotypic changes does CSP invoke through its various putative receptors and regulators and how do these changes facilitate optimized growth/survival in a biofilm environment? 2) How are these pathways regulated? 3) Can we inhibit these pathways to attenuate the biofilm phenotype of S. mutans? DMA microarray analysis will be used to identify genes and proteins that are activated by CSP. A number of mutants defective in various components of the system will be used to elucidate these complex signaling networks. Genes encoding products with altered expression in a biofilm context will be cloned and mutated using a novel allelic exchange technique; the mutants' genetic competence and biofilm forming abilities will be tested. Cell segregation and the 3D architecture of the mutant biofilms will be assessed by SEM and Confocal Scanning Laser Microscopy (CSLM). Reporter gene fusions (gfp, lacZ and luc) constructed in selected genes will be used to measure gene expression temporally in response to CSP using fluorimetric and luminetric analysis and CSLM to give insight into how the CSP signaling cascade functions to influence biofilm phenotype in real time. CSP analogs will be tested for their inhibition of the signal transduction process. The regulatory processes involved at modulating this network will be investigated using DNA-protein binding assays, protein-protein interactions and phosphorylation assays. These experiments will give insight into the signaling mechanisms used by S. mutans and potentially other biofilm-forming pathogens for optimal survival in biofilms and will hopefully lead to the design of means to control them.