The competence regulon in Streptococcus mutans biofilms

变形链球菌存在于牙菌斑的生物膜中，在那里它从饮食中产生酸 碳水化合物会导致龋齿。变形链球菌使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。这项提案涉及 细胞-细胞和环境信号激活这种“生物膜表型”的机制。法定变形链球菌 感知系统由comCDE基因编码，编码一种能力刺激肽(Csp)。 前体、组氨酸激酶和反应调节因子。我们希望回答1)哪些表型变化 CSP是否通过其各种假定的受体和调节器来激活？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)我们能抑制吗？ 这些途径削弱了变形链球菌的生物被膜表型？将使用DMA微阵列分析来 识别CSP激活的基因和蛋白质。一些在各种成分上有缺陷的突变体 将用于阐明这些复杂的信令网络。编码产品的基因 生物膜环境中改变的表达将被克隆并使用新的等位基因交换技术进行突变； 将对突变体的遗传能力和生物膜形成能力进行测试。细胞分离与3D 突变生物膜的结构将通过扫描电子显微镜和共聚焦扫描激光显微镜进行评估 (CSLM)。在选定基因中构建的报告基因融合(GFP、LacZ和Luc)将用于测量 用荧光和发光分析和CSLM方法研究CSP对基因表达的影响 深入了解CSP信号级联如何实时影响生物膜表型。CSP 将测试类似物对信号转导过程的抑制作用。监管程序 参与调节这一网络的研究将使用DNA-蛋白质结合分析，蛋白质-蛋白质 相互作用和磷酸化分析。这些实验将使我们深入了解信号机制。 被变形链球菌和其他可能形成生物膜的病原体用来在生物膜和Will中实现最佳生存 希望能导致设计出控制它们的手段。

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.