The competence regulon in Streptococcus mutans biofilms

描述：变形链球菌存在于牙菌斑的生物膜中，在那里它从饮食中的碳水化合物中产生酸来导致龋齿。变形链球菌在生物膜中使用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。这项申请阐述了细胞-细胞和环境信号激活这种生物膜表型的机制。变形链球菌群体感应系统由comCDE基因编码，编码一个能力刺激肽(CSP)前体、一个组氨酸激酶和一个反应调节因子。我们期望回答：1)CSP激活的哪些基因参与了生物被膜的形成？2)这一途径是如何调节的？3)我们能否抑制这一途径来减弱变形链球菌的生物被膜表型？4)其他环境信号是否激活了生物被膜表型？差异显示(DdPCR)和2D凝胶电泳将用于识别CSP激活的基因和蛋白质。一个同基因的comc(CSP缺失)突变体将被用来破译这一调节基因。将比较其在外源合成CSP作用下的ddPCR和2D表达谱。将使用一种新的等位基因交换技术克隆和突变编码表达改变的产物的基因；将测试突变体的遗传能力。细胞分离和突变生物膜的3D结构将通过扫描电子显微镜和共聚焦扫描激光显微镜(CSLM)进行评估。构建在这些基因中的报告基因融合(GFP、LacZ和Luc)将被用来利用荧光和光度分析和CSLM来测量CSP响应的基因表达，从而实时了解CSP信号级联功能如何影响生物膜的表型。将测试CSP类似物对信号转导过程的抑制作用。变形链球菌也有两个组分信号转导系统(TCST)来感知环境信号。生物信息学分析已在变形链球菌中鉴定出13个TCST，并构建了各自基因缺陷的突变体。使用机器人生长监测器，将检测突变体在酸、盐、过氧化氢、十二烷基硫酸钠和高糖浓度等环境胁迫下的生长能力，以阐明TCSTS的功能。这些实验将深入了解变形链球菌以及可能形成生物膜的其他细菌使用的信号机制。

DESCRIPTION: 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 application 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 of the genes activated by CSP are involved in biofilm formation? 2) How is this pathway regulated? 3) Can we inhibit this pathway to attenuate the biofilm phenotype of S. mutans? 4) Do other environmental signals activate the biofilm phenotype? Differential display (ddPCR) and 2D gel electrophoresis will be used to identify genes and proteins that are activated by the CSP. An isogenic comC (CSP-deficient) mutant will be used to decipher this regulon. Its ddPCR and 2D expression profiles with and without exogenous synthetic CSP will be compared. Genes encoding products with altered expression will be cloned and mutated using a novel allelic exchange technique; the mutants genetic competence 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 these genes will be used to measure gene expression temporally in response to CSP using fluorometric 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. S. mutans also has two component signal transduction systems (TCSTS) to sense environmental signals. Bioinformatic analyses have identified 13 TCSTS in S. mutans, and mutants defective in their individual genes were constructed. Using a robotic growth monitor, the mutants will be examined for the ability to grow in the presence of environmental stresses: acid, salts, peroxide, SDS, and high sugar concentration to elucidate the function of the TCSTS. These experiments will give insight into the signaling mechanisms used by S. mutans and potentially other biofilm-forming bacteria.