The competence regulon in Streptococcus mutans biofilms

变形链球菌生物膜的能力调节子

• 批准号: 7744647
• 负责人: Dennis G. Cvitkovitch
• 金额: $ 48.01万
• 依托单位: UNIVERSITY OF TORONTO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7744647
• 关键词: Acids; Address; Affect; Amino Acid Sequence; Architecture; Area; Attenuated; Bacteria; Base Pairing; Binding; Binding Sites; Biochemical; Biological Assay; Cell Count; Cell Density; Cell Separation; Cell Survival; Cells; Competence; Complex; Consensus Sequence; DNA; DNA Binding; DNA Footprint; DNA-Binding Proteins; Dental Plaque; Dental caries; Detection; Dietary Carbohydrates; Environment; Gene Expression; Gene Expression Profile; Gene Fusion; Gene Proteins; Genes; Genetic; Genetic Transcription; Genome; Goals; Growth; In Vitro; Individual; LacZ Genes; Laser Scanning Confocal Microscopy; Laser Scanning Microscopy; Lead; Measures; Mediating; Microarray Analysis; Microbial Biofilms; Molecular Profiling; Mutagenesis; Mutate; Operator Regions; Pathway interactions; Peptide Receptor; Peptide Signal Sequences; Peptides; Phenotype; Phosphorylation; Physiological; Process; Promoter Regions; Regulon; Relative (related person); Reporter Genes; Role; Scanning Electron Microscopy; Signal Transduction; Signal Transduction Inhibition; Signal Transduction Pathway; Site; Streptococcus mutans; System; Techniques; Testing; Time; base; design; extracellular; gel mobility shift assay; in vitro Assay; inhibitor/antagonist; insight; mRNA Expression; mutant; novel; pathogen; peptide analog; promoter; protein protein interaction; protein-histidine kinase; quorum sensing; receptor; research study; response; stress tolerance; uptake

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.

变形链球菌存在于牙菌斑的生物膜中，从饮食中产生酸 碳水化合物会引起龋齿。变形链球菌利用生物膜中的群体感应信号系统来激活 遗传能力、耐酸性并影响生物膜结构。该提案解决了 细胞间和环境信号激活这种“生物膜表型”的机制。变形链球菌法定人数 传感系统由编码能力刺激肽 (CSP) 的 comCDE 基因编码 前体、组氨酸激酶和反应调节剂。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器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）我们可以抑制吗 这些减弱变形链球菌生物膜表型的途径？ DMA 微阵列分析将用于 识别由 CSP 激活的基因和蛋白质。许多突变体的各种成分都有缺陷 该系统的功能将用于阐明这些复杂的信号网络。基因编码产物 生物膜环境中改变的表达将使用新型等位基因交换技术进行克隆和突变； 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体（gfp、lacZ 和 luc）将用于测量 使用荧光和发光分析以及 CSLM 暂时响应 CSP 的基因表达，以给出 深入了解 CSP 信号级联如何发挥作用来实时影响生物膜表型。太阳能光伏发电 将测试类似物对信号转导过程的抑制作用。监管流程 将使用DNA-蛋白质结合测定、蛋白质-蛋白质结合分析来研究参与调节该网络的过程 相互作用和磷酸化测定。这些实验将深入了解信号机制 变形链球菌和其他潜在的生物膜形成病原体利用它们在生物膜中实现最佳生存，并将 希望能够设计出控制它们的手段。