SMALL BIOEFFECTOR MOLECULES OF STREPTOCOCCUS MUTANS

变形链球菌的生物效应小分子

• 批准号: 6176151
• 负责人: Jeffrey D. Hillman
• 金额: $ 24.76万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2002-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6176151
• 关键词: Streptococcus mutans; antibiotics; bacterial genetics; bacterial proteins; bacteriocin; cell membrane; gene expression; mass spectrometry; molecular cloning; mutant; northern blottings; nuclear magnetic resonance spectroscopy; nucleic acid sequence; oral bacteria; peptide structure; pore forming protein; protein biosynthesis; protein purification; protein structure function; structural genes; thin layer chromatography; tissue /cell culture; transcription factor

Streptococcus mutans strain JH1000 is a clinical isolate which has been intensively studied because its superior ability to colonize the human oral cavity makes it a logical choice for use in the replacement therapy of dental caries. Mutant analysis was used to show that this stain's colonization potential is dependent on its production of a small, highly potent bacteriocin-like inhibitory substance (BLIS) that has a broad antimicrobial spectrum of activity. Recent studies have proven that the BLIS activity is a previously unidentified lantibiotic, which has been named mutacin 1140. In the course of identifying this activity, we also identified two N-acyl-L-homoserine lactone (AHSL)-like compounds, which are molecules involved in quorum sensing in Gram negative bacteria and which have not been previously described in Gram positive bacteria. Preliminary evidence indicates that one or both of these compounds positively regulates mutacin 1140 synthesis. Certain studies proposed in this application are designed to elucidate the structure, chemistry, and genetics of mutacin 1140 for its potential application in replacement therapy and as an antibiotic and food preservative. Other studies are designed to purify the AHSL-like molecules to enable their precise and detailed structural characterization. Physiology studies and mutant analysis will be used to confirm their role in quorum sensing. In specific aim 1 of this proposal, large scale purification methods will be used to obtain sufficient mutacin 1140 to enable complete structural identification using chemical methods and NMR spectroscopy. In specific aim 2, the mechanism of action of mutacin 1140 on target strains will be analyzed by determining its ability to form pores in their cytoplasmic membranes. In specific aim 3, we will clone and sequence all of the genes essential for mutacin 1140 synthesis and determine their structural organization. Isogenic mutants will be constructed and tested to determine the roles of the various genes in mutacin 1140 synthesis. In specific aim 4, the optimum cultivation conditions for AHSL synthesis will be determined and methods will be developed to purify them to homogeneity. They will be structurally characterized using spectroscopic methods. In specific aim 5, a reporter gene-labeled construct of JH1140 will be mutagenized with Tn917 and mutants lacking positive regulators for mutacin 1140 expression will be isolated and analyzed.

变形链球菌菌株 JH1000 是一种临床分离株，已被 因其卓越的殖民人类能力而被深入研究 口腔使其成为替代疗法的合理选择 的龋齿。 突变分析表明该染色剂 定植潜力取决于其产生的小而高的 强效细菌素样抑制物质 (BLIS)，具有广泛的作用 抗菌活性谱。 最近的研究证明， BLIS 活性是一种先前未鉴定的羊毛硫抗生素，已被 命名为mutacin 1140。在鉴定该活性的过程中，我们还 鉴定出两种 N-酰基-L-高丝氨酸内酯 (AHSL) 样化合物， 是参与革兰氏阴性细菌群体感应的分子， 以前在革兰氏阳性细菌中尚未描述过。 初步证据表明这些化合物中的一种或两种 正向调节 mutacin 1140 的合成。 本申请中提出的某些研究旨在阐明 Mutacin 1140 的结构、化学和遗传学及其潜力 在替代疗法以及作为抗生素和食品中的应用 防腐剂。 其他研究旨在纯化 AHSL 样物质 分子使其具有精确和详细的结构 表征。 将使用生理学研究和突变分析 确认它们在群体感应中的作用。 在该提案的具体目标 1 中，大规模纯化方法 将用于获得足够的突变蛋白 1140 以实现完整的 使用化学方法和核磁共振波谱进行结构鉴定。 在具体目标2中，突变蛋白1140对目标的作用机制 将通过确定其形成孔的能力来分析应变 它们的细胞质膜。 在具体目标 3 中，我们将克隆并 对突变蛋白 1140 合成所必需的所有基因进行测序，并 确定它们的结构组织。 同基因突变体将是 构建并测试以确定各种基因的作用 突变蛋白1140的合成。 具体目标4，最佳栽培 将确定 AHSL 合成的条件并确定方法 开发以将它们纯化至同质。 他们将在结构上 使用光谱方法进行表征。 在具体目标 5 中， 报告基因标记的 JH1140 构建体将用 Tn917 诱变 缺乏突变蛋白 1140 表达正调节因子的突变体将 进行隔离和分析。