SMALL BIOEFFECTOR MOLECULES OF STREPTOCOCCUS MUTANS

变形链球菌的生物效应小分子

• 批准号: 2897165
• 负责人: Jeffrey D. Hillman
• 金额: $ 32.66万
• 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/2897165
• 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JH1000菌株是一株临床分离株 因为它优越的殖民能力而被深入研究 口腔使其成为替代疗法的合理选择 有很多龋齿。突变分析表明，该菌株的 殖民潜力取决于其产量小，高度 有效的细菌素样抑制物质(BLIS)，具有广泛的 抗菌活性谱。最近的研究已经证明， BLIS的活性是一种以前未被识别的抗生素，它已经被 命名为MUTACIN 1140。在确定这一活动的过程中，我们还 鉴定了两个N-酰基-L-高丝氨酸内酯类化合物 革兰氏阴性细菌中是否有参与群体感应的分子 以前在革兰氏阳性菌中没有描述过。 初步证据表明，这两种化合物中的一种或两种 正向调节突变蛋白1140的合成。 本申请中提出的某些研究旨在阐明 突变体1140的结构、化学和遗传学研究 在替代疗法中的应用以及作为抗生素和食品的应用 防腐剂。其他研究旨在提纯AHSL样蛋白 分子能够使其精确和详细的结构 人物刻画。将使用生理学研究和突变分析 以确认它们在群体感应中的作用。 在本提案的具体目标1中，大规模提纯方法 将用于获得足够的突变体1140以实现完全 用化学方法和核磁共振波谱进行结构鉴定。 在特定目标2中，突变体1140对靶点的作用机制 将通过确定其在体内形成气孔的能力来分析菌株 它们的细胞质膜。在具体目标3中，我们将克隆和 测定突变蛋白1140合成和合成所必需的所有基因的序列 确定它们的结构组织。同基因突变株将是 构建和测试以确定不同基因在 突变素1140的合成。在具体目标4上，优化栽培 将确定合成AHSL的条件和方法 开发的目的是将它们净化成同质。他们将在结构上 用光谱学方法进行了表征。在具体目标5中，a Tn917诱变JH1140报告基因标记载体的研究 而缺乏突变体1140表达正调控因子的突变体将 被隔离和分析。