SMALL BIOEFFECTOR MOLECULES OF STREPTOCOCCUS MUTANS

变形链球菌菌株JH 1000是一种临床分离株， 因为它的上级能力， 口腔使其成为替代疗法的合理选择 龋齿。 突变分析表明， 殖民潜力取决于其生产的一个小的，高度 有效的细菌素样抑制物质（BLIS），具有广泛的 抗菌活性谱。 最近的研究表明， BLIS活性是一种以前未鉴定的lantibiotic， 命名为突变蛋白1140。 在确定这一活动的过程中，我们还 鉴定了两种N-酰基-L-高丝氨酸内酯（AHSL）样化合物， 是革兰氏阴性菌中参与群体感应的分子， 这在革兰氏阳性细菌中以前没有描述过。 初步证据表明，这些化合物中的一种或两种 正调节突变蛋白1140的合成。 本申请中提出的某些研究旨在阐明 突变蛋白1140的结构、化学和遗传学， 在替代疗法中的应用以及作为抗生素和食品 防腐剂 其他研究旨在纯化AHSL样 分子，使其精确和详细的结构 特征化 生理学研究和突变分析将被用于 来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 将被用于获得足够的突变蛋白1140，以使得能够完成 用化学方法和核磁共振波谱进行结构鉴定。 在具体目的2中，突变蛋白1140对靶向肿瘤细胞的作用机制是： 菌株将通过测定其形成孔的能力来分析， 它们的细胞质膜 在具体目标3中，我们将克隆和 对突变蛋白1140合成所必需的所有基因进行测序， 决定其组织结构。 同基因突变体将是 构建和测试，以确定各种基因在 突变蛋白1140合成。 在具体目标4中， 将确定AHSL合成的条件和方法， 将其提纯至均一。 它们将在结构上 使用光谱方法表征。 在具体目标5中， 用Tn 917诱变报告基因标记的JH 1140构建体 缺乏突变蛋白1140表达的正调节因子的突变体将 进行分离和分析

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.