SMALL BIOEFFECTOR MOLECULES OF STREPTOCOCCUS MUTANS

变形链球菌的生物效应小分子

基本信息

批准号：
6379814
负责人：
Jeffrey D. Hillman
金额：
$ 25.51万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-06-01 至 2003-05-31
项目状态：
已结题

项目摘要

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.

链球菌突变菌株JH1000是一种临床分离株，已经是深入研究，因为它具有殖民人类的卓越能力口腔使其成为替换疗法的合乎逻辑选择龋齿。突变分析被用来表明该污渍定殖潜力取决于其高度生产有效的细菌样抑制物质（BLI），具有宽活性的抗菌谱。最近的研究证明了 BLIS活动是先前未识别的甘脂蛋白，已经是命名为Mutacin 1140。在确定这项活动的过程中，我们也鉴定出两个N-酰基-L-霍森林内酯（AHSL）类似化合物，它们是参与革兰氏阴性细菌中的群体传感的分子和以前尚未在革兰氏阳性细菌中描述的。初步证据表明其中一种或两种化合物积极调节Mutacin 1140合成。本应用中提出的某些研究旨在阐明 Mutacin 1140的结构，化学和遗传学的潜力在替代疗法和抗生素和食物中的应用防腐剂。其他研究旨在净化类似AHSL 分子使其精确而详细的结构表征。将使用生理研究和突变分析确认它们在法定感应中的作用。在该提案的特定目的1中，大规模纯化方法将用于获得足够的育毒素1140以启用完整使用化学方法和NMR光谱法结构鉴定。在特定的目标2中，Mutacin 1140对靶的作用机理菌株将通过确定其形成毛孔的能力来分析菌株它们的细胞质膜。在特定的目标3中，我们将克隆和序列所有对于植物素1140合成必不可少的基因和确定其结构组织。等生突变体将是构造和测试以确定各种基因在 Mutacin 1140合成。在特定目标4中，最佳培养将确定AHSL合成条件，方法将为开发以将它们净化为同质性。他们将在结构上使用光谱法进行了表征。在特定的目标5中 JH1140的报告基因标记的构建体将用TN917诱变缺乏阳性调节剂的突变体1140表达将被隔离和分析。