Tool Compounds to Probe Multispecies Biofilms in the Human Oral Microbiome

项目摘要 拟议的研究将以Carolacton的天然产物支架为起点 用于扰乱变形链球菌(SMU)生物被膜的药物。卡罗拉克顿不同凡响：非但没有消散 建立生物膜或杀死浮游细胞，Carolacton针对过渡到生物膜状态的细胞， 而且它在非常低的浓度下是有效的。在这里，我们介绍了一种多方面的方法，包括 有机合成、分子遗传学、蛋白质组学、转录组学和微生物分析 从Carolacton开始，但作为首要目标，开发工具化合物可以 用于了解多物种环境中的SMU生物膜过程。 第一个具体目标是确定Carolacton的特定靶点(S)和最近发现的一个 对SMU生物膜也有深远影响的类似物。这种方法将利用基因， 转录学和MS蛋白质组学技术来识别与Carolacton结合的候选对象，我们从这些候选对象中 将使用生化研究来确认目标。这将阐明自然的 产品影响并允许在一般生物膜工艺中对目标进行更广泛的评估。 第二个具体目标涉及合理设计的化合物的化学合成。 敬卡罗拉克顿。建议的化合物将集中在四个主要方面：1)获得详细的结构- 对Carolacton结构的活性关系的理解；2)确定最小络合物 保持生物活性的支架；3)改善物理化学性质；4)鉴定 用于牙科复合材料的铅化合物。是高效和简明战略的核心 提出的是在我们前面描述的全合成中获得的知识。 第三个目标是研究工具化合物在单一物种中的生物学特性。 和多物种生物膜。初步结果表明，Carolacton和一种新的 人工合成的类似物对变形链球菌生物被膜具有独特和特异的表型反应。因此， 所有类似物都将使用共聚焦针对浮游细胞和生物膜细胞与变形链球菌UA159进行测试 显微镜。然后，将对两种变形链球菌的临床分离株进行分析，分析先导化合物。 和其他涉及龋齿的细菌，与大学合作。罗切斯特大学和美国大学。的 佛罗里达。同时，化合物将在变形链球菌和变形链球菌之间的混合物种生物膜中进行评估。 共生细菌(即血链球菌、戈登链球菌)，以确定病原体特定的抑制物。上一首 研究表明，信号系统受损的口腔病原体的生存能力降低。 在存在共生细菌的情况下，我们打算在这一早期工作的基础上再接再厉。

Project Summary The proposed research will interrogate the natural product scaffold of carolacton as a starting point for agents that perturb S. mutans (SMU) biofilms. Carolacton is remarkable: instead of dispersing established biofilms or killing planktonic cells, carolacton targets cells transitioning into the biofilm state, and it is effective at very low concentrations. Here we present a multi-faceted approach, including organic synthesis, molecular genetics, proteomics, transcriptomics, and microbiological assays that begins with carolacton but has as an overarching goal the development of tool compounds that can be used to understand SMU biofilm processes within a multispecies environment. The first specific aim seeks to identify the specific target(s) of carolacton and a recently discovered analog that also has profound effects on SMU biofilms. This approach will employ genetic, transcriptomic, and MS-proteomic techniques to identify candidates that bind carolacton, from which we will confirm the targets using biochemical studies. This will shed light on the processes that the natural product affects and allow for a broader evaluation of the target in general biofilm processes. The second specific aim involves the chemical synthesis of rationally designed compounds related to carolacton. Proposed compounds will focus on four main aspects: 1) obtaining a detailed structure- activity relationship understanding of the carolacton structure; 2) determining the minimally complex scaffold that retains biological activity; 3) improving the physiochemical properties; and 4) identification of a lead compound for use in dental composites. Central to the efficient and concise strategies proposed is the knowledge gained in our previously described total synthesis. The third aim will investigate the biological properties of the tool compounds both in single species and multispecies biofilms. Preliminary results have identified that both carolacton and a newly synthesized analog cause a unique and specific phenotypic response to S. mutans biofilms. Therefore, all analogs will be tested against both planktonic and biofilm cells with S. mutans UA159 using confocal microscopy. Lead compounds will then be analyzed in assays with clinical isolates of both S. mutans and other bacteria involved in dental caries in collaboration with the Univ. of Rochester and the Univ. of Florida. Concurrently, compounds will be evaluated in mixed-species biofilms between S. mutans and commensal bacteria (i.e. S. sanguinis, S. gordonii) to identify pathogen-specific inhibitors. Previous work has demonstrated that oral pathogens with compromised signaling systems are reduced viability in the presence of commensal bacteria and we intend to build on this earlier work.