Tool Compounds to Probe Multispecies Biofilms in the Human Oral Microbiome

项目摘要 拟议的研究将询问carolacton的天然产物支架作为起点 干扰S的物质变形杆菌（SMU）生物膜。Escherichiacton是显着的：而不是分散 建立生物膜或杀死增殖细胞，carolacton靶向细胞过渡到生物膜状态， 并且它在非常低的浓度下有效。在这里，我们提出了一个多方面的方法，包括 有机合成、分子遗传学、蛋白质组学、转录组学和微生物测定， 从carolacton开始，但作为一个总体目标， 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的具体目标和最近发现的 类似物，也对SMU生物膜有深远的影响。这种方法将利用遗传， 转录组学和MS-蛋白质组学技术来鉴定结合carolacton的候选物， 将通过生化研究确认目标这将揭示自然界中 产品影响并允许在一般生物膜过程中对靶标进行更广泛的评价。 第二个具体目标涉及合理设计的化合物的化学合成， 去卡罗拉克顿。提出的化合物将集中在四个主要方面：1）获得详细的结构- carolacton结构的活性关系理解; 2）确定最小复杂度 保持生物活性的支架; 3）改善理化性质;和4）鉴定 用于牙科复合材料的铅化合物。高效简洁战略的核心 所提出的是在我们先前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，卡罗拉克顿和一种新的 合成类似物引起对S.变形杆菌生物膜因此，我们认为， 所有类似物都将针对具有S.使用共聚焦显微镜 显微镜然后将在两种S的临床分离株的测定中分析先导化合物。变形 和其他与龋齿有关的细菌，与罗切斯特大学和 佛罗里达。同时，将在S.变形菌和 肠道细菌（S. sanguinis，S. Gordonii）以鉴定病原体特异性抑制剂。先前 研究表明，具有受损信号系统的口腔病原体的存活率降低， 我们打算在早期工作的基础上继续研究。

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.