Tool Compounds to Probe Multispecies Biofilms in the Human Oral Microbiome

用于探测人类口腔微生物组中多物种生物膜的工具化合物

• 批准号: 9533103
• 负责人: William M Wuest
• 金额: $ 37.01万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9533103
• 关键词: Affect; Bacteria; Binding; Biochemical; Biological; Biological Assay; Cells; Cellular Morphology; Characteristics; Chemicals; Childhood; Chronic; Clinical; Collaborations; Communities; Complex; Confocal Microscopy; Dental; Dental caries; Development; Disease; Drug Targeting; Endocarditis; Environment; Evaluation; Exhibits; Florida; Future; Genetic; Goals; Growth; Human; Investigation; Knock-out; Knowledge; Label; Laboratories; Lactams; Lead; Libraries; Light; Methods; Microbe; Microbial Biofilms; Microbiology; Molecular Genetics; Natural Products; Organic Synthesis; Organism; Phenotype; Process; Property; Proteomics; Research; Signal Transduction; Specificity; Streptococcus gordonii; Streptococcus mutans; Stress; Structure; Structure-Activity Relationship; Sum; Symbiosis; Synthesis Chemistry; System; Techniques; Testing; Therapeutic Agents; Work; analog; base; chemical stability; chemical synthesis; combat; commensal microbes; dental agent; design; drug development; experimental study; imaging probe; improved; in vivo; inhibitor/antagonist; interdisciplinary approach; interest; killings; nanomolar; oral bacteria; oral commensal; oral microbiome; oral pathogen; pathogen; pharmacophore; protein expression; response; scaffold; screening; tool; tool development; transcriptomics

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.

项目概要 拟议的研究将以 carolacton 的天然产物支架为起点 用于干扰变形链球菌 (SMU) 生物膜的物质。卡罗拉克顿 (Carolacton) 非凡：没有分散 建立生物膜或杀死浮游细胞，carolacton 目标细胞转变为生物膜状态， 并且在非常低的浓度下就有效。在这里，我们提出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、转录组学和微生物测定 从 carolacton 开始，但总体目标是开发可用于 用于了解多物种环境中的 SMU 生物膜过程。 第一个具体目标旨在确定 carolacton 的具体目标以及最近发现的 对 SMU 生物膜也有深远影响的类似物。这种方法将利用遗传、 转录组和 MS 蛋白质组技术来鉴定结合 carolacton 的候选者，从中我们 将使用生化研究确认目标。这将揭示自然过程 产品影响并允许对一般生物膜过程中的目标进行更广泛的评估。 第二个具体目标涉及合理设计的相关化合物的化学合成 到卡罗莱克顿。拟议的化合物将重点关注四个主要方面：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 保留生物活性的支架； 3）改善理化性质； 4) 身份识别 用于牙科复合材料的铅化合物的制备。高效简洁策略的核心 提出的是我们之前描述的全合成中获得的知识。 第三个目标是研究单一物种中工具化合物的生物学特性 和多物种生物膜。初步结果表明，carolacton 和一种新的 合成的类似物对变形链球菌生物膜产生独特且特异的表型反应。所以， 所有类似物都将使用共聚焦技术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。然后将在两种变形链球菌临床分离株的测定中对先导化合物进行分析 和其他与龋齿有关的细菌与大学合作。罗切斯特和大学。的 佛罗里达。同时，将在 S. mutans 和 S. mutans 之间的混合物种生物膜中评估化合物 共生细菌（即血链球菌、戈登链球菌）来识别病原体特异性抑制剂。以前的 研究表明，信号系统受损的口腔病原体的生存能力会降低 在共生细菌存在的情况下，我们打算在这项早期工作的基础上再接再厉。