Spatial Organization of the Oral Microbiome

口腔微生物组的空间组织

• 批准号: 10163156
• 负责人: Gary G Borisy
• 金额: $ 62.13万
• 依托单位: ADA FORSYTH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-12-03 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10163156
• 关键词: Address; Architecture; Attention; Biological Assay; Cell Communication; Cells; Chemistry; Communities; Complex; Corynebacterium; DNA; DNA sequencing; Data; Dental Plaque; Detection; Development; Disease; Environmental Microbiology; Fluorescent in Situ Hybridization; Gingiva; Goals; Grant; Habitats; Hard Palate; Health; Health Transition; Homeostasis; Human; Human Microbiome; Image; Imaging Techniques; Individual; Intervention; Label; Lead; Length; Maintenance; Messenger RNA; Metabolic; Metagenomics; Methods; Microbe; Molecular; Nitrate Reductases; Nitrites; Oral; Oral cavity; Oral mucous membrane structure; Pathway interactions; Peroxides; Play; Positioning Attribute; Probiotics; Process; Pyruvate Oxidase; Reaction; Research; Resolution; Ribosomal RNA; Role; Saliva; Site; Streptococcus; Structure; Surveys; Taxonomy; Techniques; Technology; Testing; Therapeutic; Therapeutic Intervention; Time; Work; active lifestyle; base; catalase; combinatorial; design; fluorescence imaging; host-microbe interactions; innovation; mRNA Expression; member; microbial; microbial community; microbiome; microbiome research; microbiota; microorganism interaction; multiplexed imaging; next generation; novel; oral biofilm; oral microbiome; single cell analysis; spectrograph; tongue dorsum; tool

ABSTRACT The human mouth is colonized by a complex microbial community that plays a key role in human health and disease. Increasingly, it is recognized that the spatial organization of microbiomes is critical to understanding the interactions of the individual taxa that comprise a community. Although next-generation DNA sequencing technology and metagenomics have revolutionized the analysis of microbial communities, these technologies require that the microbial cells first be broken open and the DNA extracted, processes in which spatial organization is lost. Thus, a major gap in our understanding is the lack of information at the spatial scale at which microbiomes live and work--the micron scale. In our previous grant period, we sought to fill this gap by using the strategy of combinatorial labeling and spectral imaging fluorescence in situ hybridization (CLASI-FISH) to analyze the micron-scale architecture of microbial communities in the healthy oral cavity--basically to determine “who lives next to who” and “who lives next to what”. This strategy was successful in that it revealed highly organized and hitherto unanticipated microbial community structures at the genus level in dental plaque and on the tongue dorsum. Much work remains to be done in characterizing these communities and ones at other oral sites as well. But it is clear that specification of community structure must be deepened to the species level. Further, it is clear that one needs to go beyond taxonomic identification. To understand the mechanistic bases of microbial relationships, it is necessary to gain data on their functional expression at the single-cell level. Also, the static images acquired after imaging fixed cells provide only snapshots in time. It is necessary to figure out ways to study the dynamics of microbial communities—how they form, develop and maintain themselves. The specific aims of this proposal are directed at these three issues. We will broaden and deepen analysis of key oral habitats so that the major microbial taxa within oral communities can be identified to species level. To test hypotheses generated from the structural results, we will develop probes for expression of key mRNA molecules at the single-cell level. To test hypotheses on the development and dynamics of microbial communities, we will develop probes for metabolic capacity in living cells using bioorthogonal click chemistry and combine their application with culture of oral microcosms. The three approaches build on the work of the previous grant period, sharing the common thread of single-cell analysis through multiplexed imaging. By revealing the precise, micron-scale structure, mRNA expression and dynamics of the oral microbiome, this research will address fundamental mechanisms and principles of community function and assembly. It will impact the broader oral and microbial research communities by developing methods, analysis tools and probes, and it should help identify novel targets for health maintenance or therapeutic intervention. ! !

抽象的 人口是由一个复杂的微生物群落殖民的，该社区在人类健康中起着关键作用 疾病。越来越多地认识到，微生物组的空间组织对于理解至关重要 包括社区的单个分类单元的互动。虽然下一代DNA测序 技术和宏基因组学彻底改变了对微生物群落的分析，这些技术 要求首先打开微生物细胞并提取DNA，其中空间的过程 组织丢失。这是我们理解的一个主要差距是在空间尺度上缺乏信息 哪些微生物组生存和工作 - 微米量表。 在上一个赠款期间，我们试图通过使用组合标签的策略和 光谱成像荧光原位杂交（CLASI-FISH）分析微观尺度结构 健康口腔中的微生物群落 - 基本上确定“谁住在谁旁边”和“谁生活 旁边是”。这种策略是成功的，因为它揭示 在牙齿斑块和舌背上的属水平的微生物群落结构。很多工作 在表征这些社区和其他口头遗址的社区和一个社区方面还有待事。但是很明显 社区结构的规范必须加深到物种水平。此外，很明显一个人需要 要了解微生物关系的机械基础，这是 需要在单细胞水平上获得其功能表达的数据。另外，获得的静态图像 成像后，固定细胞仅提供快照。有必要弄清楚研究的方法 微生物群落的动态 - 如何形成，发展和维护自己。 该提案的具体目的针对这三个问题。我们将扩大和加深分析 主要的口腔栖息地，以便可以将口腔社区内的主要微生物分类群确定为物种水平。 为了测试从结构结果产生的假设，我们将开发出表达关键mRNA的问题 分子在单细胞水平上。测试有关微生物发展和动力学的假设 社区，我们将使用生物正交点击化学来为活细胞中的代谢能力开发出问题 并将其应用与口服缩影培养结合在一起。这三种方法建立在 上一个赠款期，通过多重成像共享单细胞分析的共同线程。 通过揭示口服微生物组的精度，微米尺度结构，mRNA表达和动力学， 这项研究将解决社区功能和集会的基本机制和原理。会 通过开发方法，分析工具和 问题，它应该有助于确定健康维持或治疗干预的新目标。 呢 呢