Spatial Organization of the Oral Microbiome

口腔微生物组的空间组织

• 批准号: 10398953
• 负责人: Gary G Borisy
• 金额: $ 61.51万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10398953
• 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 人类口腔中栖息着复杂的微生物群落，对人类健康和健康发挥着关键作用。 疾病。人们越来越认识到微生物组的空间组织对于理解微生物组至关重要 组成群落的各个分类单元之间的相互作用。尽管下一代 DNA 测序 技术和宏基因组学彻底改变了微生物群落的分析，这些技术 要求首先将微生物细胞打碎并提取 DNA，该过程中的空间 组织失去了。因此，我们理解中的一个主要差距是缺乏空间尺度的信息 哪些微生物群在微米尺度上生存和工作。 在我们之前的资助期间，我们试图通过使用组合标签和 光谱成像荧光原位杂交（CLASI-FISH）分析微米级结构 健康口腔中的微生物群落——基本上是为了确定“谁住在谁旁边”和“谁住在谁旁边” 该策略的成功之处在于它揭示了高度组织性和迄今为止未曾预料到的 牙菌斑和舌背属水平的微生物群落结构。工作量很大 在描述这些群落和其他口腔场所的群落特征方面仍有待完成。但很明显的是 群落结构的规范必须深化到物种层面。此外，很明显，人们需要 超越分类学鉴定。要了解微生物关系的机制基础， 需要在单细胞水平上获得其功能表达的数据。另外，获取的静态图像 成像后，固定细胞仅及时提供快照。有必要找出研究方法 微生物群落的动态——它们如何形成、发展和维持自身。 本提案的具体目的就是针对这三个问题。我们将扩大和深化分析 关键口腔栖息地的研究，以便可以在物种水平上识别口腔群落内的主要微生物类群。 为了测试根据结构结果产生的假设，我们将开发用于关键 mRNA 表达的探针 单细胞水平的分子。检验有关微生物发育和动态的假设 社区，我们将使用生物正交点击化学开发活细胞代谢能力的探针 并将其应用与口腔微生物培养相结合。这三种方法建立在 之前的资助期，通过多重成像共享单细胞分析的共同线索。 通过揭示口腔微生物组的精确微米级结构、mRNA 表达和动态， 这项研究将探讨社区功能和集会的基本机制和原则。它将 通过开发方法、分析工具和 探针，它应该有助于确定健康维护或治疗干预的新目标。 ！ ！