Spatial Organization of the Oral Microbiome

摘要 人的口腔被一个复杂的微生物群落所定殖，该微生物群落在人类健康中起着关键作用， 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 组成群落的个体分类群之间的相互作用。虽然下一代DNA测序 技术和宏基因组学已经彻底改变了微生物群落的分析，这些技术 需要首先将微生物细胞破碎并提取DNA，在此过程中， 组织丢失了。因此，我们理解的一个主要差距是缺乏空间尺度的信息， 微生物群的生活和工作--微米尺度。 在我们之前的资助期间，我们试图通过使用组合标签策略来填补这一空白， 光谱成像荧光原位杂交（CLASI-FISH）分析的微米级架构， 健康口腔中的微生物群落--基本上是为了确定“谁住在谁的旁边”和“谁住在谁的旁边”。 旁边是什么”。这一战略是成功的，因为它揭示了高度组织和迄今为止没有预料到的 牙菌斑和舌背微生物菌群的属级结构。许多工作 在确定这些群落和其他口腔部位的群落的特征方面仍有待完成。但显然 群落结构的确定必须深入到物种水平。此外，很明显，一个人需要 超越了分类学鉴定为了理解微生物关系的机制基础， 这是获得它们在单细胞水平上的功能表达数据所必需的。另外，获取的静态图像 在成像之后，固定的细胞仅及时提供快照。有必要想办法研究 微生物群落的动态-它们如何形成，发展和维持自己。 本建议的具体目标针对这三个问题。我们将扩大和深化分析 的主要口腔栖息地，使口腔群落内的主要微生物类群可以确定到物种水平。 为了验证从结构结果中产生的假设，我们将开发用于表达关键mRNA的探针。 单细胞水平的分子。为了验证微生物的发展和动力学的假设， 社区，我们将开发探针的代谢能力，在活细胞使用生物正交点击化学 并将其应用与口腔微观文化相结合。这三种办法是在联合国 前一个授权期，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。 ! !

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. ! !