Spatial Organization of the Oral Microbiome

标题：口腔微生物群的空间组织 少年派：杰西卡·马克·韦尔奇 项目摘要/摘要 定植于口腔的细菌不仅对口腔健康，而且对全身健康都有重要作用。 和疾病。这一建议解决了尚未解决的关键问题，即如何组织口语的空间 微生物区系在微米尺度上与其功能相联系。我们之前的工作开创了口腔成像的先河 微生物群生物地理学的研究进展--标记与光谱成像-原位荧光相结合 用杂交法(Clasi-FISH)分析微生物群落的微米尺度结构 系统级别。在这个项目中，我们以这些发现为基础来研究结构-功能关系，评估 单个人类宿主内微米级联合体的复杂性，并发展多路活细胞 成像在单细胞水平上研究多物种体外生物膜的动力学。 在目标1中，我们研究了宿主-微生物组相互作用的关键参数唾液硝酸盐如何 浓度，影响舌背上细菌生物膜的结构。硝酸盐还原 舌头上细菌的能力在一氧化氮的产生中起着主要作用，一氧化氮的产生会影响 血管扩张和血压控制。反过来，唾液腺将硝酸盐分泌到唾液中会影响 舌微生物区系的新陈代谢和组成。使用志愿者的膳食硝酸盐补充剂， 我们将量化舌生物膜联合体中硝酸盐代谢类群的空间组织。 在唾液硝酸盐浓度高和低的情况下，我们将使用全基因组猎枪序列数据来 确定哪些物种和菌株对硝酸盐有反应。 在目标2中，我们解决了一个几乎完全没有探索过的问题：在微米级的社区中 微生物组，每个物种的功能是作为克隆还是作为菌株的复杂混合物？这个问题的答案是 这个问题对于学习操纵和调节口腔微生物群以及决定是否， 对于微生物组移植或建立体外生物膜模型等目的，单个菌株是 有足够的社会代表性。我们将通过对单舌的显微解剖来解决这个问题 背部直径100-200微米，随后进行全基因组鸟枪测序和 分析以评估自然口腔联合体是由克隆人还是复杂种群组成。 实时成像是分析分类单元-分类单元相互作用的一种强有力的方法，但多路传输的实时成像 具有单细胞分辨率的细菌的研究几乎完全没有在口腔微生物组的研究中。这个 目标3的目标是开发一种使用活性染料的活体成像系统，以区分活体内多达6个分类群。 简化的社区。我们将利用这个系统来研究分类单元-分类单元相互作用的动力学。 代表牙菌斑和舌头生物膜成分的协会。我们的目标是了解会员如何 在受控条件下口腔微生物群的相互反应，并评估这些是否在 体外系统发展出类似于自然群落的空间组织。

TITLE: Spatial Organization of the Oral Microbiome PI: Jessica Mark Welch Project Summary/Abstract Bacteria colonizing the human mouth play important roles not only in oral health, but in systemic health and disease. This proposal addresses the key unsolved question of how spatial organization of the oral microbiota is connected to its function at the micron scale. Our previous work pioneered the imaging of oral microbiome biogeography by developing a Combinatorial Labeling And Spectral Imaging - Fluorescence in situ Hybridization (CLASI-FISH) strategy to analyze the micron-scale architecture of microbial communities at a systems level. In this project we build on these findings to investigate structure-function relationships, assess complexity of consortia at micrometer scales within single human hosts, and develop multiplexed live cell imaging to study the dynamics of multispecies in vitro biofilms at the single-cell level. In Aim 1 we investigate how a key parameter of host-microbiome interaction, salivary nitrate concentration, affects the structure of the bacterial biofilm on the tongue dorsum. The nitrate-reducing capability of bacteria on the tongue plays a major role in the generation of nitric oxide, which impacts vasodilation and control of blood pressure. In turn, the secretion of nitrate into saliva by salivary glands affects the metabolism and composition of the tongue microbiota. Using dietary nitrate supplementation of volunteers, we will quantify the spatial organization of nitrate-metabolizing taxa in tongue biofilm consortia that developed in high and low salivary nitrate concentrations, and we will use whole-genome shotgun sequence data to determine which species and strains are responsive to nitrate. In Aim 2 we address an almost entirely unexplored question: in micron-scale neighborhoods in the oral microbiome, does each species function as a clone or as a complex mixture of strains? The answer to this question is important for learning to manipulate and modulate the oral microbiome and determining whether, for purposes such as microbiome transplants or building an in vitro biofilm model, a single strain is an adequate representative of the community. We will address this question by microdissection of single tongue dorsum consortia 100-200 micrometers in diameter, followed by whole-genome shotgun sequencing and analysis to assess whether natural oral consortia are made up of clones or complex populations. Live imaging is a powerful method of analysis of taxon-taxon interactions, but multiplexed live imaging of bacteria with single-cell resolution has been almost entirely absent from studies of the oral microbiome. The goal of Aim 3 is to develop a live-imaging system, using vital dyes, to differentiate up to 6 taxa within live simplified communities. We will use this system to investigate the dynamics of taxon-taxon interactions in consortia representing components of dental plaque and the tongue biofilm. Our goal is to learn how members of the oral microbiome respond to one another under controlled conditions, and to evaluate whether these in vitro systems develop spatial organization resembling those of natural communities.