Quantitative Imaging and Analysis of Bacterial Biofilms from the Single Cell to the Collective

从单细胞到集体的细菌生物膜的定量成像和分析

• 批准号: 9803916
• 负责人: BONNIE L BASSLER
• 金额: $ 20.25万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-10 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9803916
• 关键词: Address; Antibiotic Resistance; Bacteria; Bacterial Model; Behavior; Biological Models; Biophysics; Cell Communication; Cell Density; Cell model; Cells; Communities; Computer software; Confocal Microscopy; Custom; Development; Devices; Dimensions; Engineering; Environment; Experimental Designs; Expression Profiling; Extracellular Matrix; Filtration; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genome; Goals; Growth; Health; Human; Image; Image Analysis; Individual; Industrialization; Industry; Investigation; Lead; Liquid substance; Location; Measurement; Measures; Medical; Medicine; Microbial Biofilms; Modeling; Modernization; Morphology; Mutation; Nature; Optics; Organizational Change; Positioning Attribute; Production; Property; Proteins; Reporter; Research; Signal Transduction; Sorting - Cell Movement; Structure; Surface; System; Technology; Three-Dimensional Imaging; Time; Transport Process; Ursidae Family; Vibrio cholerae; Work; antibiotic tolerance; bacterial community; biophysical model; chronic infection; fitness; genome-wide; human pathogen; improved; individual response; insight; mutant; novel; optogenetics; pathogenic bacteria; peer; photoactivation; quantitative imaging; quorum sensing; self organization; spatiotemporal; tool; transcriptome sequencing; transcriptomics

PROJECT ABSTRACT It is now understood that in their natural environments, bacteria primarily exist in multicellular, surface-bound communities called biofilms. Biofilms cause major problems in medicine as they are inherently resistant to antibiotics and cause chronic infections; in industry, biofilms foul surfaces and clog filtration devices. Cells in biofilms display striking differences from planktonic cells, such as extracellular matrix production, a 1,000-fold increase in tolerance to antibiotics, and unique gene expression patterns that are specific to particular locations within the biofilm. Because biofilms are three dimensional, heterogeneous, and rearrange over time, investigations have been limited to optical studies of biofilm formation when only few cells are present or to gross characterization of the entire structure. We recently made a research breakthrough: We resolved the individual cells in living, growing biofilms up to a depth of 30 microns, using customized spinning-disk confocal microscopy, fluorescent reporters, and automated cell-segmentation software. This is the first time anyone has peered "into" a biofilm, to watch it develop, cell by cell, in the presence of flow, under conditions that model environmental, medical, and industrial systems. Thus, we are in a position to use three-dimensional imaging, combined with key technological advancements they propose to make in photo-activation and optogenetics, to characterize biofilms from the gene to the genome and from the cell to the collective. Central questions to be addressed for the first time include how do quorum sensing and genome-wide expression profiles vary in space and time within growing biofilms? Experimental design and interpretation of measurements will be guided by biophysical modeling. We will launch the studies with the human pathogen Vibrio cholerae, known for rapid but transient biofilm formation. Specifically, we will pioneer a comprehensive examination of biofilm formation, development, and signal transduction from the single-cell to multi-cell levels and in realistic environments that mimic the spatial, temporal, and physical constraints found in nature. The interdisciplinary work will lead to understanding of gene regulation, cell-to-cell communication, and the spatial and temporal organization of biofilms, which in turn, dictate the large- scale features and ecological fitness of these multicellular systems. The proposal is unusually interdisciplinary: it teams Bassler, a microbiologist who is a leader in quorum sensing and biofilms, with Stone, an engineer whose focus is imaging, fluid dynamics, and the modeling of transport processes, and Wingreen, a theoretical biophysicist who models bacterial signaling circuits and biofilm development. The approach of direct imaging, beyond connecting genetics to biophysics, promises new insights relevant to understanding and manipulating biofilms with the goal of improving human health.

项目摘要 现在人们知道，在自然环境中，细菌主要以多细胞、表面结合的形式存在。 称为生物膜的群落。生物膜在医学上造成重大问题，因为它们具有固有的抗性 抗生素并引起慢性感染；在工业中，生物膜会污染表面并堵塞过滤装置。细胞在 生物膜与浮游细胞表现出显着差异，例如细胞外基质的产生，是浮游细胞的 1,000 倍 对抗生素的耐受性增加，以及特定位置特有的独特基因表达模式 生物膜内。因为生物膜是三维的、异质的，并且会随着时间的推移而重新排列， 研究仅限于生物膜形成的光学研究，当只存在很少的细胞或粗略的细胞时 整个结构的表征。我们最近取得了一项研究突破：我们解决了个体 使用定制的转盘共聚焦显微镜观察活体生长的生物膜中的细胞，深度可达 30 微米， 荧光报告基因和自动细胞分割软件。这是第一次有人“窥视” 生物膜，在流动的情况下，在模拟环境的条件下，观察它逐个细胞的发育， 医疗和工业系统。因此，我们可以使用三维成像，结合关键 他们建议在光激活和光遗传学方面取得技术进步，以表征生物膜 从基因到基因组，从细胞到集体。首先要解决的核心问题 时间包括群体感应和全基因组表达谱在生长过程中如何随空间和时间变化 生物膜？实验设计和测量结果的解释将由生物物理模型指导。我们 将启动针对人类病原体霍乱弧菌的研究，霍乱弧菌以快速但短暂的生物膜形成而闻名。 具体来说，我们将率先对生物膜的形成、发展和信号进行全面检查 从单细胞水平到多细胞水平的转导，以及在模拟空间、时间、 以及自然界中发现的物理限制。跨学科的工作将导致对基因调控的理解， 细胞间的通讯，以及生物膜的空间和时间组织，这反过来又决定了大 这些多细胞系统的规模特征和生态适应性。该提案异常跨学科： 该团队由微生物学家巴斯勒 (Bassler) 和工程师斯通 (​​Stone) 组成，巴斯勒是群体感应和生物膜领域的领军人物。 重点是成像、流体动力学和运输过程的建模，而 Wingreen 是一位理论研究人员 模拟细菌信号回路和生物膜发育的生物物理学家。直接成像方法， 除了将遗传学与生物物理学联系起来之外，还有望带来与理解和操纵相关的新见解 生物膜以改善人类健康为目标。