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 现在可以理解，在自然环境中，细菌主要存在于多细胞，表面结合 社区称为生物膜。生物膜在医学上引起主要问题，因为它们固有地抵抗 抗生素并引起慢性感染；在行业中，生物膜污垢表面和堵塞过滤器件。细胞中的 生物膜显示出与浮游细胞的明显差异，例如细胞外基质产生，1,000倍 对抗生素的耐受性增加以及特定于特定位置的独特基因表达模式 在生物膜内。由于生物膜是三维，异质和随着时间的重新排列的，所以 当仅出现细胞或粗体时，研究仅限于生物膜形成的光学研究 整个结构的表征。我们最近取得了研究突破：我们解决了个人 使用定制的旋转盘共聚焦显微镜，生存的细胞，生长至30微米的深度为30微米 荧光记者和自动细胞分割软件。这是任何人第一次凝视着“ 一个生物膜观察其发展的生物膜，在有流动的情况下，在对环境的条件下进行流动的情况， 医疗和工业系统。因此，我们可以使用三维成像，并结合键 他们建议在光激活和光遗传学方面做出的技术进步，以表征生物膜 从基因到基因组，从细胞到集体。第一个要解决的中心问题 时间包括法定感测和全基因组表达图如何在生长中的空间和时间上有所不同 生物膜？实验设计和测量的解释将由生物物理建模指导。我们 将通过人类病原体弧菌霍乱（以快速但短暂的生物膜形成而闻名）进行研究。 具体而言，我们将对生物膜形成，开发和信号进行全面检查 从单细胞到多细胞水平的转导，在模仿空间，时间， 和自然界中发现的身体约束。跨学科的工作将导致对基因调节的理解， 细胞间通信以及生物膜的空间和时间组织，这反过来决定了大型 这些多细胞系统的规模特征和生态适应性。该提议异常跨学科： IT团队是一名微生物学家巴斯勒（Bassler），他是法定人物感知和生物膜的领导者，与Stone（Stone），一名工程师 焦点是成像，流体动力学和运输过程的建模，而Wingreen（理论上） 生物物理学家对细菌信号通路和生物膜发育进行建模。直接成像的方法， 除了将遗传学与生物物理学联系起来，还承诺与理解和操纵有关的新见解 生物膜的目的是改善人类健康。