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

项目摘要 现在人们知道，在它们的自然环境中，细菌主要存在于多细胞、表面结合的环境中 被称为生物膜的社区。生物被膜在医学上造成重大问题，因为它们天生就对 抗生素和导致慢性感染；在工业中，生物膜污染表面和堵塞过滤设备。单元格 生物膜显示出与浮游细胞的显著不同，例如细胞外基质的产生是浮游细胞的1000倍 增加对抗生素的耐受性，以及特定部位特有的独特基因表达模式 在生物膜内。因为生物膜是三维的，异质的，随着时间的推移会重新排列， 研究仅限于对生物膜形成的光学研究，当只有很少的细胞存在或粗大时 对整个结构的表征。我们最近取得了一项研究突破：我们解决了个人 使用定制的旋转圆盘共聚焦显微镜，在30微米深的活的、正在生长的生物膜中的细胞， 荧光记者和自动细胞分割软件。这是第一次有人“窥探” 一个生物膜，在流动的存在下，在模拟环境的条件下，一个细胞一个细胞地观察它的发展， 医疗和工业系统。因此，我们能够使用三维成像，结合关键技术 他们计划在光激活和光遗传学方面取得的技术进步，以表征生物膜 从基因到基因组，从细胞到集体。第一个问题要解决的中心问题 时间包括群体感应和全基因组表达谱在生长过程中如何在空间和时间上发生变化 生物膜？实验设计和测量解释将由生物物理模型指导。我们 将启动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被膜形成而闻名。 具体地说，我们将率先全面检查生物膜的形成、发育和信号。 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约束。跨学科的工作将导致对基因调控的理解， 细胞间的交流，以及生物膜的空间和时间组织，这反过来又决定了大的- 这些多细胞系统的规模特征和生态适宜性。这项提议异乎寻常地是跨学科的： 它的团队包括巴斯勒和斯通，巴斯勒是一名微生物学家，是群体感应和生物膜领域的领导者，斯通是一名工程师，他的 重点是成像、流体动力学和输运过程的建模，而温人则是一种理论 为细菌信号回路和生物膜发育建模的生物物理学家。直接成像的方法， 除了将遗传学与生物物理学联系起来外，它还承诺提供与理解和操纵相关的新见解 以改善人类健康为目标的生物膜。

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.