Environmental regulation of gene expression dissected by microfluidics

描述(申请人提供)：人类牙菌斑生物膜是一个物理和化学复杂的环境，居住着许多细菌物种，包括变形链球菌，它被认为是人类龋齿的主要病因。变形链球菌有许多导致其毒力的行为，它通过使用从环境中接收到的化学线索来调节和激活这些行为。然而，变形链球菌在生物膜内不同位置的局部环境可能在化学和物理上是不同的，这取决于pH、氧气浓度和营养有效性的巨大变化，以及允许细菌交流和调节毒力行为的不同定植物种和不同浓度的小分子。该项目试图确定这种微环境的多样性如何影响变异链球菌的核心毒力行为，即所谓的遗传能力。能力是指摄取细胞外DNA的能力，而调节变形链球菌能力的遗传网络与调节有机体几乎所有其他致龋性特征的机制密切交织在一起。这些能力基因对环境条件和接收到的化学信号的性质极其敏感。能力基因在一群细胞中的表达可以是一致的，也可以只涉及细菌的一部分激活。因此，一个广泛的科学目标是了解变形链球菌如何处理或解释环境信号以调节能力和其他毒力行为，生物膜中的微环境如何影响这一过程，以及能力基因在整个口腔生物膜中如何在细胞对细胞水平上被激活。本项目将专注于识别和了解控制变形链球菌能力的遗传“开关”，探索pH、氧气浓度和碳水化合物可获得性如何影响多肽信号对能力的调节，并研究和模拟变形链球菌生物膜内多肽信号调节能力的方式。该项目将通过一种新颖的微流控、单细胞方法来实现这些目标。这种方法允许将多个明确定义的环境输入提供给变形链球菌细胞的亚群，同时对这些亚群的能力基因激活情况进行测量和定量建模。该项目将提供有关变形链球菌能力如何在口腔生物膜中空间分布以及何种化学条件引发这种行为和相关毒力行为的详细信息。成功实现这些目标将通过提高对变形链球菌如何导致牙病的理解，促进人类口腔健康的研究。

DESCRIPTION (provided by applicant): The human dental plaque biofilm is a physically and chemically complex environment that is inhabited by a many bacterial species, including Streptococcus mutans, which is regarded as the primary etiological cause of human dental caries. S. mutans has a number of behaviors that give rise to its virulence, and it regulates and activates these behaviors through the use of chemical cues received from its environment. However, the local environment of S. mutans at different locations within a biofilm may be chemically and physically different, subject to substantial variation in pH, oxygen concentration and nutrient availability, as well as different balances of colonizing species and different concentrations of the small molecules that allow the bacteria to communicate and regulate virulence behaviors. This project seeks to determine how this diversity of microenvironments affects the centrally important S. mutans virulence behavior known as genetic competence. Competence is the ability to take up extracellular DNA, and the genetic network that regulates competence in S. mutans is closely intertwined with the mechanisms that regulate virtually every other cariogenic trait of the organism. The competence genes are extremely sensitive to environmental conditions and to the nature of the chemical signals received. Expression of competence genes across a population of cells can be uniform or can involve activation of only a subset of the bacteria. Therefore a broad scientific goal is to understand how S. mutans processes or interprets environmental signals to regulate competence and other virulence behaviors, how microenvironments in the biofilm affect this processing, and how competence genes are activated at the cell-to-cell level throughout an oral biofilm. This project will focus o identifying and understanding the genetic "switches" that control S. mutans competence, exploring how pH, oxygen concentration, and carbohydrate availability affect the regulation of competence by peptide signals, and studying and modeling the ways that competence is regulated by peptide signals inside an S. mutans biofilm. The project will accomplish these goals through a novel microfluidic, single-cell approach. This method allows multiple, well-defined environmental inputs to be supplied to subpopulations of S. mutans cells while the profile of competence gene activation across those subpopulations is measured and modeled quantitatively. The project will yield detailed information about how S. mutans competence is spatially distributed in an oral biofilm and what kinds of chemical conditions trigger this and related virulence behaviors. Success in achieving these objectives will advance research in human oral health by improving the understanding of how Streptococcus mutans causes dental disease.