Understanding molecular accumulation in single cells via microfluidics and omics

所有活着的有机体都与环境交换分子。生物体努力吸收糖、氨基酸和离子等生存所必需的分子，同时试图排除有毒分子，如有毒废物和药物。为了实现这一目标，构成有机体的细胞被膜包围，这些膜充当不需要的分子的物理屏障，从而实现受控的分子交换。这些膜是由跨越蛋白质的脂类组成的，这些蛋白质形成了几种不同的物理路径，分子通过膜进行运输。了解这些途径如何帮助一些细胞减少它们从环境中吸收的有毒化合物的数量是生物学中的一个基本问题。事实上，即使具有相同的基因构成，细胞之间也存在着重要的差异。例如，在我们肠道中常见的大肠杆菌群体中，一些细菌的生长速度比其他细菌慢得多。更令人惊讶的是，种群中的一些细菌能够在大量杀死其余种群的抗生素药物中存活下来。相比之下，人们对细胞间吸收化合物能力的差异以及环境如何影响这种能力知之甚少。这个项目将通过确定两个基因相同的细胞如何积累不同数量的给定化合物来填补我们知识中的这一关键空白。这一知识将为通过外部控制分子积累来操纵遗传相同细胞群体的表型结构开辟道路。为了实现这一目标，我们将开发和使用一种新的跨学科方法组合，利用单细胞微生物学(Pagliara)、数学(Tsaneva-Atanasova)和组学(Jeffries)的互补专业知识。我们将使用革兰氏阴性细菌，如大肠杆菌作为模式生物，使用抗生素作为模式运输分子物种，来优化这种方法。这一选择一方面是由可用于研究细菌的生物学、生物物理和建模工具所决定的，另一方面是出于提高抗生素治疗效果的迫切需要。我们将使用具有数百个微室的微流体设备，每个微室能够分离出一种细菌。这些设备将使我们能够捕获和培养数百种细菌；通过使用显微镜和数学方法，我们将测量种群中每个细胞吸收的抗生素的量。因此，这些测量将有助于研究种群内细胞间药物摄取的差异，从而识别出药物积累减少的个体。然后，我们将分析只吸收少量抗生素的细菌的RNA和蛋白质含量。这将使我们能够确定哪些机制有助于这些细菌排除抗生素，从而在抗生素治疗中存活下来。然后，我们将使用这些信息来操纵这些细菌的膜特性，以增加进入每个细菌的药物的量。这些研究将使我们能够确定在具有相同遗传物质的细胞内吸收分子的能力的基本多样性，并了解单个细菌使用哪些途径来实现这种多样性。这将为药物治疗提供指导，从而造福于我们的社会。我们将开发的新方法将很容易转移到其他细菌和真菌以及癌细胞上。更广泛地说，这些方法将通过使用选择性地针对执行特定功能的亚群的化合物，为操纵克隆种群的表型结构开辟道路。总体而言，我们的项目将在微生物学、微生物生态学、药理学和工业过程中产生广泛的影响。

All living organisms exchange molecules with the environment. Organisms strive to take up molecules essential for subsistence such as sugars, amino acids and ions while simultaneously attempting to exclude poisonous molecules such as toxic waste and drugs. To achieve this aim, the cells constituting an organism are surrounded by membranes that act as physical barriers for unwanted molecules allowing for a controlled molecular exchange. These membranes are made up of lipids that are spanned by proteins that form several different physical pathways for molecular transport across the membrane. Understanding how these pathways help some cells to reduce the amount of toxic compounds they take up from the environment is a fundamental question in biology. In fact, there are important differences between cells even with the same genetic make-up. For example, in a population of Escherichia coli, commonly found in our intestine, some bacteria grow much slower than others. Even more surprisingly, some bacteria within the population are able to survive a quantity of antibiotic drugs that kills the rest of the population. In contrast, little is known about cell-to-cell differences in the ability to take up compounds and how the environment affects such capabilities.This project will fill this crucial gap in our knowledge by determining how can two genetically identical cells accumulate substantially different quantities of a given compound. This knowledge will open the way to the manipulation of the phenotypic structure of a population of genetically identical cells by externally controlling molecular accumulation.To achieve this aim we will develop and use a novel combination of cross-disciplinary approaches drawing on complementary expertise in single cell microbiology (Pagliara), mathematics (Tsaneva-Atanasova) and omics (Jeffries). We will optimise such approaches using gram-negative bacteria, such as Escherichia coli, as model organisms and antibiotics as model transported molecular species. This choice is dictated on one hand by the repertoire of biological, biophysical and modelling tools available for investigating bacteria and the urgent need for improving the efficacy of antibiotic treatment on the other hand.We will use microfluidic devices with hundreds of microscopic chambers each capable to isolate a single bacterium. These devices will allow us to capture and grow hundreds of bacteria; by using microscopy and mathematical approaches we will measure the amount of antibiotic that is taken up by each cell within the population. These measurements therefore will enable studying the cell-to-cell differences in drug uptake within the population and thus identifying individuals that show reduced drug accumulation.We will then analyse the content of RNA and proteins of bacteria that take up only small quantities of antibiotics. This will allow us to determine which mechanisms help these bacteria to exclude antibiotics and thus survive antibiotic treatment. We will then use this information to manipulate the properties of the membrane of these bacteria in order to increase the amount of drugs that enter in each bacterium.These studies will allow us to identify the fundamental diversity in the capability to take up molecules within cells with identical genetic material and to understand which pathways are used by individual bacteria to achieve this diversity. This will benefit our society by providing guidelines for pharmacotherapy. The novel approaches that we will develop will be readily transferable to other bacteria and fungi as well as cancer cells. More broadly, these approaches will open the way to the manipulation of the phenotypic structure of a clonal population by using compounds that selectively target subpopulations performing specific functions. Overall our project will have wide implications in microbiology, microbial ecology, pharmacology and industrial processes.