Understanding molecular accumulation in single cells via microfluidics and omics

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.

Molecular exchange across cellular membranes is at the basis of life and has been investigated via ensemble measurements. However, single-cell technologies have demonstrated that clonal populations are heterogeneous in physiological parameters such as growth rate and resistance to stress. In contrast, little is known about the heterogeneity of molecular accumulation within a clonal population as a result of molecules entering, leaving or being degraded by each cell.

This project will determine how can two genetically identical cells accumulate substantially different quantities of a given compound. We will use gram-negative bacteria as model organisms, (auto)fluorescent antibiotics as model transported compounds and microfluidics-enabled single-cell microscopy to identify bacteria displaying reduced drug accumulation. These studies will allow us to rapidly predict the outcome of antibiotic treatment by measuring drug accumulation and to determine the impact of the environment and the cell cycle on the heterogeneity in antibiotic accumulation. Furthermore, we will use fluorescent-activated cell-sorting, transcriptomics and proteomics to determine the molecular mechanisms underpinning reduced antibiotic accumulation in bacterial subsets.

Taken together these studies will allow us to reveal the fundamental diversity in molecular accumulation within clonal populations and to understand which strategies are used by bacterial subsets to achieve this diversity. Moreover, the novel approaches developed in this project will open the way to the manipulation of the phenotypic structure of a clonal population by selectively controlling molecular exchange across cellular membranes. Therefore, overall our project will have wide implications for clinical researchers and the healthcare sector, industrial processes and agriculture, microbial ecology and evolution.