Spatio-temporal dynamics of mutation avoidance and antimicrobial resistance

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Spontaneous mutations are the basis of evolutionary innovation. They are also central to diseases in higher organisms and the root of some of the most pressing medical problems that we face: antimicrobial resistance and cancer. Mutations are usually detrimental for microbial cells. Therefore, cells have evolved to control mutations very tightly and DNA mutation rates are remarkably low. Yet mutations can be beneficial, for instance, a single point mutation in the right gene helps a cell to gain antibiotic resistance. Cells thus need mutations but just not too many. We need deep understanding of how the number of mutations fluctuate with the internal and external cellular environment. Addressing such dynamical aspects of mutations in the natural environment is vital for understanding the survival, adaptation and evolution of all cells. My previous work found that the rate of mutations is regulated by the environment associated with the cell-density: mutation rates decrease in dense populations up to 20-fold. Specifically, I discovered that crucial effectors of this environmental dependence of mutations are enzymes that enable cells to avoid mutations. In this Fellowship I shall observe mutations in individual microbes growing in their native environment, community. Only studying individual cells growing in a dense community will give us an understanding of mutation dynamics in the real-world. To accomplish my aims, I will combine live fluorescence microscopy, microfluidics, statistical modelling and interact with outstanding researchers from other disciplines. I shall use the Escherichia coli K-12 model system and observe mobility of DNA repair proteins, which will enable me to count mutations in individual cells. This will tell us about cell-to-cell variation in the number of mutations and how such a heterogeneity depends on micro-environments generated in the community. I will also quantify the molecular diffusion of mutation avoidance protein MutT in these micro-environments. I will establish the potential link between MutT dynamics and downstream processes involved in the generation of mutations. I will do all this not only in cells during normal community growth, but also in cells that survive the antibiotic treatment without obtaining the genetic resistance. These tolerant and persistent cells start to divide again, after the antibiotic is removed. My study will determine dynamics of mutations in these survivor cells and how micro-environments, generated in the aftermath of the antibiotic treatment, affect their fate. Understanding factors that affect a cell's capability to avoid and repair mutations is essential to better predict the development of mutation-based resistance in microbial communities and to exploit that understanding to combat antimicrobial resistance.Generated knowledge, in years 1-4, will be used in years 5-7 to test how various drug candidates impact the mutation dynamics and fate of cells that survive antibiotic treatment. I will also apply these developed experimental approach to more clinically relevant strains.