The aim of the proposed research is to advance our understanding of factors that determine how genetic changes - mutations – affect Biological strength (e.g growth rates, competitive advantage or disadvantage) of microorganisms. Mutations create genetic diversity on which evolution acts. At the same time they may detrimentally affect protein structural integrity, stability and possible interactions with other proteins and other components of cells. We aim here to determine how the interplay between effect of mutations on proteins and cellular response to mutated proteins determines the fitness outcome of mutations.
Our approach is based on the view of the cell as a dynamic system where several players (chaperones, which take care of protein folding, proteases, which degrade incorrectly folded or aggregated proteins, feedback loops which detect that some proteins are lacking and crank up their production in response) determine concentrations of active functional proteins in the cytoplasm. We developed a unique genetic tool, which makes it possible to incorporate carefully planned mutations of our choice directly on a bacterial chromosome, to challenge cells with mutations that have known effect on molecular properties of proteins. We measure fitness of mutant strains as well as intracellular abundance of mutated proteins and evaluate the intracellular homeostatis response to mutations. Through genetic manipulations, which allow controlling various branches of homeostatic response (chaperons, proteases, feedbacks), we will discover how main components of cell machinery synergistically respond to mutations and define their fitness effect.
A new theoretical model based on kinetic analysis of protein turnover in living cells, which includes action of chaperones, proteases, conformational transitions in proteins and their aggregation, will guide the experimental exploration. This research will advance our knowledge of how Physics and Biology interplay to sculpt fitness landscape of microorganisms and, in a longer perspective, provide tools to control bacterial adaptation to antibiotics and other external factors.
The work will be carried out at Harvard by a team of talented postdocs and graduate students. The lab is well equipped to carry out proposed research – it has all necessary equipment and tools.
Project outcome and impact of result
Project outcome and impact of results :
• Successful completion of this project will provide a novel powerful and predictive model of bacterial cell cytoplasm
• The results of this reserach will be publsihed in open access scholarly journals. Dr Shakhnovich lectures extensively in universities around the country and abroad. He will present this research to members of scientific community and general public.
• This work will result in a better understanding of the mechanisms of bacterial resistance and is likely to lead to the development of new antibiotics with novel mode of action. In particular our preliminary data shows that some mutations cause significant cellular response leading to fitness rescue. One approach then could be to develop an antibiotic ''cocktail'', which affects simultaneously the target protein and the key component of the signal that causes cellular rescue. That way we can significantly decrease the ability of bacteria to evolve resistance through mutations.
• This work will generate massive amount of data, which will be available to public via free dabases hosted at Harvard. The database will contain Biophysical data on mutant proteins and associated fitness data on strains making it possible for researchers and public to analyze it and draw their own, improved, genotype-phenotype relations. In additioin plasmids and mutant bacterial strains will be made available to researchers in Academia and public institutions free of charge.
We plan to develop a ''game of evolution'' for public enjoyment and education. This will be a user-friendly program, freely available on the web, that simulates bacterial populations in a multiscale model based the genome-based model of evolution that has been developed in Shakhnovich lab. A player will be challenged to find evolutinary routes by which bacteria survives in challenging environments and to observe how their genomes evolve in response to environmental and man-made challenges.
Professor of Chemistry and Chemical Biology, Harvard University
Cambridge, MA, USA