CAREER: The Molecular Basis of Persister Cell and Biofilm Formation by the E. Coli Protein MqsR

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Intellectual Merit:Research on biofilms, bacterial communities familiar to everyone as they coat our teeth at night, has been ongoing for many years. However, even today, the only certain way to remove biofilms is by mechanical force, i.e. a toothbrush. While brushing our teeth is routine, removal of biofilms from ships, pipes and medical devices, and other surfaces, is much more difficult and expensive. The formation of biofilms is one of the major defense and survival mechanisms utilized by bacteria. However, a detailed understanding of how biofilms assemble and are regulated at a molecular level is only rudimentarily understood. The formation of bacterial persisters, a genetically identical sub-population of metabolically quiescent cells that express protein toxins and exhibit multidrug tolerance, is at the core of biofilm formation. However, persistence is also one of the most poorly understood mechanisms used by bacteria to survive environmental stress. Recently, the Escherichia coli protein MqsR (B3022, YgiU) was identified as a key persistence factor, as it is the most highly upregulated gene in persisters. Because its sequence does not resemble that of any characterized protein, its molecular function, i.e. how it is regulated at a molecular level and especially how it drives the formation of the persister phenotype, is currently unknown. Accordingly, the long-term objective of this CAREER project is to elucidate the molecular mechanisms that lead to the persister state, with a focus on understanding the protein activities that lead to persister formation. In addition, a thorough understanding of how these activities are regulated under normal and stressful conditions and how they can be blocked for the development of novel agents that inhibit the formation of the persister cell phenotype will be determined. Specifically, the following questions will be answered: 1) What is the 3-dimensional structure, and thus the function, of the MqsR toxin and how is its toxicity mitigated by its interaction with MqsA (B3021, YgiT)? 2) How do MqsA and the MqsR:MqsA complex differentially regulate E. coli gene transcription? and 3) How does MqsR toxicity lead to biofilm and persister cell formation? Taken together, these studies will define the molecular mechanism of MqsR and provide essential new insights into how MqsR controls bacterial persistence and biofilm formation.Broader impacts:Biofilms, complex communities of bacteria that are highly resistant to antimicrobials and cost the world economy billions of dollars every year, are extraordinarily enriched in persister cells. A molecular understanding of the function and regulation of the proteins that play a key role in persistence, like MqsR, will provide novel targets needed for development of new chemical agents that target biofilms. In this CAREER project, multiple research disciplines, including structural biology, biochemistry, and genetics, will be integrated to provide projects for both undergraduate and graduate students that reveal the interdisciplinary nature of scientific research. The cornerstone of the educational program is a multi-component Protein Science Workshop (PSW) that will provide Community College of Rhode Island (CCRI) students with the opportunity to expand their research experiences. It is composed of lectures and laboratories at Brown University and RI-EPSCoR facilities and will expose the CCRI students to state-of-the-art research and technologies. In addition, each year, one CCRI student will be invited to carry-out their own independent research project. The long-term goal of the collaboration with CCRI, the first of its kind in Rhode Island, is to attract and develop a new generation of scientists from the often forgotten pool of adult students who are returning to school in order to obtain the knowledge and skills that will allow them to pursue higher education and/or entry into the biotechnology workforce.