Toxin Activation by the Protein Quality Control System in Bacteria

Bacteria use a variety of cellular pathways to survive environmental stress, viral (phage) infection, and exposure to antibiotic compounds. One protective mechanism is through the action of toxin-antitoxin systems, which help bacteria withstand these pressures by making a toxin that slows the bacterium’s growth until conditions improve. In order to understand how bacteria induce a toxin-antitoxin system to activate the toxin and survive stress, this project will focus on determining how protein maintenance factors, including chaperone proteins and proteases, mediate antitoxin removal. In addition to benefits from better understanding of bacterial responses to stress for use in biotechnology and possible medical understanding, this work will train students in cell and molecular biology research to prepare them for future careers in the science and biomedical engineering field. The project will also provide a platform to disseminate information to the scientific community through lectures, laboratories, and a seminar series in synthetic biology. Toxin-antitoxin systems in bacteria comprise modular genetic elements in which the encoded toxin inhibits bacterial growth, but toxin activity is regulated by an encoded cognate antitoxin. Functionally acting as regulatory switches, TA systems are poised to activate upon exposure to stress. TA system activation occurs as free toxin becomes uncomplexed from the neutralizing antitoxin, but the mechanism by which this occurs in the cell is not understood. This project will use the model Type 2 TA system MqsRA to elucidate the mechanism of toxin activation in Escherichia coli in response to stress, and will determine the role of proteases, ribosomes, and ribosome-associated chaperones in toxin activation. The project aims will: (1) Identify recognition determinants for proteolytic degradation and determine if Lon and ClpXP recognize overlapping regions of MqsA antitoxin; (2) Determine the role of cochaperones, including SecB, in turnover, and; (3) Evaluate translation-coupled degradation as a new paradigm for TA activation by means of isolation of ribosome nascent chain (RNC) complexes, mass spectrometry, and in vitro translation. The use of reconstituted proteolysis systems and traceable fluorescent fusion proteins will uncover recognition principles underlying antitoxin turnover and assess the contributions of a variety of cellular factors and cochaperones on toxin activation. This combination of biochemical, proteomics, and genetic techniques provides an innovative approach to understand cellular responses to stress, protein synthesis, and quality-control systems. This project is jointly funded by the NSF/BIO/MCB Cell Dynamics & Function Program and the NSF/BIO/MCB Division. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.