The objective of this research program is to use a combination of genetic, biochemical, and structural techniques to study the mechanism of signal transduction in the PhoP-PhoQ two-component regulatory system. The PhoP-PhoQ system is required for virulence and/or regulatory stress responses in enteric bacteria. The focus will be on the PhoQ sensor-transmitter; how extracellular signals are recognized and how this information is transduced to the cytoplasm. To identify determinants of PhoQ structure and function, the plan is to isolate and construct mutations that alter receptor function. The applicant will characterize the spectral properties, thermodynamic stability, and oligomeric form of mutant variants in vitro to ascertain the nature of their phenotypes in vivo. The applicant has grown diffraction-quality crystals of the PhoQ sensor domain and will collaborate with Dr. Wayne Hendrickson of Columbia University to solve the structure. Structural information, coupled with a detailed genetic and biophysical analysis of the PhoQ sensor, may provide a picture of receptor function at a molecular level that does not exist for any other two-component system. Also, the intent is to test a model in which ligand binding causes a conformational change that influences the oligomeric state and/or orientation of an a-helical coiled-coil that, in turn, mediates the regulatory response. The applicant will also identify and study other components of the signal transduction pathway. Improving the understanding of two-component signal transduction is important to understanding the biological processes in which they participate. These studies may have implications for bacterial pathogenesis because structural and functional information could aid in screening and/or designing inhibitors of this system. Other components of the signal transduction pathway could serve as additional targets of inhibitors. This approach to combating bacterial infection is an attractive possible alternative to antibiotics as drug resistance becomes more widespread.
