Establishing the Thermodynamic and Kinetic Thresholds for Bacterial Protein Secretion via the Type 3 Secretion System

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Although the delivery of bacterial effector proteins into the cytoplasm of a host cell is a fundamental mechanism in pathogen infection, little is presently known regarding the detailed workings of the widely conserved type 3 secretion system (TTSS). Gaining an in-depth understanding of the molecular mechanisms employed by bacteria to deliver specific proteins at the right time and the right place will provide fundamental insights into the engineering of proteins for bacterial delivery and for developing novel strategies for disrupting pathogen infection. The main objectives of this project are addressed in the following questions: How are proteins able to be injected in a functional form into the host after having been requisitely unfolded in order to navigate the narrow lumen of the needle-like protrusion of the TTSS? What are the fundamental requirements that a given protein must meet in order to be successfully translocated from a bacterial pathogen into the host cytoplasm? This project employs fluorescence and NMR spectroscopies to investigate relationships between intracellular bacterial pH and the intrinsic folding equilibrium of effector proteins, with the overall goal of establishing the thermodynamic and kinetic parameters that facilitate coordinated effector delivery into the host cell. The outcomes of this research project should provide fundamental insights into how novel proteins might be engineered for bacterial delivery into host targets, and for developing novel strategies for disrupting a major mechanism of pathogen infection. The results of this project will help fill a large gap in our understanding of how bacteria deliver proteins to host cells. Results will be broadly disseminated through journal publications, the PIs Web page, and presentations at major meetings. The PI also teaches a large Proteins course (BioBM 631) in which current research results are incorporated. This project will strengthen networks with the Cornell plant pathology labs of Alan Collmer and Greg Martin, and provide training for graduate and undergraduate students in a highly interactive research environment that connects the disparate fields of plant biology, microbiology, biophysics, and protein NMR spectroscopy. Recent participation of an underrepresented minority Leadership Alliance student in the PIs lab will assist in future recruitment within this target population. The PI is also a Mentor for Assistant Professor Barbara Lyons in the INBRE program at New Mexico State University (NMSU), a Minority Serving Institution. Through the Leadership Alliance and INBRE programs, this project will provide research opportunities for and mentorship of underrepresented minority students in science.