Type 3 secretion systems (T3SSs) are expressed by approximately 30 different bacterial pathogens. They are essential for the virulence of these organisms in humans, yet the mechanisms required for T3SS function are incompletely understood. T3SSs form translocon pores in the membranes of mammalian cells. This pore is required for the T3SS to translocate bacterial effector proteins across the mammalian membrane into the cell cytosol. The molecular mechanisms required for the pore to support effector translocation are poorly described. Shigella is a bacterial pathogen that requires a T3SS both to invade cells and then to spread into adjacent cells. At invasion, the translocon pore interacts with mammalian intermediate filaments to support T3SS function. In contrast, my preliminary data show Shigella spread into neighboring cells occurs independent of intermediate filaments. These results suggest translocon pore function is different at discrete stages of Shigella infection. The overall goals of this project are to investigate how the translocon pore supports Shigella infection. I propose the following specific aims:
Aim 1 : To investigate the mechanisms by which interaction of IpaC with intermediate filaments leads to docking.
Aim 2 : To test whether the IpaC coiled-coil domain participates in processes required for docking.
Aim 3 : To characterize the function of the translocon pore during cell-to-cell spread of Shigella. During Phase 1 of this project, I will develop techniques and approaches to investigate the bacterial proteins that comprise the Shigella flexneri translocon pore. Phase 1 of this award will be carried out in the laboratory of Dr. Marcia Goldberg at Massachusetts General Hospital and Harvard Medical School. A career development plan will be implemented emphasizing additional training and mentoring to prepare me for and during the independent phase of this project. As an independent investigator, I will combine my previous training with new skills to complete the remaining research objectives. This proposal is designed to allow me to achieve my long- term research goals and to attain an independently funded faculty position that focuses on defining bacterial virulence mechanisms and on exploiting these mechanisms to develop novel therapeutics to block infection. !
Many bacterial pathogens require specialized bacterial machinery to deliver proteins into human cells; this machinery is required for these bacteria to cause disease in humans. This proposal aims to define how bacterial protein delivery functions. Due to the near ubiquitous requirement of bacterial protein delivery to establish disease, this proposal is highly likely to define general mechanisms in these processes and general knowledge about how bacterial pathogens establish infection.
