Kontrolle und Untersuchung des Effektorexports durch das bakterielle Typ 3 Injektionssystem mittels Optogenetik

The type III secretion system (T3SS) is a needle-like structure that is used by many pathogenic Gram-negative bacteria to translocate effector proteins from the bacterial cytosol into host cells. It has been successfully applied to deliver non-native cargo into various host cells for different purposes such as vaccination or immunotherapy. However, bacterial T3SS are not restricted to specific target cells and inject effector proteins into any eukaryotic host cell upon contact. Lack of target specificity is therefore a main obstacle in the further development and application of the T3SS as a specific protein delivery tool. Previous studies have shown that the cytosolic complex of the T3SS acts as a highly dynamic interface, in which parts permanently exchange and shuttle between the cytosol and the T3SS, and that this exchange is directly linked to protein secretion function. These findings raise a new possibility to control the T3SS activity via specific sequestration and release of cytosolic T3SS components. Optogenetic applications, up to now mainly established in eukaryotic cell research, provide a new toolbox to achieve precise control over protein interactions with light. In my PhD work, I incorporated optogenetic interaction switches to bacteria, with the aim to specifically control cellular events. The combination of optogenetic interaction switches with a dynamic essential cytosolic component of the T3SS enables reversible spatial and temporal control of the T3SS function and resulting in the application LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS). This enhances the use of the T3SS as a specific tool for protein delivery into eukaryotic cells and enables broad applications. We present in this work that the secretion of native effector proteins and non-native cargo proteins, as well as the translocation of cargos into eukaryotic host cells can be efficiently controlled by light in the engineered strains. As a direct biological application, we present the delivery of pro-apoptotic cargos into cancer cells. I further utilized the optogenetic protein association and dissociation principle to investigate dynamic cellular events of the T3SS with focus on the dynamics of the cytosolic complex and its proposed link to effector shuttling and sorting events.