Investigating the regulation of the type III secretion system using optogenetic tools

The foodborne pathogen Yersinia enterocolitica is the causative agent of gastrointestinal infections. It harbors a type III secretion system (T3SS) that enables the bacterium to translocate toxic effector proteins into eukaryotic target cells. These effector proteins help the bacterium to manipulate the host immune system and to prevent phagocytosis, leading to a disease called yersiniosis. This thesis examined small accessory proteins that are crucial for the regulation and function of the T3SS. In the first project, I had a look at the proteins YscX and YscY. Without these proteins, the T3SS needle cannot be built, resulting in non-pathogenic Yersiniae. Inside the bacterial cell, the YscX-YscY heterodimer binds at the structural T3SS component SctV to form a tripartite complex that regulates protein export. In this thesis, I discovered that domains of YscX, which do not interact with YscY, are crucial for functional T3SS secretion. We found specific conserved amino acid residues of YscX and SctV whose mutations lacked secretion, implicating that these residues are involved in the formation of the tripartite complex. By optogenetic sequestration of YscY from its site of action during T3SS assembly, I confirmed that it is an essential protein in the process of T3SS formation. A second T3SS regulatory unit is the gatekeeper complex, which consists of YopN, TyeA, and the chaperones YscB and SycN. So far, it is assumed that YopN prevents effector secretion by blocking the export pore like a plug, which is then opened by the release of YopN upon host cell contact. However, I found that in live cell fluorescence microscopy experiments, all gatekeeper proteins localize in the bacterial cytosol in secreting and non-secreting conditions, indicating no plugging mechanism. To examine a possible cytosolic mode of action, I utilized optogenetic interaction switches. In order to expand the possibilities for light-dependent protein control, I established a PopZ-based polar anchor that forms a phase-separated compartment at the cell pole. I saw a loss of gatekeeper function when they were sequestered to this polar compartment, indicating that they cannot interact with other regulatory proteins anymore. This suggested that the gatekeepers are embedded in a cytosolic interaction network with other proteins to fulfill their regulatory function. Thus, we conducted protein-protein interaction studies and found consistently enriched interactions with the translocator proteins that form the needle tip (SctA) and pore in the eukaryotic cell membrane (SctB, SctE). Our data support a model where gatekeeper proteins would regulate the switch to effector export via a cytosolic mechanism and in association with the translocator proteins. These findings gave insights into critical post-translational mechanisms of T3SS regulation. Our results show and confirm that for Yersinia virulence, T3SS assembly and substrate export are precisely regulated, disclosing potential targets for future therapeutics. I also demonstrated that post-translational optogenetic control was applicable to various T3SS proteins to study their roles over time in T3SS assembly and secretion. Thus, this versatile tool can be expanded to other processes within bacterial cells. In a third project, I utilized the T3SS as a protein delivery tool for heterologous substrates. In collaboration with the group of Prof. Dr. Oliver Hantschel, we successfully demonstrated, that monobody proteins can be delivered into various eukaryotic cancer cells via the Yersinia T3SS. These monobodies act as antagonists of oncoproteins, supporting T3SS-dependent protein delivery as a tool for possible therapeutic use. This study was published in 2024.