Date Published: December 26, 2018
Publisher: Public Library of Science
Author(s): Theresa Nauth, Franziska Huschka, Michaela Schweizer, Jens B. Bosse, Andreas Diepold, Antonio Virgilio Failla, Anika Steffen, Theresia E. B. Stradal, Manuel Wolters, Martin Aepfelbacher, Brian K Coombes.
Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery—also named injectisome—assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their formation have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology, we provide novel information about the spatiotemporal organization of T3SS translocons and their regulation by host cell factors.
Bacterial type III secretion systems (T3SSs) are molecular machines also termed injectisomes that translocate proteins of bacterial origin (i.e. effectors) into host cells. T3SSs are essential virulence factors of numerous human, animal and plant pathogens including Chlamydia, Pseudomonas, EPEC and EHEC, Salmonella, Shigella and Yersinia [1, 2]. Based on sequence identity among structural components nine T3SS families were classified . Whereas the assembly process, structure and function of the T3SSs are highly conserved, the biochemical activities of the translocated effectors often are multifaceted and reflect the infection strategies of the individual pathogens . Because of their uniqueness in bacteria on one hand and central role for bacterial pathogenicity on the other hand T3SSs have been considered as targets for novel antiinfective strategies [5–8]. In addition, the ability of T3SSs to inject immunogenic proteins into immune cells has been exploited for experimental vaccination strategies .
The two hydrophobic translocators present in most T3SSs and the pore complex/translocon that these proteins form in host cell membranes are particularly difficult to investigate. This is amongst others due to the highly elaborate transit of the translocators from the bacterial interior, where they have to be in a soluble form, through the T3SS needle, when they are in an unfolded state, up to their dynamic interaction with the needle tip. The tip complex is supposed to orchestrate integration of the translocators into the host cell membrane, a process that presumably is accompanied by refolding and heteromultimeric assembly of the proteins. Because it is localized at the interface of the bacterial T3SS and the target cell membrane, the translocon is unavoidably controlled by host cell factors that determine the composition and function of cell membranes.