Abstract
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.
Highlights
Bacterial type III secretion systems (T3SSs) are molecular machines termed injectisomes that translocate proteins of bacterial origin into host cells
Animal and plant pathogenic bacteria employ a molecular machine termed injectisome to inject their toxins into host cells
Injectisomes are highly similar between the different bacterial pathogens and most of their overall structure is well established at the molecular level
Summary
Bacterial type III secretion systems (T3SSs) are molecular machines termed injectisomes that translocate proteins of bacterial origin (i.e. effectors) into host cells. 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 [4]. 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 [5,6,7,8]. The ability of T3SSs to inject immunogenic proteins into immune cells has been exploited for experimental vaccination strategies [9]
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