Search

Yersinia enterocolitica employs a type three secretion system (T3SS) to translocate immunosuppressive effector proteins into host cells. To this end, the T3SS assembles a translocon/pore complex composed of the translocator proteins YopB and YopD in host cell membranes serving as an entry port for the effectors. The translocon is formed in a Yersinia-containing pre-phagosomal compartment that is connected to the extracellular space. As the phagosome matures, the translocon and the membrane damage it causes are recognized by the cell-autonomous immune system. We infected cells in the presence of fluorophore-labeled ALFA-tag-binding nanobodies with a Y. enterocolitica strain expressing YopD labeled with an ALFA-tag. Thereby we could record the integration of YopD into translocons and its intracellular fate in living host cells. YopD was integrated into translocons around 2 min after uptake of the bacteria into a phosphatidylinositol-4,5-bisphosphate enriched pre-phagosomal compartment and remained there for 27 min on average. Damaging of the phagosomal membrane as visualized with recruitment of GFP-tagged galectin-3 occurred in the mean around 14 min after translocon formation. Shortly after recruitment of galectin-3, guanylate-binding protein 1 (GBP-1) was recruited to phagosomes, which was accompanied by a decrease in the signal intensity of translocons, suggesting their degradation. In sum, we were able for the first time to film the spatiotemporal dynamics of Yersinia T3SS translocon formation and degradation and its sensing by components of the cell-autonomous immune system.

Type 3 secretion systems (T3SS) are essential virulence factors of numerous bacterial pathogens and inject effector proteins into host cells. The needle-like T3SS machinery consists of more than 20 components, has a length of around 100 nm and a diameter of up to 30 nm according to EM studies. Its intrabacterial components are highly dynamic and in permanent exchange with other bacterial structures. Therefore, a temporally and spatially resolved visualization of the T3SS using fluorescence microscopy techniques has been challenging. In the present study, novel labeling strategies were combined with super-resolution microscopy such as STED, STORM and MINFLUX. MINFLUX nanoscopy allowed to visualize components of the T3SS machinery such as the dynamic sorting platform component YscL and the extrabacterial pore protein YopD at unprecedented resolutions. The presented results represent the basis for an in depth investigation of T3SS structure and function and therefore gain new insights into the infection process of human pathogens in order to develop novel treatment and prevention strategies.