Live imaging of Yersinia translocon formation and immune recognition in host cells

Abstract

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 or disassembly. 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.

Super‐resolution fluorescence microscopy for investigating bacterial cell biology

“…To minimize the label error for MINFLUX nanoscopy, an ALFA‐tag was introduced into YopD's extracellular domain (giving rise to YopD‐ALFA). It was demonstrated that the ALFA‐tag did not compromise the central functions of YopD during protein translocation by the Y. enterocolitica T3SS (Rudolph et al., 2022). MINFLUX nanoscopy allowed to visualize single YopD‐ALFA molecules bound by fluorescent nanobodies in Yersinia translocation pores.…”

MINFLUX imaging of a bacterial molecular machine at nanometer resolution

“…WA-314 YopD-ALFA was generated using a CRISPR-Cas12a-assisted recombineering approach as described previously [50,51].…”

“…This also offers the advantage of flexible approaches, for example, when the same molecule is to be studied using different nanoscopy techniques or imaging modalities (e.g., multicolor imaging or single molecule tracking). We have recently established in situ modification of injectisome proteins with a short peptide (ALFA) tag [43]. The ALFA tag can bind with high affinity to different fluorophore-labeled nanobodies (NbALFA) [42].…”

“…The ALFA tag can bind with high affinity to different fluorophore-labeled nanobodies (NbALFA) [42]. ALFA-tag/NbALFA pairs enable the imaging of proteins within molecular complexes in living and fixed cells [42,43] and are well suited for advanced super-resolution techniques such as MINFLUX due to their small size.…”

Application of ALFA-Tagging in the Nematode Model Organisms Caenorhabditis elegans and Pristionchus pacificus

“…Since its introduction in 2019, ALFA-tagging has been applied in various systems [ 3 , 4 , 5 ], but to the best of our knowledge not yet in nematodes. Nematodes represent one of the major animal phyla with an estimated species number of more than a million [ 6 ], with free-living, as well as animal and plant parasitic species [ 7 ].…”