Visualization of translocons in Yersinia type III protein secretion machines during host cell infection

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.

Self-Labeling Enzyme Tags for Analyses of Translocation of Type III Secretion System Effector Proteins

“…SRM has been recently applied to the study of the T3SS translocon assembly during infection by Y. enterocolitica (17) and to analysis of the distribution of SPI2-T3SS effector protein SseJ in STM-infected cells (18). Due to the requirement for immunostaining, these analyses were restricted to fixed cells.…”

Life After Secretion—Yersinia enterocolitica Rapidly Toggles Effector Secretion and Can Resume Cell Division in Response to Changing External Conditions

“…The T3SS protein complex, commonly named the injectisome, is comprised of an extracellular needle, a series of membrane rings embedding an export apparatus, and several cytosolic components (Figure 1A). The needle creates a continuous channel that connects the bacterium to the host cytoplasm with the help of translocator proteins that form a needle tip and a pore within the host cell membrane (Håkansson et al, 1996; Nauth et al, 2018; Park et al, 2018), thus allowing the translocation of effector proteins. The membrane rings anchor the injectisome in the peptidoglycan and the inner and outer membrane, while the export apparatus facilitates the transfer of effectors from the bacterial cytosol through the needle (Kuhlen et al, 2018).…”

Differences of the tumour cell glycocalyx affect binding of capsaicin-loaded chitosan nanocapsules

“…Stimulated emission depletion microscopy and confocal microscopy were carried out in sequential line scanning mode using an Abberior stimulated emission depletion expert line microscope as previously reported 74 . Briefly, cells were seeded on glass coverslips and cultivated until they reached confluency.…”