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PLoS Pathog. 2018 Dec 26;14(12):e1007527. doi: 10.1371/journal.ppat.1007527. eCollection 2018 Dec.

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

Author information

1
Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany.
2
Center for Molecular Neurobiology (ZMNH), University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany.
3
Heinrich-Pette-Institute (HPI), Leibniz-Institute for Experimental Virology, Hamburg, Germany.
4
Department of Ecophysiology, Max-Planck-Institute for Terrestrial Microbiology, Marburg, Germany.
5
UKE Microscopy Imaging Facility, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany.
6
Department of Cell Biology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.

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.

PMID:
30586431
PMCID:
PMC6324820
DOI:
10.1371/journal.ppat.1007527
[Indexed for MEDLINE]
Free PMC Article

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