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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Humans
  • Microscopy, Fluorescence
  • Type III Secretion Systems*
  • Yersinia Infections / transmission*
  • Yersinia enterocolitica / pathogenicity*

Substances

  • Type III Secretion Systems

Grant support

The investigators FH and MA were supported by CUI RFB 2.1 Aepfelbacher of the excellence cluster “The Hamburg Centre for Ultrafast Imaging - Structure, Dynamics and Control of Matter at the Atomic Scale” of the Deutsche Forschungsgemeinschaft (DFG). http://www.cui.uni-hamburg.de/ MA was supported by the Joachim Herz Stiftung, Hamburg, Germany. https://www.joachim-herz-stiftung.de/ TS was supported by HGF impulse fund W2/W3-066. https://www.helmholtz.de/ueber_uns/die_gemeinschaft/impuls_und_vernetzungsfonds/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.