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T3S injectisome needle complex structures in four distinct states reveal the basis of membrane coupling and assembly

Abstract

The bacterial injectisome is a syringe-shaped macromolecular nanomachine utilized by many pathogenic Gram-negative bacteria, including the causative agents of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. Bacterial proteins destined for self-assembly and host-cell targeting are translocated by the injectisome in a process known as type III secretion (T3S). The core structure is the ~4 MDa needle complex (NC), built on a foundation of three highly oligomerized ring-forming proteins that create a hollow scaffold spanning the bacterial inner membrane (IM) (24-mer ring-forming proteins PrgH and PrgK in the Salmonella enterica serovar Typhimurium Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS)) and outer membrane (OM) (15-mer InvG, a member of the broadly conserved secretin pore family). An internalized helical needle projects from the NC and bacterium, ultimately forming a continuous passage to the host, for delivery of virulence effectors. Here, we have captured snapshots of the entire prototypical SPI-1 NC in four distinct needle assembly states, including near-atomic resolution, and local reconstructions in the absence and presence of the needle. These structures reveal the precise localization and molecular interactions of the internalized SpaPQR ‘export apparatus’ complex, which is intimately encapsulated and stabilized within the IM rings in the manner of a nanodisc, and to which the PrgJ rod directly binds and functions as an initiator and anchor of needle polymerization. We also describe the molecular details of the extensive and continuous coupling interface between the OM secretin and IM rings, which is remarkably facilitated by a localized 16-mer stoichiometry in the periplasmic-most coupling domain of the otherwise 15-mer InvG oligomer.

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Fig. 1: Cryo-EM snapshots capture the molecular basis of needle complex assembly.
Fig. 2: Recruitment of an extra secretin monomer in the periplasm locally alters ring stoichiometry and facilitates multivalent symmetrical coupling of the OM and IM T3S components.
Fig. 3: Molecular interactions that govern NC assembly.
Fig. 4: The SpaPQR complex forms an assembly platform for PrgJ and PrgI needle polymerization.
Fig. 5: The structural snapshots capture the transitions associated with needle assembly and secretin gating.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Cryo-EM maps and atomic coordinates have been deposited with the EMDB and PDB with the following accession codes EMDB ID: EMD-20310, EMD-20311, EMD-20312, EMD-20313, EMD-20314, EMD-20315, EMD-20316, EMD-20317, EMD-20556 and PDB ID: 6PEE, 6PEM, 6PEP, 6Q14, 6Q15 and 6Q16.

References

  1. Abrusci, P. et al. Architecture of the major component of the type III secretion system export apparatus. Nat. Struct. Mol. Biol. 20, 99–104 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Worrall, L. J. et al. Near-atomic-resolution cryo-EM analysis of the Salmonella T3S injectisome basal body. Nature 540, 597–601 (2016).

    Article  CAS  PubMed  Google Scholar 

  3. Wagner, S. et al. Organization and coordinated assembly of the type III secretion export apparatus. Proc. Natl Acad. Sci. USA 107, 17745–17750 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Schraidt, O. & Marlovits, T. C. Three-dimensional model of Salmonella’s needle complex at subnanometer resolution. Science 331, 1192–1195 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Zarivach, R., Vuckovic, M., Deng, W., Finlay, B. B. & Strynadka, N. C. J. Structural analysis of a prototypical ATPase from the type III secretion system. Nat. Struct. Mol. Biol. 14, 131–137 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Yip, C. K. et al. Structural characterization of the molecular platform for type III secretion system assembly. Nature 435, 702–707 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Spreter, T. et al. A conserved structural motif mediates formation of the periplasmic rings in the type III secretion system. Nat. Struct. Mol. Biol. 16, 468–476 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bergeron, J. R. C. et al. A refined model of the prototypical Salmonella SPI-1 T3SS basal body reveals the molecular basis for its assembly. PLoS Pathog. 9, e1003307 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bergeron, J. R. C. et al. The modular structure of the inner-membrane ring component PrgK facilitates assembly of the type III secretion system basal body. Structure 23, 161–172 (2015).

    Article  CAS  PubMed  Google Scholar 

  10. Majewski, D. D., Worrall, L. J. & Strynadka, N. C. Secretins revealed: structural insights into the giant gated outer membrane portals of bacteria. Curr. Opin. Struct. Biol. 51, 61–72 (2018).

    Article  CAS  PubMed  Google Scholar 

  11. Dietsche, T. et al. Structural and functional characterization of the bacterial type III secretion export apparatus. PLoS Pathog. 12, 1–25 (2016).

    Article  CAS  Google Scholar 

  12. Kuhlen, L. et al. Structure of the core of the type III secretion system export apparatus. Nat. Struct. Mol. Biol. 25, 583–590 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zilkenat, S. et al. Determination of the stoichiometry of the complete bacterial type III secretion needle complex using a combined quantitative proteomic approach. Mol. Cell. Proteom. 15, 1598–1609 (2016).

    Article  CAS  Google Scholar 

  14. Hu, B., Lara-Tejero, M., Kong, Q., Galán, J. E. & Liu, J. In situ molecular architecture of the Salmonella Type III secretion machine. Cell 168, 1065–1074 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hu, J. et al. Cryo-EM analysis of the T3S injectisome reveals the structure of the needle and open secretin. Nat. Commun. 9, 1–11 (2018).

    Article  CAS  Google Scholar 

  16. Bai, X., Rajendra, E., Yang, G., Shi, Y. & Scheres, S. H. W. Sampling the conformational space of the catalytic subunit of human γ-secretase. eLife 4, e11182 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Schraidt, O. et al. Topology and organization of the Salmonella typhimurium type III secretion needle complex components. PLoS Pathog. 6, e1000824 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Sanowar, S. et al. Interactions of the transmembrane polymeric rings of the Salmonella enterica serovar Typhimurium type III secretion system. mBio 1, 1–8 (2010).

    Article  CAS  Google Scholar 

  19. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    Article  CAS  PubMed  Google Scholar 

  20. Erhardt, M. et al. Mechanism of type-III protein secretion: regulation of FlhA conformation by a functionally critical charged-residue cluster. Mol. Microbiol. 104, 234–249 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ward, E. et al. Type-III secretion pore formed by flagellar protein FliP. Mol. Microbiol. 107, 94–103 (2018).

    Article  CAS  PubMed  Google Scholar 

  22. Loquet, A. et al. Atomic model of the type III secretion system needle. Nature 486, 276–279 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Radics, J., Königsmaier, L. & Marlovits, T. C. Structure of a pathogenic type 3 secretion system in action. Nat. Struct. Mol. Biol. 21, 82–87 (2014).

    Article  CAS  PubMed  Google Scholar 

  24. Marlovits, T. C. et al. Structural insights into the assembly of the type III secretion needle complex. Science 306, 1040–1042 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sal-man, N., Deng, W. & Finlay, B. B. EscI: a crucial component of the type III secretion system forms the inner rod structure in enteropathogenic Escherichia coli. Biochem J. 125, 119–125 (2012).

    Article  CAS  Google Scholar 

  26. Zhong, D. et al. The Salmonella type III secretion system inner rod protein PrgJ is partially folded. J. Biol. Chem. 287, 25303–25311 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lefebre, M. D. & Galan, J. E. The inner rod protein controls substrate switching and needle length in a Salmonella type III secretion system. Proc. Natl Acad. Sci. USA 111, 817–822 (2014).

    Article  CAS  PubMed  Google Scholar 

  28. El Hajjami, N. et al. The inner-rod component of Shigella flexneri type 3 secretion system, MxiI, is involved in the transmission of the secretion activation signal by its interaction with MxiC. Microbiologyopen 7, 1–11 (2018).

    Article  CAS  Google Scholar 

  29. Wee, D. H. & Hughes, K. T. Molecular ruler determines needle length for the Salmonella Spi-1 injectisome. Proc. Natl Acad. Sci. USA 2015, 1–6 (2015).

    Google Scholar 

  30. Diepold, A., Amstutz, M., Sorg, I., Jenal, U. & Cornelis, G. R. Deciphering the assembly of the Yersinia type III secretion injectisome. EMBO J. 29, 1928–1940 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Diepold, A. & Wagner, S. Assembly of the bacterial type III secretion machinery. FEMS Microbiol. Rev. 38, 802–822 (2014).

    Article  CAS  PubMed  Google Scholar 

  32. Berger, C., Robin, G. P., Bonas, U. & Koebnik, R. Membrane topology of conserved components of the type III secretion system from the plant pathogen Xanthomonas campestris pv. vesicatoria. Microbiology 156, 1963–1974 (2010).

    Article  CAS  PubMed  Google Scholar 

  33. van Arnam, J. S., McMurry, J. L., Kihara, M. & Macnab, R. M. Analysis of an engineered Salmonella flagellar fusion protein, FliR-FlhB. J. Bacteriol. 186, 2495–2498 (2004).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Zarivach, R. et al. Structural analysis of the essential self-cleaving type III secretion proteins EscU and SpaS. Nature 453, 124–127 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Yan, Z., Yin, M., Xu, D., Zhu, Y. & Li, X. Structural insights into the secretin translocation channel in the type II secretion system. Nat. Struct. Mol. Biol. 24, 177–183 (2017).

    Article  CAS  PubMed  Google Scholar 

  36. Hay, I. D., Belousoff, M. J. & Lithgow, T. Structural basis of type 2 secretion system engagement between the inner and outer bacterial membranes. mBio 8, 1–6 (2017).

    Article  Google Scholar 

  37. Hu, B. et al. Visualization of the type III secretion sorting platform of Shigella flexneri. Proc. Natl Acad. Sci. USA 112, 1047–1052 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kowal, J. et al. Structure of the dodecameric Yersinia enterocolitica secretin YscC and its trypsin-resistant core. Structure 21, 2152–2161 (2013).

    Article  CAS  PubMed  Google Scholar 

  39. Koo, J., Burrows, L. L. & Lynne Howell, P. Decoding the roles of pilotins and accessory proteins in secretin escort services. FEMS Microbiol. Lett. 328, 1–12 (2012).

    Article  CAS  PubMed  Google Scholar 

  40. Chernyatina, A. & Harry, L. Architecture of a bacterial type II secretion system. Preprint at https://www.biorxiv.org/content/10.1101/397794 (2018).

  41. Guilvout, I. et al. Independent domain assembly in a trapped folding intermediate of multimeric outer membrane secretins. Structure 22, 582–589 (2014).

    Article  CAS  PubMed  Google Scholar 

  42. Burkinshaw, B. J. et al. Structural analysis of a specialized type III secretion system peptidoglycan-cleaving enzyme. J. Biol. Chem. 290, 10406–10417 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Marlovits, T. C. et al. Assembly of the inner rod determines needle length in the type III secretion injectisome. Nature 441, 637–640 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Wagner, S., Stenta, M., Metzger, L. C., Dal, M. & Cornelis, G. R. Length control of the injectisome needle requires only one molecule of Yop secretion protein P (YscP). Proc. Natl Acad. Sci. USA 107, 13860–13865 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Erhardt, M., Singer, H. M., Wee, D. H., Keener, J. P. & Hughes, K. T. An infrequent molecular ruler controls flagellar hook length in Salmonella enterica. EMBO J. 30, 2948–2961 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Kenjale, R. et al. The needle component of the type III secreton of Shigella regulates the activity of the secretion apparatus. J. Biol. Chem. 280, 42929–42937 (2005).

    Article  CAS  PubMed  Google Scholar 

  47. Deng, W. et al. Assembly, structure, function and regulation of type III secretion systems. Nat. Rev. Microbiol. 15, 323–337 (2017).

    Article  CAS  PubMed  Google Scholar 

  48. Kimbrough, T. G. & Miller, S. I. Contribution of Salmonella typhimurium type III secretion components to needle complex formation. Proc. Natl Acad. Sci. USA 97, 11008–11013 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Eichelberg, K. & Galán, J. E. Differential regulation of Salmonella typhimurium type III secreted proteins by pathogenicity island 1 (SPI-1)-encoded transcriptional activators InvF and HilA. Infect. Immun. 67, 4099–4105 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, 1–22 (2018).

    Article  Google Scholar 

  53. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. CryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  54. Chen, S. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  56. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. Sect. D 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  57. Wang, R. Y. R. et al. Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta. eLife 5, 1–22 (2016).

    Google Scholar 

  58. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. Sect. D 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  59. Barad, B. A. et al. EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. Sect. D 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  61. The PyMOL Molecular Graphics System v.2.0 (Schrödinger, LLC, 2017).

  62. Zimmermann, L. et al. A completely reimplemented MPI bioinformatics toolkit with a new HHpred server at its core. J. Mol. Biol. 430, 2237–2243 (2018).

    Article  CAS  PubMed  Google Scholar 

  63. Edwards, R. A., Keller, L. H. & Schi, D. M. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene 207, 149–157 (1998).

    Article  CAS  PubMed  Google Scholar 

  64. Ferrie, L. et al. Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery. J. Bacteriol. 192, 6418–6427 (2010).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Miller for providing S. Typhimurium deletion strains and plasmids, as well as the InvG antibody. This work was funded by operating grants from CIHR, to N.C.J.S. and B.B.F. and the Howard Hughes International Senior Scholar Program, to N.C.J.S. B.B.F. is the UBC Peter Wall Distinguished Professor. N.C.J.S. is a Tier I Canada Research Chair in Antibiotic Discovery.

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M.V. performed all cloning and protein purification. C.H. performed single-particle cryo-EM grid preparation and data collection with assistance from Z.Y. J.H. performed data processing and map generation with assistance from L.J.W. and C.E.A. L.J.W. performed model building, refinement and structural analysis with help from J.H. W.D. made the Salmonella deletion mutants and expression strains and carried out secretion assays with assistance from B.B.F. L.J.W., J.H. and N.C.J.S. wrote the manuscript with input from all.

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Correspondence to Natalie C. J. Strynadka.

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Supplementary Information

Supplementary Table 1, Supplementary Figs. 1–10, Supplementary Video Legends and Supplementary References.

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Supplementary Video 1

Allosteric step of InvG periplasmic gate opening.

Supplementary Video 2

Steric step of InvG periplasmic gate opening.

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Hu, J., Worrall, L.J., Vuckovic, M. et al. T3S injectisome needle complex structures in four distinct states reveal the basis of membrane coupling and assembly. Nat Microbiol 4, 2010–2019 (2019). https://doi.org/10.1038/s41564-019-0545-z

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