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Architecture of the major component of the type III secretion system export apparatus

Nature Structural & Molecular Biology volume 20, pages 99–104 (2013)Cite this article

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Abstract

Type III secretion systems (T3SSs) are bacterial membrane–embedded nanomachines designed to export specifically targeted proteins from the bacterial cytoplasm. Secretion through T3SS is governed by a subset of inner membrane proteins termed the 'export apparatus'. We show that a key member of the Shigella flexneri export apparatus, MxiA, assembles into a ring essential for secretion in vivo. The ring-forming interfaces are well-conserved in both nonflagellar and flagellar homologs, implying that the ring is an evolutionarily conserved feature in these systems. Electron cryo-tomography revealed a T3SS-associated cytoplasmic torus of size and shape corresponding to those of the MxiA ring aligned to the secretion channel located between the secretion pore and the ATPase complex. This defines the molecular architecture of the dominant component of the export apparatus and allows us to propose a model for the molecular mechanisms controlling secretion.

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Figure 1: MxiAC crystallized as a nonameric ring.
Figure 2: In vitro and in vivo analysis of the MxiA ring.
Figure 3: Altering the inner and outer surfaces of the MxiA ring.
Figure 4: MxiAC surface conservation.
Figure 5: Model for MxiA architecture in the T3SS.
Figure 6: Mechanistic model of T3SS secretion.

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References

  1. Cornelis, G.R. The type III secretion injectisome, a complex nanomachine for intracellular 'toxin' delivery. Biol. Chem. 391, 745–751 (2010).

    Article  CAS  Google Scholar 

  2. Patel, J.C. & Galan, J.E. Manipulation of the host actin cytoskeleton by Salmonella–all in the name of entry. Curr. Opin. Microbiol. 8, 10–15 (2005).

    Article  CAS  Google Scholar 

  3. Blocker, A.J. et al. What's the point of the type III secretion system needle? Proc. Natl. Acad. Sci. USA 105, 6507–6513 (2008).

    Article  CAS  Google Scholar 

  4. Erhardt, M., Namba, K. & Hughes, K.T. Bacterial nanomachines: the flagellum and type III injectisome. Cold Spring Harb. Perspect. Biol. 2, a000299 (2010).

    Article  CAS  Google Scholar 

  5. Marlovits, T.C. & Stebbins, C.E. Type III secretion systems shape up as they ship out. Curr. Opin. Microbiol. 13, 47–52 (2010).

    Article  CAS  Google Scholar 

  6. Deane, J.E., Abrusci, P., Johnson, S. & Lea, S.M. Timing is everything: the regulation of type III secretion. Cell. Mol. Life Sci. 67, 1065–1075 (2010).

    Article  CAS  Google Scholar 

  7. Diepold, A., Wiesand, U. & Cornelis, G.R. The assembly of the export apparatus (YscR,S,T,U,V) of the Yersinia type III secretion apparatus occurs independently of other structural components and involves the formation of an YscV oligomer. Mol. Microbiol. 82, 502–514 (2011).

    Article  Google Scholar 

  8. 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  Google Scholar 

  9. Li, H. & Sourjik, V. Assembly and stability of flagellar motor in Escherichia coli. Mol. Microbiol. 80, 886–899 (2011).

    Article  CAS  Google Scholar 

  10. Minamino, T., Imada, K. & Namba, K. Mechanisms of type III protein export for bacterial flagellar assembly. Mol. Biosyst. 4, 1105–1115 (2008).

    Article  CAS  Google Scholar 

  11. Minamino, T., Imada, K. & Namba, K. Molecular motors of the bacterial flagella. Curr. Opin. Struct. Biol. 18, 693–701 (2008).

    Article  CAS  Google Scholar 

  12. Diepold, A. et al. Deciphering the assembly of the Yersinia type III secretion injectisome. EMBO J. 29, 1928–1940 (2010).

    Article  CAS  Google Scholar 

  13. Zhu, K., Gonzalez-Pedrajo, B. & Macnab, R.M. Interactions among membrane and soluble components of the flagellar export apparatus of Salmonella. Biochemistry 41, 9516–9524 (2002).

    Article  CAS  Google Scholar 

  14. McMurry, J.L., Van Arnam, J.S., Kihara, M. & Macnab, R.M. Analysis of the cytoplasmic domains of Salmonella FlhA and interactions with components of the flagellar export machinery. J. Bacteriol. 186, 7586–7592 (2004).

    Article  CAS  Google Scholar 

  15. Bange, G. et al. FlhA provides the adaptor for coordinated delivery of late flagella building blocks to the type III secretion system. Proc. Natl. Acad. Sci. USA 107, 11295–11300 (2010).

    Article  CAS  Google Scholar 

  16. Minamino, T. et al. Interaction of a bacterial flagellar chaperone FlgN with FlhA is required for efficient export of its cognate substrates. Mol. Microbiol. 83, 775–788 (2012).

    Article  CAS  Google Scholar 

  17. Worrall, L.J., Vuckovic, M. & Strynadka, N.C. Crystal structure of the C-terminal domain of the Salmonella type III secretion system export apparatus protein InvA. Prot. Sci. 19, 1091–1096 (2010).

    Article  CAS  Google Scholar 

  18. Saijo-Hamano, Y. et al. Structure of the cytoplasmic domain of FlhA and implication for flagellar type III protein export. Mol. Microbiol. 76, 260–268 (2010).

    Article  CAS  Google Scholar 

  19. Moore, S.A. & Jia, Y. Structure of the cytoplasmic domain of the flagellar secretion apparatus component FlhA from Helicobacter pylori. J. Biol. Chem. 285, 21060–21069 (2010).

    Article  CAS  Google Scholar 

  20. Lilic, M., Quezada, C.M. & Stebbins, C.E. A conserved domain in type III secretion links the cytoplasmic domain of InvA to elements of the basal body. Acta Crystallogr. D Biol. Crystallogr. 66, 709–713 (2010).

    Article  CAS  Google Scholar 

  21. Andrews, G.P. & Maurelli, A.T. mxiA of Shigella flexneri 2a, which facilitates export of invasion plasmid antigens, encodes a homolog of the low-calcium-response protein, LcrD, of Yersinia pestis. Infect. Immun. 60, 3287–3295 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Ashida, H. et al. Shigella deploy multiple countermeasures against host innate immune responses. Curr. Opin. Microbiol. 14, 16–23 (2011).

    Article  CAS  Google Scholar 

  23. 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  Google Scholar 

  24. Worrall, L.J., Lameignere, E. & Strynadka, N.C. Structural overview of the bacterial injectisome. Curr. Opin. Microbiol. 14, 3–8 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Sanowar, S. et al. Interactions of the transmembrane polymeric rings of the Salmonella enterica serovar Typhimurium type III secretion system. mBio 1, e00158-10 (2010).

    Article  Google Scholar 

  27. Lee, L.K., Ginsburg, M.A., Crovace, C., Donohoe, M. & Stock, D. Structure of the torque ring of the flagellar motor and the molecular basis for rotational switching. Nature 466, 996–1000 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  29. Hodgkinson, J.L. et al. Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout. Nat. Struct. Mol. Biol. 16, 477–485 (2009).

    Article  CAS  Google Scholar 

  30. Goodsell, D.S. & Olson, A.J. Structural symmetry and protein function. Annu. Rev. Biophys. Biomol. Struct. 29, 105–153 (2000).

    Article  CAS  Google Scholar 

  31. Thomas, D.R., Francis, N.R., Xu, C. & DeRosier, D.J. The three-dimensional structure of the flagellar rotor from a clockwise-locked mutant of Salmonella enterica serovar Typhimurium. J. Bacteriol. 188, 7039–7048 (2006).

    Article  CAS  Google Scholar 

  32. Chen, S. et al. Structural diversity of bacterial flagellar motors. EMBO J. 30, 2972–2981 (2011).

    Article  CAS  Google Scholar 

  33. Liu, J. et al. Intact flagellar motor of Borrelia burgdorferi revealed by cryo-electron tomography: evidence for stator ring curvature and rotor/C-ring assembly flexion. J. Bacteriol. 191, 5026–5036 (2009).

    Article  CAS  Google Scholar 

  34. Minamino, T. et al. Roles of the extreme N-terminal region of FliH for efficient localization of the FliH-FliI complex to the bacterial flagellar type III export apparatus. Mol. Microbiol. 74, 1471–1483 (2009).

    Article  CAS  Google Scholar 

  35. Ibuki, T. et al. Common architecture of the flagellar type III protein export apparatus and F- and V-type ATPases. Nat. Struct. Mol. Biol. 18, 277–282 (2011).

    Article  CAS  Google Scholar 

  36. Pallen, M.J., Bailey, C.M. & Beatson, S.A. Evolutionary links between FliH/YscL-like proteins from bacterial type III secretion systems and second-stalk components of the FoF1 and vacuolar ATPases. Prot. Sci. 15, 935–941 (2006).

    Article  CAS  Google Scholar 

  37. Imada, K., Minamino, T., Tahara, A. & Namba, K. Structural similarity between the flagellar type III ATPase FliI and F1-ATPase subunits. Proc. Natl. Acad. Sci. USA 104, 485–490 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  39. Minamino, T., Morimoto, Y.V., Hara, N. & Namba, K. An energy transduction mechanism used in bacterial flagellar type III protein export. Nature Commun. 2, 475 (2011).

    Article  Google Scholar 

  40. Paul, K., Erhardt, M., Hirano, T., Blair, D.F. & Hughes, K.T. Energy source of flagellar type III secretion. Nature 451, 489–492 (2008).

    Article  CAS  Google Scholar 

  41. Minamino, T. & Namba, K. Distinct roles of the FliI ATPase and proton motive force in bacterial flagellar protein export. Nature 451, 485–488 (2008).

    Article  CAS  Google Scholar 

  42. Wilharm, G. et al. Yersinia enterocolitica type III secretion depends on the proton motive force but not on the flagellar motor components MotA and MotB. Infect. Immun. 72, 4004–4009 (2004).

    Article  CAS  Google Scholar 

  43. Galperin, M., Dibrov, P.A. & Glagolev, A.N. delta mu H+ is required for flagellar growth in Escherichia coli. FEBS Lett. 143, 319–322 (1982).

    Article  CAS  Google Scholar 

  44. Hara, N., Namba, K. & Minamino, T. Genetic Characterization of Conserved Charged Residues in the Bacterial Flagellar Type III Export Protein FlhA. PLoS ONE 6, e22417 (2011).

    Article  CAS  Google Scholar 

  45. Minamino, T., Kinoshita, M., Imada, K. & Namba, K. Interaction between FliI ATPase and a flagellar chaperone FliT during bacterial flagellar protein export. Mol. Microbiol. 83, 168–178 (2012).

    Article  CAS  Google Scholar 

  46. Akeda, Y. & Galan, J.E. Chaperone release and unfolding of substrates in type III secretion. Nature 437, 911–915 (2005).

    Article  CAS  Google Scholar 

  47. Thomas, J., Stafford, G.P. & Hughes, C. Docking of cytosolic chaperone-substrate complexes at the membrane ATPase during flagellar type III protein export. Proc. Natl. Acad. Sci. USA 101, 3945–3950 (2004).

    Article  CAS  Google Scholar 

  48. Berg, H.C. The rotary motor of bacterial flagella. Annu. Rev. Biochem. 72, 19–54 (2003).

    Article  CAS  Google Scholar 

  49. Murphy, G.E., Leadbetter, J.R. & Jensen, G.J. In situ structure of the complete Treponema primitia flagellar motor. Nature 442, 1062–1064 (2006).

    Article  CAS  Google Scholar 

  50. Blocker, A. et al. Structure and composition of the Shigella flexneri “needle complex”, a part of its type III secreton. Mol. Microbiol. 39, 652–663 (2001).

    Article  CAS  Google Scholar 

  51. Wolf, S., Freier, E., Potschies, M., Hofmann, E. & Gerwert, K. Directional proton transfer in membrane proteins achieved through protonated protein-bound water molecules: a proton diode. Angew. Chem. Int. Ed. Engl. 49, 6889–6893 (2010).

    Article  CAS  Google Scholar 

  52. Miroux, B. & Walker, J.E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298 (1996).

    Article  CAS  Google Scholar 

  53. Solano, C. et al. Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella. Proc. Natl. Acad. Sci. USA 106, 7997–8002 (2009).

    Article  CAS  Google Scholar 

  54. Hendrixson, D.R. & DiRita, V.J. Transcription of sigma54-dependent but not sigma28-dependent flagellar genes in Campylobacter jejuni is associated with formation of the flagellar secretory apparatus. Mol. Microbiol. 50, 687–702 (2003).

    Article  CAS  Google Scholar 

  55. 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  Google Scholar 

  56. Marteyn, B. et al. Modulation of Shigella virulence in response to available oxygen in vivo. Nature 465, 355–358 (2010).

    Article  CAS  Google Scholar 

  57. Rayment, I. Reductive alkylation of lysine residues to alter crystallization properties of proteins. Methods Enzymol. 276, 171–179 (1997).

    Article  CAS  Google Scholar 

  58. Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).

    Article  CAS  Google Scholar 

  59. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

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

    Article  CAS  Google Scholar 

  61. Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D Biol. Crystallogr. 60, 2210–2221 (2004).

    Article  CAS  Google Scholar 

  62. Agulleiro, J.I. & Fernandez, J.J. Fast tomographic reconstruction on multicore computers. Bioinformatics 27, 582–583 (2011).

    Article  CAS  Google Scholar 

  63. Nicastro, D. et al. The molecular architecture of axonemes revealed by cryoelectron tomography. Science 313, 944–948 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank N.C. Strynadka for access to InvA coordinates ahead of publication, P. Sansonetti (Institute Pasteur, Paris) for providing the polyclonal antibodies to Ipa proteins, I. Lasa (Universidad Pública de Navarra, Pamplona) for the pKO3blue plasmid, L. De Colibus, C. King and members of the Lea group for general assistance, the staff of the protein crystallography beamlines at the European Synchrotron Radiation Source, Grenoble (France) and DIAMOND facility, Didcot (UK) for assistance in data collection, and S.K. Mazmanian for use of the microaerobic chamber. P.A. is funded by grant 083599/Z/07/Z and J.E.D. by grant WT083599MA, both from the Wellcome Trust to S.M.L.; S.J. is funded by grant G0900888 from the UK Medical Research Council to S.M.L. and C.M.T.; P.R. is funded by the Oxford Martin School Vaccine Design Institute of which S.M.L. is codirector. M.V.-I. is funded by FP7 Marie Curie EIMID-IAPP-217768 grant. M.B. and G.J.J. were supported by the Howard Hughes Medical Institute.

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  1. Morgan D Beeby, Pietro Roversi & Janet E Deane

    Present address: Present addresses: Division of Molecular Biosciences, Imperial College London, London, UK (M.D.B.), Center for Cooperative Research in Biosciences, Parque Tecnológico De Bizkaia, Bilbao, Spain (P.R.) and Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK (J.E.D.).,

Authors and Affiliations

  1. Sir William Dunn School of Pathology, Oxford University, Oxford, UK

    Patrizia Abrusci, Marta Vergara-Irigaray, Steven Johnson, Pietro Roversi, Miriam E Friede, Janet E Deane, Christoph M Tang & Susan M Lea

  2. Centre for Molecular Microbiology and Infection, Imperial College London, London, UK

    Marta Vergara-Irigaray

  3. Department of Biology, California Institute of Technology, Pasadena, California, USA

    Morgan D Beeby & Grant J Jensen

  4. Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California, USA

    Morgan D Beeby & Grant J Jensen

  5. Department of Microbiology, UT Southwestern Medical Center, Dallas, Texas, USA

    David R Hendrixson

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Contributions

J.E.D., S.J. and S.M.L. initiated the project, which P.A. later joined; S.M.L. and C.M.T. supervised the project. M.E.F. and J.E.D. designed the MxiAC expression vector and did protein expression and stability trials. P.A. performed the large-scale purification, methylation, crosslinking and SPR of MxiAC and its mutants. S.J. performed the MALS experiments. P.A. designed the 'export apparatus' co-expression vectors and purified the recombinant complex. P.A. crystallized MxiAC and optimized crystals for data collection, and S.M.L. soaked and handled crystals for data collection. P.A., P.R. and S.M.L. contributed to the data collection, structure determination and model building. C.M.T. and M.V.-I. designed and performed the complementation and invasion assays in S. flexneri. D.R.H. created the C. jejuni strains, and M.D.B. and G.J.J. designed and performed cryo-EM tomography. P.A., S.J. and S.M.L. analyzed data and wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Susan M Lea.

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Abrusci, P., Vergara-Irigaray, M., Johnson, S. et al. Architecture of the major component of the type III secretion system export apparatus. Nat Struct Mol Biol 20, 99–104 (2013). https://doi.org/10.1038/nsmb.2452

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