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Secretion of type III effectors into host cells in real time

Nature Methods volume 2, pages 959–965 (2005)Cite this article

Abstract

Type III secretion (T3S) systems are key features of many gram-negative bacteria that translocate T3S effector proteins directly into eukaryotic cells. There, T3S effectors exert many effects, such as cellular invasion or modulation of host immune responses. Studying spatiotemporal orchestrated secretion of various effectors has been difficult without disrupting their functions. Here we developed a new approach using Shigella flexneri T3S as a model to investigate bacterial translocation of individual effectors via multidimensional time–lapse microscopy. We demonstrate that direct fluorescent labeling of tetracysteine motif–tagged effectors IpaB and IpaC is possible in situ without loss of function. Studying the T3S kinetics of IpaB and IpaC ejection from individual bacteria, we found that the entire pools of IpaB and IpaC were released concurrently upon host cell contact, and that 50% of each effector was secreted in 240 s. This method allows an unprecedented analysis of the spatiotemporal events during T3S.

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Figure 1: IpaB-4Cys and IpaC-4Cys complement ipaB and ipaC strains, respectively.
Figure 2: FLAsH labeling of of IpaB-4Cys and IpaC-4Cys inside S. flexneri.
Figure 3: Effects of the FlAsH concentration on the labeling and invasiveness of S. flexneri.
Figure 4: IpaB and IpaC localization at bacterially induced actin foci after bacterial protein secretion in fixed samples.
Figure 5: Kinetics of IpaB and IpaC secretion by real-time microscopy.

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References

  1. Hueck, C.J. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62, 379–433 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Cornelis, G.R. & Van Gijsegem, F. Assembly and function of type III secretory systems. Annu. Rev. Microbiol. 54, 735–774 (2000).

    Article  CAS  PubMed  Google Scholar 

  3. Lafont, F., Tran Van Nhieu, G., Hanada, K., Sansonetti, P. & van der Goot, F.G. Initial steps of Shigella infection depend on the cholesterol/sphingolipid raft-mediated CD44-IpaB interaction. EMBO J. 21, 4449–4457 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Gruenheid, S. & Finlay, B.B. Microbial pathogenesis and cytoskeletal function. Nature 422, 775–781 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Sansonetti, P.J. Rupture, invasion and inflammatory destruction of the intestinal barrier by Shigella, making sense of prokaryote-eukaryote cross-talks. FEMS Microbiol. Rev. 25, 3–14 (2001).

    CAS  PubMed  Google Scholar 

  6. Sory, M.P. & Cornelis, G.R. Translocation of a hybrid YopE–adenylate cyclase from Yersinia enterocolitica into HeLa cells. Mol. Microbiol. 14, 583–594 (1994).

    Article  CAS  PubMed  Google Scholar 

  7. Day, J.B., Ferracci, F. & Plano, G.V. Translocation of YopE and YopN into eukaryotic cells by Yersinia pestis yopN, tyeA, sycN, yscB and lcrG deletion mutants measured using a phosphorylatable peptide tag and phosphospecific antibodies. Mol. Microbiol. 47, 807–823 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Charpentier, X. & Oswald, E. Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter. J. Bacteriol. 186, 5486–5495 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Rosqvist, R., Magnusson, K.E. & Wolf-Watz, H. Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells. EMBO J. 13, 964–972 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Mounier, J., Bahrani, F.K. & Sansonetti, P.J. Secretion of Shigella flexneri Ipa invasins on contact with epithelial cells and subsequent entry of the bacterium into cells are growth stage dependent. Infect. Immun. 65, 774–782 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Cain, R.J., Hayward, R.D. & Koronakis, V. The target cell plasma membrane is a critical interface for Salmonella cell entry effector-host interplay. Mol. Microbiol. 54, 887–904 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Jacobi, C.A. et al. In vitro and in vivo expression studies of yopE from Yersinia enterocolitica using the gfp reporter gene. Mol. Microbiol. 30, 865–882 (1998).

    Article  CAS  PubMed  Google Scholar 

  13. Buchrieser, C. et al. The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri. Mol. Microbiol. 38, 760–771 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Menard, R., Sansonetti, P. & Parsot, C. The secretion of the Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD. EMBO J. 13, 5293–5302 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Blocker, A. et al. The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes. J. Cell Biol. 147, 683–693 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Tran Van Nhieu, G., Caron, E., Hall, A. & Sansonetti, P.J. IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells. EMBO J. 18, 3249–3262 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ohya, K., Handa, Y., Ogawa, M., Suzuki, M. & Sasakawa, C. IpgB1 is a novel Shigella effector protein involved in bacterial invasion of host cells: Its activity to promote membrane ruffling via RAC1 and CDC42 activation. J. Biol. Chem. 280, 24022–24034 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Yoshida, S. et al. Shigella deliver an effector protein to trigger host microtubule destabilization, which promotes Rac1 activity and efficient bacterial internalization. EMBO J. 21, 2923–2935 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bourdet-Sicard, R. et al. Binding of the Shigella protein IpaA to vinculin induces F-actin depolymerization. EMBO J. 18, 5853–5862 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Adams, S.R. et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124, 6063–6076 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Tsien, R.Y. Building and breeding molecules to spy on cells and tumors. FEBS Lett. 579, 927–932 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Gaietta, G. et al. Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 503–507 (2002).

    Article  CAS  PubMed  Google Scholar 

  23. Ignatova, Z. & Gierasch, L.M. Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling. Proc. Natl. Acad. Sci. USA 101, 523–528 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. Cambronne, E.D., Sorg, J.A. & Schneewind, O. Binding of SycH chaperone to YscM1 and YscM2 activates effector yop expression in Yersinia enterocolitica. J. Bacteriol. 186, 829–841 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Guzman, L.M., Belin, D., Carson, M.J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Barzu, S., Benjelloun-Touimi, Z., Phalipon, A., Sansonetti, P. & Parsot, C. Functional analysis of the Shigella flexneri IpaC invasin by insertional mutagenesis. Infect. Immun. 65, 1599–1605 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Schuch, R., Sandlin, R.C. & Maurelli, A.T. A system for identifying post-invasion functions of invasion genes: requirements for the Mxi-Spa type III secretion pathway of Shigella flexneri in intercellular dissemination. Mol. Microbiol. 34, 675–689 (1999).

    Article  CAS  PubMed  Google Scholar 

  28. Clerc, P.L., Ryter, A., Mounier, J. & Sansonetti, P.J. Plasmid-mediated early killing of eucaryotic cells by Shigella flexneri as studied by infection of J774 macrophages. Infect. Immun. 55, 521–527 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Allaoui, A., Sansonetti, P.J. & Parsot, C. MxiD, an outer membrane protein necessary for the secretion of the Shigella flexneri lpa invasins. Mol. Microbiol. 7, 59–68 (1993).

    Article  CAS  PubMed  Google Scholar 

  30. Dumenil, G. et al. Interferon alpha inhibits a Src-mediated pathway necessary for Shigella-induced cytoskeletal rearrangements in epithelial cells. J. Cell Biol. 143, 1003–1012 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Schlumberger, M.C. et al. Real-time imaging of type III secretion: Salmonella SipA injection into host cells. Proc. Natl. Acad. Sci. USA 102, 12548–12553 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Parsot, C., Menard, R., Gounon, P. & Sansonetti, P.J. Enhanced secretion through the Shigella flexneri Mxi-Spa translocon leads to assembly of extracellular proteins into macromolecular structures. Mol. Microbiol. 16, 291–300 (1995).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank C. Parsot for reagents and helpful discussions, and we acknowledge all other members of P. Sansonetti's laboratory for their support. Furthermore, we thank P. Roux, M. Marchand and S. Shorte at the “Plate-forme d'Imagerie Dynamique” (PFID) at the Institut Pasteur for invaluable experimental help, discussion and advice on data processing. Finally, we are grateful to M. Mavris and J. Rohde for critically reading the manuscript, and to K. Schauer for help with the figure layout. P.S. is a Howard Hughes International Scholar, and J.E. received successive fellowships from the Fondation pour la Recherche Médicale (FRM), the European Molecular Biology Organization (EMBO) and the Human Frontiers Science Program (HFSP).

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  1. INSERM U389, Institut Pasteur, 28 rue du Docteur Roux, Paris, 75724 Cedex 15, France

    Jost Enninga, Joëlle Mounier, Philippe Sansonetti & Guy Tran Van Nhieu

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Correspondence to Jost Enninga or Guy Tran Van Nhieu.

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Supplementary information

Supplementary Fig. 1

Fusion between IpaC and EGFP prevents the secretion of the resulting protein chimera and of other type III effectors. (PDF 519 kb)

Supplementary Fig. 2

Fluorescent labelling of bacterial proteins with biarsenic components. (PDF 578 kb)

Supplementary Fig. 3

Fluorescent labelling of intrabacterial IpaB–4Cys and IpaC–4Cys is dependent on the FlAsH concentration. (PDF 789 kb)

Supplementary Fig. 4

IpaB–4Cys or IpaC–4Cys are secreted with similar efficiencies independently of FlAsH-incorporation. (PDF 961 kb)

Supplementary Fig. 5

Ethanedithiol (EDT) negatively affects T3S mediated cellular invasion by Shigella flexneri. (PDF 414 kb)

Supplementary Fig. 6

The appearance of actin foci formation occurs with similar kinetics for M90T, ipaB/IpaB–4Cys, and ipaC/IpaC–4Cys. (PDF 221 kb)

Supplementary Video 1

Time lapse of IpaB secretion. (MOV 616 kb)

Supplementary Methods (PDF 78 kb)

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Enninga, J., Mounier, J., Sansonetti, P. et al. Secretion of type III effectors into host cells in real time. Nat Methods 2, 959–965 (2005). https://doi.org/10.1038/nmeth804

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