Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2

Abstract

The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Retro-2 binds directly to Sec16A.
Fig. 2: Depletion of Sec16A phenocopies Retro-2 effects.
Fig. 3: Retro-2 treatment slows the anterograde transport of Syn5.
Fig. 4: Syn5 SNARE complex formation is not affected by Retro-2.
Fig. 5: Syn5 interacts with GPP130.
Fig. 6: Syn5-GPP130 interaction is required for STxB retrograde trafficking.

Data and code availability

The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier project accession no. PXD015642. Imaris and Matlab scripts for EEA1 quantification are available on request. All other data supporting the findings of this study are available within the paper and its supplementary information files.

References

  1. 1.

    Johannes, L. & Römer, W. Shiga toxins—from cell biology to biomedical applications. Nat. Rev. Microbiol. 8, 105–116 (2010).

    CAS  PubMed  Google Scholar 

  2. 2.

    Tarr, P. I., Gordon, C. A. & Chandler, W. L. Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome. Lancet 365, 1073–1086 (2005).

    CAS  PubMed  Google Scholar 

  3. 3.

    Endo, Y. et al. Site of action of a Vero toxin (VT2) from Escherichia coli O157:H7 and of Shiga toxin on eukaryotic ribosomes. RNA N-glycosidase activity of the toxins. Eur. J. Biochem. 171, 45–50 (1988).

    CAS  PubMed  Google Scholar 

  4. 4.

    Fraser, M. E., Chernaia, M. M., Kozlov, Y. V. & James, M. N. Crystal structure of the holotoxin from Shigella dysenteriae at 2.5 Å resolution. Nat. Struct. Biol. 1, 59–64 (1994).

    CAS  PubMed  Google Scholar 

  5. 5.

    Ling, H. et al. Structure of Shiga-like toxin I B-pentamer complexed with an analogue of its receptor Gb3. Biochemistry 37, 1777–1788 (1998).

    CAS  PubMed  Google Scholar 

  6. 6.

    Sandvig, K., Olsnes, S., Brown, J. E., Petersen, O. W. & van Deurs, B. Endocytosis from coated pits of Shiga toxin: a glycolipid-binding protein from Shigella dysenteriae 1. J. Cell Biol. 108, 1331–1343 (1989).

    CAS  PubMed  Google Scholar 

  7. 7.

    Römer, W. et al. Shiga toxin induces tubular membrane invaginations for its uptake into cells. Nature 450, 670–675 (2007).

    PubMed  Google Scholar 

  8. 8.

    Mallard, F. et al. Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform. J. Cell Biol. 156, 653–664 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Sandvig, K. et al. Retrograde transport of endocytosed Shiga toxin to the endoplasmic reticulum. Nature 358, 510–512 (1992).

    CAS  PubMed  Google Scholar 

  10. 10.

    Spooner, R. A. & Lord, J. M. How ricin and Shiga toxin reach the cytosol of target cells: retrotranslocation from the endoplasmic reticulum. Curr. Top. Microbiol. Immunol. 357, 19–40 (2012).

    CAS  PubMed  Google Scholar 

  11. 11.

    Johannes, L. & Popoff, V. Tracing the retrograde route in protein trafficking. Cell 135, 1175–1187 (2008).

    CAS  PubMed  Google Scholar 

  12. 12.

    Sandvig, K., Skotland, T., van Deurs, B. & Klokk, T. I. Retrograde transport of protein toxins through the Golgi apparatus. Histochem. Cell Biol. 140, 317–326 (2013).

    CAS  PubMed  Google Scholar 

  13. 13.

    Bujny, M. V., Popoff, V., Johannes, L. & Cullen, P. J. The retromer component, sorting nexin-1, is required for efficient early endosome-to-trans Golgi network retrograde transport of Shiga toxin. J. Cell Sci. 120, 2010–2021 (2007).

    CAS  PubMed  Google Scholar 

  14. 14.

    Popoff, V. et al. The retromer complex and clathrin define a post-early endosomal retrograde exit site. J. Cell Sci. 120, 2022–2031 (2007).

    CAS  PubMed  Google Scholar 

  15. 15.

    Utskarpen, A., Slagsvold, H. H., Dyve, A. B., Skanland, S. S. & Sandvig, K. SNX1 and SNX2 mediate retrograde transport of Shiga toxin. Biochem. Biophys. Res. Commun. 358, 566–570 (2007).

    CAS  PubMed  Google Scholar 

  16. 16.

    Mukhopadhyay, S. & Linstedt, A. D. Manganese blocks intracellular trafficking of Shiga toxin and protects against Shiga toxicosis. Science 335, 332–335 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Mukhopadhyay, S., Redler, B. & Linstedt, A. D. Shiga toxin binding site for host cell receptor GPP130 reveals unexpected divergence in toxin trafficking mechanisms. Mol. Biol. Cell 24, 2311–2318 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Tai, G. et al. Participation of syntaxin 5/Ykt6/GS28/GS15 SNARE complex in transport from the early/recycling endosome to the TGN. Mol. Biol. Cell 15, 4011–4022 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Hui, N. et al. An isoform of the Golgi t-SNARE, syntaxin 5, with an endoplasmic reticulum retrieval signal. Mol. Biol. Cell 8, 1777–1787 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Mancias, J. D. & Goldberg, J. Structural basis of cargo membrane protein discrimination by the human COPII coat machinery. Embo J. 27, 2918–2928 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Gillet, D., Barbier, J., Johannes, L., Cintrat, J. -C. & Noël, R. New compounds having a protective activity against toxins with intracellular activity. Patent WO2014060586A1 (2012).

  22. 22.

    Mukhopadhyay, S. & Linstedt, A. D. Retrograde trafficking of AB(5) toxins: mechanisms to therapeutics. J. Mol. Med. (Berl.) 91, 1131–1141 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Stechmann, B. et al. Inhibition of retrograde transport protects mice from lethal ricin challenges. Cell 141, 231–242 (2010).

    CAS  PubMed  Google Scholar 

  24. 24.

    Secher, T. et al. Retrograde trafficking inhibitors of Shiga toxins reduces morbidity and mortality of mice infected with enterohemorrhagic Escherichia coli (STEC). Antimicrob. Agents Chemother. 59, 5010–5013 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Canton, J. & Kima, P. E. Targeting host syntaxin-5 preferentially blocks Leishmania parasitophorous vacuole development in infected cells and limits experimental Leishmania infections. Am. J. Pathol. 181, 1348–1355 (2012).

    CAS  PubMed  Google Scholar 

  26. 26.

    Gupta, N. et al. Inhibitors of retrograde trafficking active against ricin and Shiga toxins also protect cells from several viruses, Leishmania and Chlamydiales. Chem. Biol. Interact. 267, 96–103 (2017).

    CAS  PubMed  Google Scholar 

  27. 27.

    Carney, D. W. et al. Structural optimization of a retrograde trafficking inhibitor that protects cells from infections by human polyoma- and papillomaviruses. Bioorg. Med. Chem. 22, 4836–4847 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Dai, W. et al. Antiviral effects of Retro-2(cycl) and Retro-2.1 against Enterovirus 71 in vitro and in vivo. Antivir. Res. 144, 311–321 (2017).

    CAS  PubMed  Google Scholar 

  29. 29.

    Dai, W. W. Antiviral effect of Retro-2.1 against herpes simplex virus type 2. J. Microbiol. Biotechnol. 28, 849–859 (2018).

    CAS  PubMed  Google Scholar 

  30. 30.

    Cruz, L. et al. Potent inhibition of human cytomegalovirus by modulation of cellular SNARE syntaxin 5. J. Virol. 91, e01637-16 (2017).

    PubMed  Google Scholar 

  31. 31.

    Shtanko, O. et al. Retro-2 and its dihydroquinazolinone derivatives inhibit filovirus infection. Antivir. Res. 149, 154–163 (2018).

    CAS  PubMed  Google Scholar 

  32. 32.

    Harrison, K. et al. Vaccinia virus uses retromer-independent cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions during virus morphogenesis. J. Virol. 90, 10120–10132 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Sivan, G., Weisberg, A. S., Americo, J. L. & Moss, B. Retrograde transport from early endosomes to the trans-Golgi network enables membrane wrapping and egress of vaccinia virus virions. J. Virol. 90, 8891–8905 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Noel, R. et al. N-methyldihydroquinazolinone derivatives of Retro-2 with enhanced efficacy against Shiga toxin. J. Med. Chem. 56, 3404–3413 (2013).

    CAS  PubMed  Google Scholar 

  35. 35.

    Whittle, J. R. & Schwartz, T. U. Structure of the Sec13–Sec16 edge element, a template for assembly of the COPII vesicle coat. J. Cell Biol. 190, 347–361 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Wälchli, S. et al. The mitogen-activated protein kinase p38 links Shiga toxin-dependent signaling and trafficking. Mol. Biol. Cell. 19, 95–104 (2008).

    PubMed  PubMed Central  Google Scholar 

  37. 37.

    Hughes, H. & Stephens, D. J. Assembly, organization, and function of the COPII coat. Histochem. Cell Biol. 129, 129–151 (2008).

    CAS  PubMed  Google Scholar 

  38. 38.

    Xu, Y., Martin, S., James, D. E. & Hong, W. GS15 forms a SNARE complex with syntaxin 5, GS28, and Ykt6 and is implicated in traffic in the early cisternae of the Golgi apparatus. Mol. Biol. Cell 13, 3493–3507 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Chen, Y. A. & Scheller, R. H. SNARE-mediated membrane fusion. Nat. Rev. Mol. Cell Biol. 2, 98–106 (2001).

    CAS  PubMed  Google Scholar 

  40. 40.

    Hong, W. SNAREs and traffic. Biochim. Biophys. Acta 1744, 493–517 (2005).

    PubMed  Google Scholar 

  41. 41.

    Amessou, M. et al. Syntaxin 16 and syntaxin 5 control retrograde transport of several exogenous and endogenous cargo proteins. J. Cell. Sci. 120, 1457–1468 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Puri, S., Bachert, C., Fimmel, C. J. & Linstedt, A. D. Cycling of early Golgi proteins via the cell surface and endosomes upon lumenal pH disruption. Traffic 3, 641–653 (2002).

    CAS  PubMed  Google Scholar 

  43. 43.

    Natarajan, R. & Linstedt, A. D. A cycling cis-Golgi protein mediates endosome-to-Golgi traffic. Mol. Biol. Cell 15, 4798–4806 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Linstedt, A. D., Mehta, A., Suhan, J., Reggio, H. & Hauri, H. P. Sequence and overexpression of GPP130/GIMPc: evidence for saturable pH-sensitive targeting of a type II early Golgi membrane protein. Mol. Biol. Cell 8, 1073–1087 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Ledger, P. W., Uchida, N. & Tanzer, M. L. Immunocytochemical localization of procollagen and fibronectin in human fibroblasts: effects of the monovalent ionophore, monensin. J. Cell Biol. 87, 663–671 (1980).

    CAS  PubMed  Google Scholar 

  46. 46.

    Mollenhauer, H. H., Morre, D. J. & Rowe, L. D. Alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity. Biochim. Biophys. Acta 1031, 225–246 (1990).

    CAS  PubMed  Google Scholar 

  47. 47.

    Cañeque, T., Muller, S. & Rodriguez, R. Visualizing biologically active small molecules in cells using click chemistry. Nat. Rev. Chem 2, 202–215 (2018).

    Google Scholar 

  48. 48.

    Adolf, F., Rhiel, M., Reckmann, I. & Wieland, F. T. Sec24C/D-isoform-specific sorting of the preassembled ER-Golgi Q-SNARE complex. Mol. Biol. Cell 27, 2697–2707 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Campbell, J. L. & Schekman, R. Selective packaging of cargo molecules into endoplasmic reticulum-derived COPII vesicles. Proc. Natl Acad. Sci. USA 94, 837–842 (1997).

    CAS  PubMed  Google Scholar 

  50. 50.

    Gupta, N. et al. (S)-N-methyldihydroquinazolinones are the active enantiomers of Retro-2 derived compounds against toxins. ACS Med. Chem. Lett. 5, 94–97 (2014).

    CAS  PubMed  Google Scholar 

  51. 51.

    Mallard, F. et al. Direct pathway from early/recycling endosomes to the Golgi apparatus revealed through the study of Shiga toxin B-fragment transport. J. Cell Biol. 143, 973–990 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Jordan, M., Schallhorn, A. & Wurm, F. M. Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 24, 596–601 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Poullet, P., Carpentier, S. & Barillot, E. myProMS, a web server for management and validation of mass spectrometry-based proteomic data. Proteomics 7, 2553–2556 (2007).

    CAS  PubMed  Google Scholar 

  54. 54.

    Valot, B., Langella, O., Nano, E. & Zivy, M. MassChroQ: a versatile tool for mass spectrometry quantification. Proteomics 11, 3572–3577 (2011).

    CAS  PubMed  Google Scholar 

  55. 55.

    Vizcaino, J. A. et al. 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 44, 11033 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Soderberg, O. et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Methods 3, 995–1000 (2006).

    PubMed  Google Scholar 

  57. 57.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    CAS  Google Scholar 

  58. 58.

    Boncompain, G. & Perez, F. Synchronizing protein transport in the secretory pathway. Curr. Protoc. Cell Biol. 57, 15.19.1–15.19.16 (2012).

    Google Scholar 

  59. 59.

    Venditti, R. et al. Sedlin controls the ER export of procollagen by regulating the Sar1 cycle. Science 337, 1668–1672 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank R. Rodriguez (cell imaging of Retro-2.1 and target identification), G. Boncompain (RUSH), C. Viaris De Lesegno (PLA), R. Onclercq-Delic and S. Bombard (intoxication assays), C. Brewee and B. Sancerne (recombinant protein production and cytotoxicity assays), D. Buisson (purification of 15) and E. Chirkin (synthesis of new batches of chemicals) for help with the indicated experiments. We thank V. Sabatet from the Laboratoire de Spectrométrie de Masse Protéomique for myProMS assistance. We acknowledge support from grants from the Agence Nationale pour la Recherche (ANR-11-BSV2-0018 and ANR-14-CE16-0004-03 to L.J., J.B., J.-C.C. and D.G. and ANR-19-CE13-0001-01 to L.J.), the Human Frontier Science Program (RGP0029-2014 to L.J.), the European Research Council (advanced grant no. 340485 to L.J.), the Swedish Research Council (K2015-99X-22877-01-6 to L.J., J.-C.C. and D.G.), the Joint Ministerial Program of R&D against CBRNE Risks (D.G., J.B., J.-C.C. and L.J.), the CEA (D.G., J.B. and J.-C.C.), the Île de France Region DIM Malinf initiative (grant no. 140101 to D.G., J.B. and L.J.), the Région Île-de-France (D.L.) and the Fondation pour la Recherche Médicale (D.L.). The Gillet and Cintrat teams are members of LabEx LERMIT (ANR-10-LABX-33) and the Johannes team is a member of Labex CelTisPhyBio (11-LBX-0038) and Idex Paris Sciences et Lettres (ANR-10-IDEX-0001–02 PSL). We also acknowledge the Cell and Tissue Imaging (PICT-IBiSA) and Nikon Imaging Centre, Institut Curie, a member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04).

Author information

Affiliations

Authors

Contributions

L.J. and D.G. conceived and designed the study. S.J.R., H.-F.R. and M.D.G.-C. performed the click chemistry immunofluorescence experiments and M.D.G.-C. and S.J.R. performed the click chemistry pulldown experiments. S.J.R. performed the in vitro Retro-2 pulldown, the SNARE PLA, the SNARE relocalization, the Syn5-RUSH assay and the GPP130 rescue analysis. S.J.R., M.D.G.-C., J.B. and L.T. performed the intoxication assays. The BLI assay was performed by J.B. and R.S. Purification of the Syn5 and GPP130 variants, the monensin study and the in vitro Syn5 pulldown of the GPP130 variants were performed by C.B. and A.D.L. S.J.R. and M.D.G.-C. performed the Sec16A and syntaxin-5 proteomics analysis and immunofluorescence. A.F. performed the Sec23 kinetic studies, the GFP-Sec16A pulldown and the STxB, GPP130 and Syn5 immunofluorescence with quantification. C.A.V.-C. wrote the scripts and automated the EEA1 colocalization methods. A.C., M.M. and J.-C.C. designed and performed the chemical synthesis of the azide-functionalized Retro-2 derivatives, and J.B. and L.T. characterized their anti-Shiga toxin activity. J.M., S.P. and L.T. prepared and characterized the recombinant Sec16A1266–1678/Sec13 protein complex. F.D. carried out the MS work, and D.L. supervised the MS and proteomic data analysis. S.J.R. and L.J. wrote the paper. A.F., J.-C.C., J.B., D.G., A.D.L. and C.L. critically revised the manuscript and aided in the design and analysis of experiments.

Corresponding authors

Correspondence to Daniel Gillet or Ludger Johannes.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Tables 1–2, Figs. 1–7 and Supplementary Note.

Reporting Summary

Supplementary Dataset 1

Proteomics quantification results GFP-Sec16A vehicle versus EGFP.

Supplementary Dataset 2

Proteomics quantification results GFP-Sec16A Retro-2 versus GFP-Sec16A vehicle.

Supplementary Dataset 3

Proteomics quantification results GFP-STX5 versus EGFP.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Forrester, A., Rathjen, S.J., Daniela Garcia-Castillo, M. et al. Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2. Nat Chem Biol 16, 327–336 (2020). https://doi.org/10.1038/s41589-020-0474-4

Download citation

Further reading