AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP


AB5 toxins are produced by pathogenic bacteria and consist of enzymatic A subunits that corrupt essential eukaryotic cell functions, and pentameric B subunits that mediate uptake into the target cell. AB5 toxins include the Shiga, cholera and pertussis toxins and a recently discovered fourth family, subtilase cytotoxin, which is produced by certain Shiga toxigenic strains of Escherichia coli. Here we show that the extreme cytotoxicity of this toxin for eukaryotic cells is due to a specific single-site cleavage of the essential endoplasmic reticulum chaperone BiP/GRP78. The A subunit is a subtilase-like serine protease; structural studies revealed an unusually deep active-site cleft, which accounts for its exquisite substrate specificity. A single amino-acid substitution in the BiP target site prevented cleavage, and co-expression of this resistant protein protected transfected cells against the toxin. BiP is a master regulator of endoplasmic reticulum function, and its cleavage by subtilase cytotoxin represents a previously unknown trigger for cell death.

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Figure 1: SubAB cleaves BiP in Vero cells.
Figure 2: Co-localization of SubAB and BiP.
Figure 3: In vivo cleavage of BiP and induction of CHOP, and in vitro cleavage of purified BiP.
Figure 4: Structural comparison of SubA with subtilisin Carlsberg.
Figure 5: Expression of SubAB-resistant BiP D416 prevents cytotoxicity.


  1. 1

    Fan, E., Merritt, E. A., Verlinde, C. L. M. J. & Hol, W. G. J. AB5 toxins: structures and inhibitor design. Curr. Opin. Struct. Biol. 10, 680–686 (2000)

    CAS  Article  Google Scholar 

  2. 2

    Paton, J. C. & Paton, A. W. Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clin. Microbiol. Rev. 11, 450–479 (1998)

    CAS  Article  Google Scholar 

  3. 3

    Paton, A. W., Srimanote, P., Talbot, U. M., Wang, H. & Paton, J. C. A new family of potent AB5 cytotoxins produced by Shiga toxigenic Escherichia coli. J. Exp. Med. 200, 35–46 (2004)

    CAS  Article  Google Scholar 

  4. 4

    Paton, A. W. & Paton, J. C. Multiplex PCR for direct detection of Shiga toxigenic Escherichia coli producing the novel subtilase cytotoxin. J. Clin. Microbiol. 43, 2944–2947 (2005)

    CAS  Article  Google Scholar 

  5. 5

    Siezen, R. J. & Leunissen, J. A. M. Subtilases: The superfamily of subtilisin-like serine proteases. Protein Sci. 6, 501–523 (1997)

    CAS  Article  Google Scholar 

  6. 6

    Kim, P. S. & Arvan, P. Endocrinopathies in the family of endoplasmic reticulum (ER) storage diseases: disorders of protein trafficking and the role of ER molecular chaperones. Endocr. Rev. 19, 173–202 (1998)

    CAS  PubMed  Google Scholar 

  7. 7

    Hendershot, L. M. The ER chaperone BiP is a master regulator of ER function. Mt. Sinai J. Med. 71, 289–297 (2004)

    PubMed  Google Scholar 

  8. 8

    Gething, M. J. Role and regulation of the ER chaperone BiP. Semin. Cell Dev. Biol. 10, 465–472 (1999)

    CAS  Article  Google Scholar 

  9. 9

    Hamman, B. D., Hendershot, L. M. & Johnson, A. E. BiP maintains the permeability barrier of the ER membrane by sealing the lumenal end of the translocon pore before and early in translocation. Cell 92, 747–758 (1998)

    CAS  Article  Google Scholar 

  10. 10

    Lee, A. S. The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods 35, 373–381 (2005)

    CAS  Article  Google Scholar 

  11. 11

    Rao, R. V., Ellerby, H. M. & Bredesen, D. E. Coupling endoplasmic reticulum stress to the cell death program. Cell Death Differ. 11, 372–380 (2004)

    CAS  Article  Google Scholar 

  12. 12

    Rao, R. V. et al. Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78. FEBS Lett. 514, 122–128 (2002)

    CAS  Article  Google Scholar 

  13. 13

    Jiang, J., Prasad, K., Lafer, E. M. & Sousa, R. Structural basis of interdomain communication in the Hsc70 chaperone. Mol. Cell 20, 513–524 (2005)

    CAS  Article  Google Scholar 

  14. 14

    Le Bonniec, B. F., Guinto, E. R. & Esmon, C. T. Interaction of thrombin des-ETW with antithrombin III, the Kunitz inhibitors, thrombomodulin and protein C. Structural link between the autolysis loop and the Tyr-Pro-Pro-Trp insertion of thrombin. J. Biol. Chem. 267, 19341–19348 (1992)

    CAS  PubMed  Google Scholar 

  15. 15

    Lencer, W. I. & Tsai, B. The intracellular voyage of cholera toxin: going retro. Trends Biochem. Sci. 28, 639–645 (2003)

    CAS  Article  Google Scholar 

  16. 16

    Yu, M. & Haslam, D. B. Shiga toxin is transported from the endoplasmic reticulum following interaction with the luminal chaperone HEDJ/ERdj3. Infect. Immun. 73, 2524–2532 (2005)

    CAS  Article  Google Scholar 

  17. 17

    Macario, A. J. L. & Conway de Macario, E. Sick chaperones, cellular stress and disease. N. Engl. J. Med. 353, 1489–1501 (2005)

    CAS  Article  Google Scholar 

  18. 18

    Talbot, U. M., Paton, J. C. & Paton, A. W. Protective immunization of mice with an active-site mutant of subtilase cytotoxin of Shiga toxin-producing Escherichia coli. Infect. Immun. 73, 4432–4436 (2005)

    CAS  Article  Google Scholar 

  19. 19

    Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970)

    ADS  CAS  Article  Google Scholar 

  20. 20

    Towbin, H., Staehelin, T. & Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl Acad. Sci. USA 76, 4350–4354 (1979)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Maniatis, T., Fritsch, E. F. & Sambrook, J. Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982)

  22. 22

    Kozutsumi, Y. et al. Identification of immunoglobulin heavy chain binding protein as glucose-regulated protein 78 on the basis of amino acid sequence, immunological cross-reactivity, and functional activity. J. Cell Sci. Suppl. 11, 115–137 (1989)

    CAS  Article  Google Scholar 

  23. 23

    Yanisch-Perron, C., Vieira, J. & Messing, J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mp18 and pUC19 vectors. Gene 33, 103–119 (1985)

    CAS  Article  Google Scholar 

  24. 24

    Hobbs, S., Jitrapakdee, S. & Wallace, J. C. Development of a bicistronic vector driven by the human polypeptide chain elongation factor 1α promoter for creation of stable mammalian cell lines that express very high levels of recombinant proteins. Biochem. Biophys. Res. Commun. 252, 368–372 (1998)

    CAS  Article  Google Scholar 

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We thank M.-J. Gething and L. Helfenbaum for materials and advice, J. Wallace for discussions and L. Zaker-Tabrizi for technical assistance. This research was supported by a Program Grant from the Australian National Health and Medical Research Council (NHMRC) (to A.W.P and J.C.P). J.R. is supported by an Australian Research Council Professorial and Federation Fellowship and T.B. by a NHMRC Peter Doherty Fellowship. C.M.T is supported by grants from the National Institutes of Health, USA. Author Contributions A.W.P. designed, performed and interpreted experiments, and contributed to writing the manuscript. T.B., J.C.W., M.C.J.W. and J.R. crystallized SubA, solved the structure and contributed to manuscript preparation. C.M.T. contributed to experimental design and interpretation and writing of the manuscript. U.M.T. performed experiments. J.C.P. contributed to design and interpretation of experiments, project management and writing of the manuscript.

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Correspondence to Adrienne W. Paton or James C. Paton.

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The coordinates and structure factors for SubA have been deposited in the Protein Data Bank and assigned the deposition code 2iy9. Reprints and permissions information is available at www.nature.com/reprints. The authors declare that they have no competing financial interests.

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Paton, A., Beddoe, T., Thorpe, C. et al. AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP. Nature 443, 548–552 (2006). https://doi.org/10.1038/nature05124

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