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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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)
Paton, J. C. & Paton, A. W. Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clin. Microbiol. Rev. 11, 450–479 (1998)
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)
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)
Siezen, R. J. & Leunissen, J. A. M. Subtilases: The superfamily of subtilisin-like serine proteases. Protein Sci. 6, 501–523 (1997)
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)
Hendershot, L. M. The ER chaperone BiP is a master regulator of ER function. Mt. Sinai J. Med. 71, 289–297 (2004)
Gething, M. J. Role and regulation of the ER chaperone BiP. Semin. Cell Dev. Biol. 10, 465–472 (1999)
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)
Lee, A. S. The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods 35, 373–381 (2005)
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)
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)
Jiang, J., Prasad, K., Lafer, E. M. & Sousa, R. Structural basis of interdomain communication in the Hsc70 chaperone. Mol. Cell 20, 513–524 (2005)
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)
Lencer, W. I. & Tsai, B. The intracellular voyage of cholera toxin: going retro. Trends Biochem. Sci. 28, 639–645 (2003)
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)
Macario, A. J. L. & Conway de Macario, E. Sick chaperones, cellular stress and disease. N. Engl. J. Med. 353, 1489–1501 (2005)
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)
Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970)
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)
Maniatis, T., Fritsch, E. F. & Sambrook, J. Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982)
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)
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)
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)
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.
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.
About this article
Cite this article
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
Life Sciences (2020)
NOD1 and NOD2 Activation by Diverse Stimuli: a Possible Role for Sensing Pathogen-Induced Endoplasmic Reticulum Stress
Infection and Immunity (2020)
Function and mutual interaction of BiP‐, PERK‐, and IRE1α‐dependent signalling pathways in vascular tumours
The Journal of Pathology (2020)
Preparation of Biodegradable PLGA-Nanoparticles Used for pH-Sensitive Intracellular Delivery of an Anti-inflammatory Bacterial Toxin to Macrophages
Chemical and Pharmaceutical Bulletin (2020)
Unbiased Profiling of the Human Proinsulin Biosynthetic Interaction Network Reveals a Role for Peroxiredoxin 4 in Proinsulin Folding