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Crystal structure of Clostridium difficile toxin A

Nature Microbiology volume 1, Article number: 15002 (2016) Cite this article

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Abstract

Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon1,2. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host3,4. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.

Clostridium difficile is the leading cause of health-care-associated infection in the USA, with clinical outcomes that range from mild diarrhoea to pseudomembranous colitis, toxic megacolon and death5. Patients typically respond to treatment with antibiotics such as vancomycin or metronidazole, but recurrence occurs in 25–30% of patients6 and, in 2011, the infection was linked to 29,000 US deaths5. Because CDI is a toxin-mediated disease, a structural and mechanistic understanding of toxin function is a significant priority for the development of novel anti-toxin therapeutics.

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Figure 1: Structure of TcdA.
Figure 2: Zinc is required for autoprocessing activity.
Figure 3: The delivery domain provides an extended scaffold for an α-helical hydrophobic stretch involved in pore formation.

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Acknowledgements

This research was supported by NIAID of the National Institutes of Health (award no. R01AI095755 to D.B.L.) and NIGMS (award no. R01GM042569 to D.P.G.). The authors thank staff at the LS-CAT beamline for help with data collection. Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under contract no. DE-AC02-06CH11357. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant no. 085P1000817). Use of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the US DOE, Office of Science, Office of Basic Energy Sciences (contract no. DE-AC02-98CH10886). Operations at the NSLS beamline X3B were supported by NIH P30-EB009998.

Author information

Author notes

  1. Nicole M. Chumbler and Stacey A. Rutherford: These authors contributed equally to this work.

Authors and Affiliations

  1. Chemical and Physical Biology Program, Vanderbilt University School of Medicine, Nashville, 37232, Tennessee, USA

    Nicole M. Chumbler, Benjamin W. Spiller & D. Borden Lacy

  2. Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, 37232, Tennessee, USA

    Stacey A. Rutherford, Melissa A. Farrow, Benjamin W. Spiller & D. Borden Lacy

  3. Department of Biochemistry, University of Toronto and the Molecular Structure & Function Research Institute at The Hospital for Sick Children, Toronto, M5S 1A8, Ontario, Canada

    Zhifen Zhang & Roman A. Melnyk

  4. Interdisciplinary Graduate Program in Biochemistry, Indiana University, Bloomington, 47405, Indiana, USA

    John P. Lisher

  5. Department of Chemistry, Indiana University, Bloomington, 47405, Indiana, USA

    John P. Lisher & David P. Giedroc

  6. Case Western Reserve University Center for Synchrotron Biosciences, National Synchrotron Light Source, Building 725, Brookhaven National Laboratory, New York, 11973, USA

    Erik Farquhar

  7. Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, 37212, Tennessee, USA

    Benjamin W. Spiller

  8. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, 37205, Tennessee, USA

    D. Borden Lacy

  9. The Veterans Affairs Tennessee Valley Healthcare System, Nashville, 37212, Tennessee, USA

    D. Borden Lacy

Authors
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Contributions

S.A.R. crystallized TcdA1832. S.A.R., B.W.S. and D.B.L. determined the TcdA1832 structure. S.A.R., N.M.C. and M.A.F. generated expression clones and purified proteins. N.M.C. and M.A.F. conducted autoprocessing, Rac1 glucosylation and cell binding assays. Z.Z. conducted viability and Rb+ release assays. N.M.C. prepared samples for ICP-MS assays and J.P.L. performed the assays. E.F. performed XAS measurements. All authors were involved in data analysis and assisted in editing the manuscript. D.B.L. wrote the paper.

Corresponding author

Correspondence to D. Borden Lacy.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Tables 1–4, Figures 1–5 and References (PDF 1613 kb)

Supplementary Video 1

A cartoon representation of the TcdA1832 crystal structure coloured as in Figure 1 and rotating about the vertical axis. (MOV 9685 kb)

Supplementary Video 2

A hypothetical trajectory of movement between the apo- and InsP6-bound structures of the TcdA APD highlights significant structural changes in the InsP6 binding site, the beta-flap, and the APD active site. The trajectory for the protein (coloured as in Figure 2) was calculated in Chimera (ref. 36). (MOV 4776 kb)

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Chumbler, N., Rutherford, S., Zhang, Z. et al. Crystal structure of Clostridium difficile toxin A. Nat Microbiol 1, 15002 (2016). https://doi.org/10.1038/nmicrobiol.2015.2

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