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Nature Medicine
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Host S-nitrosylation inhibits clostridial small molecule–activated glucosylating toxins

Nature Medicinevolume 17, pages1136–1141 (2011)Cite this article

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Abstract

The global prevalence of severeClostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins1,2,3,4. Virulence is dependent on the autoactivation of a toxin cysteine protease5,6,7,8,9, which is promoted by the allosteric cofactor inositol hexakisphosphate (InsP6)10,11,12,13,14,15,16,17. Host mechanisms that protect against such exotoxins are poorly understood. It is increasingly appreciated that the pleiotropic functions attributed to nitric oxide (NO), including host immunity, are in large part mediated by S-nitrosylation of proteins18,19. Here we show thatC. difficile toxins are S-nitrosylated by the infected host and that S-nitrosylation attenuates virulence by inhibiting toxin self-cleavage and cell entry. Notably, InsP6- and inositol pyrophosphate (InsP7)-induced conformational changes in the toxin enabled host S-nitrosothiols to transnitrosylate the toxin catalytic cysteine, which forms part of a structurally conserved nitrosylation motif. Moreover, treatment with exogenous InsP6 enhanced the therapeutic actions of oral S-nitrosothiols in mouse models ofC. difficile infection. Allostery in bacterial proteins has thus been successfully exploited in the evolutionary development of nitrosothiol-based innate immunity and may provide an avenue to new therapeutic approaches.

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Figure 1:C. difficile toxins are S-nitrosylatedin vivo.
Figure 2: Toxin S-nitrosylation is allosterically regulated by inositolphosphate.
Figure 3: A catalytic-site motif for S-nitrosylation.
Figure 4: GSNO-based therapy forC. difficile infection.

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Acknowledgements

This work was supported by the Eli & Edith Broad Foundation, the John S. Dunn Gulf Coast Consortium for Chemical Genomics Robert A. Welch Collaborative Grant Program, the Howard Hughes Medical Institute and grants from the US National Institutes of Health National Institute of Allergy and Infectious Diseases (R01AI088748, N01AI30050), National Institute of Diabetes and Digestive and Kidney Diseases (R01DK084509, K01DK076549; R21-DK078032-01), National Heart, Lung, and Blood Institute (R01-HL059130, R01-HL091876, R01-HL095463, P01-HL075443-06A, NO1-HV-00245) and 1UL1RR029876-01. We thank D. Powell, S. Weinman, C.S. Schein and G. Prestwich for their critiques.

Author information

Authors and Affiliations

  1. Department of Gastroenterology & Hepatology, University of Texas Medical Branch, Galveston, Texas, USA

    Tor C Savidge, Petri Urvil, Kausar Ali, Aproteem Choudhury, Vinay Acharya & Irina Pinchuk

  2. Department of Biochemistry & Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA

    Numan Oezguen, John E Wiktorowicz & Werner Braun

  3. Department of Microbiology & Immunology, University of Texas Medical Branch, Galveston, Texas, USA

    Alfredo G Torres

  4. National Heart, Lung and Blood Institute Proteomics Center, University of Texas Medical Branch, Galveston, Texas, USA

    Robert D English & John E Wiktorowicz

  5. Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA

    Michael Loeffelholz

  6. Department of Basic Sciences, The Commonwealth Medical College, Scranton, Pennsylvania, USA

    Raj Kumar

  7. Division of Infectious Diseases, Department of Biomedical Sciences, Tufts University Cummings School of Veterinary Medicine, North Grafton, Massachusetts, USA

    Lianfa Shi, Weijia Nie & Hanping Feng

  8. Institute for Transformative Molecular Medicine, Case Western Reserve University and University Hospitals, Cleveland, Ohio, USA

    Bo Herman, Alfred Hausladen & Jonathan S Stamler

  9. Department of Medicine, Case Western Reserve University and University Hospitals Case Medical Center, Cleveland, Ohio, USA

    Bo Herman, Alfred Hausladen & Jonathan S Stamler

  10. Division of Digestive Diseases, University of California–Los Angeles, Los Angeles, California, USA

    Charalabos Pothoulakis

Authors
  1. Tor C Savidge
  2. Petri Urvil
  3. Numan Oezguen
  4. Kausar Ali
  5. Aproteem Choudhury
  6. Vinay Acharya
  7. Irina Pinchuk
  8. Alfredo G Torres
  9. Robert D English
  10. John E Wiktorowicz
  11. Michael Loeffelholz
  12. Raj Kumar
  13. Lianfa Shi
  14. Weijia Nie
  15. Werner Braun
  16. Bo Herman
  17. Alfred Hausladen
  18. Hanping Feng
  19. Jonathan S Stamler
  20. Charalabos Pothoulakis

Contributions

T.C.S. designed the study, performed InsP6, GSNO and cytotoxicity assays, BIACORE analysis, and wrote the paper; P.U. performed the toxin S-nitrosylation and InsP6 binding studies; N.O. and W.B. performed the toxin structural modeling and molecular docking simulations; I.P. performed the SNO immunofluorescence; K.A., A.C. and V.A. performed toxin autocleavage, InsP7 phosphorylation and UDP-glucosylation assays; A.G.T. performed animal toxin studies; R.D.E. performed the mass spectrometry; J.E.W. performed the cysteine saturation labeling studies; M.L. provided clinical specimens; R.K. performed the CD spectral analysis; L.S., W.N. and H.F. developed the toxin mutants, performed InsP6 cleavage and stool cytotoxicity assays and animals studies; B.H., A.H. and J.S.S. performed or oversaw the measurements of GSNO and SNO proteins; J.S.S. assisted with the study design and writing of the paper; C.P. prepared holotoxins, performed animal toxin studies and assisted with study design and manuscript editing.

Corresponding author

Correspondence toTor C Savidge.

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Competing interests

C.P. is a paid consultant with Merck and Optimer Pharmaceuticals and a paid speaker for the Postgraduate Institute for Medicine. J.S.S. has a small financial interest in N30 Pharma, Adamas Pharma, Vindica LLC, SabrePharm and LifeHealth, early-stage companies in development of nitric oxide–related therapeutics.

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Savidge, T., Urvil, P., Oezguen, N.et al. Host S-nitrosylation inhibits clostridial small molecule–activated glucosylating toxins.Nat Med17, 1136–1141 (2011). https://doi.org/10.1038/nm.2405

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