The global prevalence of severe Clostridium 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 that C. 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 of C. 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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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.
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 Med 17, 1136–1141 (2011). https://doi.org/10.1038/nm.2405
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