Clostridium difficile is a bacterial pathogen that causes serious and potentially fatal inflammatory disease of the colon. Anti-virulence strategies to treat C. difficile infection are attractive options to antibiotic therapy because the human microbiota is spared and damage to host tissue is minimized. TcdA and TcdB are the major virulence factors produced by C. difficile and mediate host cytotoxicity by glycosylating and inactivating Rho GTPases using UDP-glucose as a glucosyl donor, leading to cell rounding and cell death. We used kinetic isotope effects (KIEs) and transition state (TS) theory to solve the TS structure of the glycosyltransferase domain of TcdB (TcdB-GTD). This permits the design of TS analogues which are potent enzyme inhibitors. KIE analysis supported the formation of a late, dissociative glucocation-like TS where positive charge develops on the anomeric carbon. Iminosugars mimic glucocationic TS’s as they contain cationic nitrogen groups at or near the anomeric carbon. We tested imimosugars for inhibition of TcdB-GTD and TcdA-GTD. Isofagomine and noeuromycin were the most potent iminosugars identified and display uncompetitive inhibition, suggesting that both iminosugars act by binding to the enzyme-UDP complex. Isothermal titration calorimetry experiments supported this finding, where isofagomine and noeuromycin could only bind to TcdB-GTD in the presence of UDP with Kd = 66.9 nM and Kd = 179.7 nM respectively. X-Ray crystal structures of TcdB-GTD in complex with UDP and either isofagomine or noeuromycin, revealed that both iminosugars form an ion-pair interaction between the cationic nitrogen atom of the iminosugar and the β-phosphate of UDP. Finally, we confirmed that both iminosugars protected Tcd-induced rounding of IMR90 cells as visualized by light microscopy. We also showed through western blot analysis of IMR90 cell lysates that both iminosugars prevent Tcd induced toxicity by preventing the glycosylation of Rho GTPases, thereby demonstrating potential as new treatments for C. difficile.

Inhibition of clostridium difficile TcdA and TcdB toxins with transition state analogues

ACS Spring Meeting 2021

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