Characterization and development of new anti‐toxins targeting Clostridium difficile

Abstract

Clostridium difficile is a bacterial pathogen that causes serious and potentially fatal inflammatory disease of the colon. One strategy for targeting C. difficile infection is to development treatments that target bacterial virulence. 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. The Tcd toxins, TcdA and TcdB, are the major virulence factors produced by C. difficile and mediate host cytotoxicity by glucosylating and inactivating Rho family GTPases using UDP-glucose as a glucosyl donor. Glucosylation of Rho GTPases leads to cell rounding and eventually cell death. We used kinetic isotope effects (KIEs) and transition state theory to solve the transition state structure of the glycosyltransferase domain of TcdB (TcdB-GTD). This permits the design of transition state analogues which are potent enzyme inhibitors. KIEs compare the chemical steps of enzymatic reaction rates with isotopically labeled substrates and unlabeled substrates and provide critical information on the catalytic mechanism, bond lengths and geometry at the transition state. KIE analysis on the glycohydrolase reaction of TcdB-GTD supported the formation of a late, dissociative glucocation-like transition state where positive charge develops on the anomeric carbon. Glucocationic transition states are best mimicked by cationic nitrogen groups at or near the anomeric carbon. Iminosugars contain a nitrogen atom in the sugar ring in place of oxygen and mimic glucocationic transition states. We tested iminosugars for inhibition of TcdB-GTD and TcdA-GTD. Isofagomine (Ki TcdB-GTD = 1.4 µM and Ki TcdA-GTD = 0.24 µM) and noeuromycin (Ki TcdB-GTD = 10.6 µM and Ki TcdA-GTD = 4.7 µM) were the most potent iminosugars identified. Isofagomine and noeuromycin were shown to exhibit an uncompetitive mechanism of inhibition suggesting that both iminosugars act by binding to the enzyme-UDP complex. Isothermal titration calorimetry experiments with TcdB-GTD supported this finding where isofagomine and noeuromycin could only bind to TcdB-GTD in the presence of UDP with Kd = 0.133 µM and Kd = 0.4 µM respectively. In addition, the X-ray crystal structure of TcdB-GTD in complex with either Isofagomine or noeuromycin with UDP revealed the formation of an ion-pair interaction between the cationic nitrogen atom of the iminosugar and the β-phosphate of UDP. Finally, we confirmed that isofagomine and noeuromycin could prevent Tcd toxin-induced cytotoxicity of IMR90 cells. Firstly, both iminosugars were able to prevent Tcd toxin-induced cell rounding as visualized by light microscopy. Secondly, Western blot analysis of Tcd toxin treated IMR90 cell lysates revealed that both isofagomine and noeuromycin could prevent glucosylation of Rac1 by Tcd toxin. Isofagomine and noeuromycin are effective in preventing Tcd toxin action. Transition state analogues against TcdA and TcdB have the potential to be used as treatments for C. difficile infections.