Structure of the full-length Clostridium difficile toxin

1Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA, USA. 2Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY, USA. 3Department of Microbial Pathogenesis, University of Maryland Baltimore, Baltimore, MD, USA. 4Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA, USA. 5NE-CAT and Department of Chemistry and Chemical Biology, Cornell University, Argonne National Laboratory, Argonne, IL, USA. *e-mail: r.jin@uci.edu C lostridium difficile is classified as one of the top three urgent antibiotic resistance threats by the Centers for Disease Control and Prevention. The pathology of CDI is primarily mediated by two homologous exotoxins, TcdA and TcdB, which target and disrupt the colonic epithelium, leading to diarrhea and colitis1–5. While the relative roles of these two toxins in the pathogenesis of CDI are not completely understood, recent studies have shown that TcdB is more virulent than TcdA and more important for inducing the host inflammatory and innate immune responses5–8. Notably, a TcdB-neutralizing antibody (bezlotoxumab) was recently approved by the US Food and Drug Administration as a prevention against recurrent infection9. However, more effective therapies for CDI are desperately needed. TcdA (~308 kDa) and TcdB (~270 kDa) contain four functional domains: an N-terminal glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a central delivery and receptor-binding domain (DRBD), and a CROPs domain (Fig. 1a). Toxins bind to cell surface receptors via the DRBD and the CROPs and enter the cells via endocytosis10–14. Acidification in the endosome triggers conformational changes in the toxins that prompt the DRBD to form a pore and deliver the GTD and the CPD across the endosomal membrane15–18. In the cytosol, the CPD is activated by eukaryote-specific inositol hexakisphosphate (InsP6) and subsequently undergoes autoproteolysis to release the GTD. The GTD then glucosylates small GTPases of the Rho family, including Rho, Rac, and Cdc42, and inhibits their functions19–23. Numerous structures have been reported for fragments of TcdA and TcdB, which have provided tremendous insight into the functions of these toxin domains10,12,13,24–28. However, it remains unknown how individual domains interact within the supertertiary structure of the holotoxin and how the holotoxin dynamically responds in a precise stepwise manner to the environmental and cellular cues, such as low pH and InsP6, that lead to intoxication. Here we report the crystal structure of TcdB holotoxin at 3.87-Å resolution, which was crystallized at an endosomal pH (pH 5.2) and displays an architecture that is distinct from that of the prior model derived from EM29. To probe the structural dynamics of TcdB in solution, we used a combination of small-angle X-ray scattering (SAXS), single-molecule fluorescence resonance energy transfer (smFRET), and cross-linking mass spectrometry (XL-MS). These experiments consistently demonstrated that the elongated CROPs of TcdB showed pH-dependent structural flexibility, which may help TcdB modulate its activity in response to environmental pH change. Furthermore, we reveal novel structural mechanisms underlying neutralization of TcdB by three antibodies. These findings collectively reveal new strategies for developing therapeutics and vaccines for treatment of CDI.