Pore formation by the CDTb component of the Clostridioides difficile binary toxin is Ca2+-dependent
Clostridioides difficile infection (CDI) is one of the five most urgent bacterial threats in the United States. Furthermore, hypervirulent CDI strains express a third toxin termed the C. difficile binary toxin (CDT), and its molecular mechanism for entering host cells is not fully elucidated. Like other AB-type binary toxins, CDT enters host cells via endosomes. Here we show via surface plasmon resonance and electrochemical impedance spectroscopy that the cell-binding component of CDT, termed CDTb, binds and form pores in lipid bilayers in the absence of its enzymatic component, CDTa. This occurs upon lowering free Ca<sup>2+</sup> ion concentration, and not by decreasing pH, as found for other binary toxins (i.e., anthrax). Cryogenic electron microscopy (CryoEM), X-ray crystallography, and nuclear magnetic resonance (NMR) studies show that dissociation of Ca<sup>2+</sup> from a single site in receptor binding domain 1 (RBD1) of CDTb triggers conformational exchange in CDTb. These and structure/function studies of a Ca<sup>2+</sup>-binding double mutant targeting RBD1 (i.e., D623A/D734A) support a model in which dissociation of Ca<sup>2+</sup> from RBD1 induces dynamic properties in CDTb that enable it to bind and form pores in lipid bilayers.
Authors
- Richard R. Rustandi (ORCID: https://orcid.org/0000-0002-2287-5188)
- Edwin Pozharski (ORCID: https://orcid.org/0000-0001-7012-5376)
- David J. Weber (ORCID: https://orcid.org/0000-0002-8824-1110)
- Kristen M. Varney (ORCID: https://orcid.org/0000-0003-3283-4950)
- Spiridon E. Sevdalis (ORCID: https://orcid.org/0009-0009-7846-1177)
- Raquel Godoy‐Ruiz (ORCID: https://orcid.org/0000-0003-4569-0781)
- Kaylin A. Adipietro
- Mary E. Cook (ORCID: https://orcid.org/0000-0002-6298-2586)
- Dan Hunter (ORCID: https://orcid.org/0000-0002-0907-1820)
- Amédée des Georges (ORCID: https://orcid.org/0000-0002-9704-3781)
- Dinendra L. Abeyawardhane (ORCID: https://orcid.org/0000-0003-3002-7792)
- Izza F. Nawaz
- Alejandro X. Spittel
- Vitalii I Silin
Institutions
- The Graduate Center, CUNY (US)
- City College of New York (US)
- University of Maryland, Baltimore (US)
- Merck & Co., Inc., Rahway, NJ, USA (United States) (US)
- National Institute of Standards and Technology (US)
- City University of New York (US)
- CUNY Advanced Science Research Center (US)
- Research Institute for Bioscience and Biotechnology (NP)
- Advanced Bioscience Laboratories (United States) (US)
Publication Details
- Journal
- Communications Biology
- Published
- 2025-06-09
- DOI
- https://doi.org/10.1038/s42003-025-08343-x
- Citations
- 2
- Primary Topic
- Clostridium difficile and Clostridium perfringens research
- Type
- article
- Field-Weighted Citation Impact
- 2.79
Funders
- National Institutes of Health
- National Institute of Standards and Technology
- National Cancer Institute
- National Institute of Diabetes and Digestive and Kidney Diseases
- Center for Nanoscale Science and Technology