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<p><i>Clostridioides difficile</i> 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 <i>C. difficile</i> 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.</p><p></p>

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Pore formation by the CDTb component of the Clostridioides difficile binary toxin is Ca2+-dependent

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年6月9日

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  • Dinendra L. Abeyawardhane,
  • Spiridon E. Sevdalis,
  • Kaylin A. Adipietro,
  • Raquel Godoy-Ruiz,
  • Kristen M. Varney,
  • Izza F. Nawaz,
  • Alejandro X. Spittel,
  • Daniel Hunter,
  • Richard R. Rustandi,
  • Vitalii I. Silin,
  • Amedee des Georges,
  • Mary E. Cook,
  • Edwin Pozharski,
  • David J. Weber

摘要

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 Ca2+ 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 Ca2+ from a single site in receptor binding domain 1 (RBD1) of CDTb triggers conformational exchange in CDTb. These and structure/function studies of a Ca2+-binding double mutant targeting RBD1 (i.e., D623A/D734A) support a model in which dissociation of Ca2+ from RBD1 induces dynamic properties in CDTb that enable it to bind and form pores in lipid bilayers.