Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly

Abstract

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.

1HN, 13C, and 15N resonance assignments of the Clostridioides difficile receptor binding domain 2 (CDTb, residues 757–876)

“…b The secondary structure of RBD2 predicted from the chemical shift index method is illustrated and consists of 8 beta strands (β1, I767-N775; β2, T781-A789; β3, Q802-T809; β4, K816-N827; β5, T837-N841; β6, I852-Y855; β7, K857-I863; β8, R868-V875) shown with blue arrows and 2 alpha helices (α1, D758-A764; α2, K795-Y800) highlighted in red. The remaining regions of RBD2 are predicted to exist as random coil and are shown with a black line in the secondary structure representation CSI analyses predicts that the RBD domain has eight beta strands (I767-N775; T781-A789; Q802-T809; K816-N827; T837-N841; I852-Y855; K857-I863; R868-V875) and 2 alpha helices (D758-A764; K795-Y800), which is fully consistent with the X-ray and cryoEM structures reported previously (Xu et al 2020). In summary, the chemical shift values for backbone and sidechain resonances of RBD2 obtained here were deposited in the Biological Magnetic Resonance Bank database (http://www.bmrb.wisc.edu) under accession number 28,131, and these data will be important for nextstage NMR studies that map RBD2 biomolecular interactions and for developing inhibitors targeting CDTb.…”

Human α-Defensin-5 Efficiently Neutralizes Clostridioides difficile Toxins TcdA, TcdB, and CDT

“…It was demonstrated that also CDTb forms pores in lipid bilayer membranes in vitro (Kronhardt et al, 2017). More recently, it was shown that CDTb forms di-heptamer like structures (Xu et al, 2020). Furthermore, when applied to cells, CDTb alone (in the absence of CDTa) is able to cause dramatic changes in cell morphology and cell viability (Kronhardt et al, 2017).…”

The Importance of Therapeutically Targeting the Binary Toxin from Clostridioides difficile

“…Likewise, the ‘A’ subunit binds to the ‘B’ oligomer complex in all binary toxins prior to being endocytosed and trafficked into endosomes. While the ‘A’ subunit is monomeric for each binary toxin, the oligomeric structure has been shown to consist of varying compositions including heptameric, octameric, as well as di-heptamer associations of singly processed ‘B’ subunits [ 19 , 20 ]. Likewise, it is hypothesized that a conformational shift in the ‘B’ subunit assembly, facilitated by the acidic pH of the endosomal compartment, induces translocation of the ‘A’ subunit into the host cytosol where the host NAD + /NADPH acts as a donor for catalytic transfer of ADP-ribose to monomeric G-actin.…”

“…Limited chymotrypsin proteolysis of pro-CDTb is sufficient to remove the signaling peptide (SD; residues 1–43) and the activation domain (AD; residues 44–211) yielding an oligomeric and active form of CDTb (residues 212–876; Figure 3 A,B) [ 15 ]. Unlike the heptameric and octomeric states identified for the cell-binding components of other bacterial binary toxins such as anthrax and iota toxins, CDTb was shown by single-particle cryoEM micrsoscopy to fold into two unique di-heptameric structures (~1.0 MDa) including a symmetric and an asymmetric dimer of heptamers, solved at resolutions of 3.14 Å ( Sym CDTb) and 2.84 Å ( Asym CDTb), respectively [ 20 ]. A corroborative study confirmed these data at a lower resolution and were given names as ‘short’ and ‘long’ particles, corresponding to the asymmetric ( Asym CDTb) and symmetric ( Sym CDTb) di-heptamers, respectively ( Figure 3 C and 3D) [ 37 ].…”