The Importance of Therapeutically Targeting the Binary Toxin from Clostridioides difficile

Abstract

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell's cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.

Keywords: CDT; CDTa; CDTb; Clostridioides difficile; binary toxin; infectious disease; protein structural biology.

Figure 1

Schematic representation of the AB toxins causing C. difficile infection. The large enterotoxins (TcdA/Toxin A and TcdB/Toxin B) are composed of an N-terminal glycosylating enzymatic domain (green), an autocatalytic processing domain (yellow), a delivery and/or pore-forming domain for translocation (purple), and a binding domain with combined repetitive oligopeptides known as ‘CROPs’ (blue). The binary toxin, C. difficile transferase (CDT), consists of two independently produced components, CDTa and CDTb. The N-terminal domain of the enzymatic component (CDTa) binds to the binding component (CDTb) while the C-terminal domain of CDTa causes toxic ADP-ribosyltransferase activity within the host cell.

Figure 2

X-ray Crystal Structure of CDTa modified from PDB file: 2WN8. The N-terminal region (residues 1–215, orange) is the CDTb-binding domain. The C-terminal region (residues 224–420, pale green) is the catalytic domain with ADP-ribosyltransferase activity. The ARTT loop (residues 377–387, blue) is crucial for substrate binding and ADP-ribosylation as the active site residues are located around this region (inset). However, Glu385 and Glu387 in the ARTT loop are not directly involved in the catalytic site formation. A small peptide loop (residues 216–223, black) connects both termini.

Figure 3

Structure of CDTb. (A) The sequence of the pro-CDTb before proteolytic cleavage of signaling domain (SD) and activation domain (AD). Activated CDTb consists of residues 212–876. (B) Structure of activated ^AsymCDTb monomer corresponding to each domain in the sequence; HD1 (red), βBD (green), HD2 (purple), HD3 (yellow), RBD1 (blue), and RBD2 (cyan). Linker regions 1 and 2 are shown in grey and the green spheres represent Ca²⁺ ions in HD1 and RBD1. (C) Structure of activated ^AsymCDTb di-heptameric assembly highlighting the arrangement of individual domains of the monomer. ^AsymCDTb structure is modified from PDB file: 6UWR. (D) Structure of activated ^SymCDTb di-heptameric assembly with corresponding domains of the monomer. The βBD is not elongated to represent the pore-forming unit and therefore ^SymCDTb is identified as a pre-pore state. ^SymCDTb structure is modified from PDB file: 6UWT.

Figure 4

The putative mechanism for cell entry of CDT. ^SymCDTb can acts as a pre-pore state while ^AsymCDTb is a potential pre-insertion state. The binding of CDTa to CDTb via the N-terminal region may trigger the decoupling of heptameric units. The resulting CDT complex can engage with the cell surface receptors (LSR or CD44) embedded in the cell membrane, localized to lipid rafts, and this phenomenon is potentially facilitated by glycan binding.