Clostridioides difficile toxins: mechanisms of action and antitoxin therapeutics

Abstract

Clostridioides difficile is a Gram-positive anaerobe that can cause a spectrum of disorders that range in severity from mild diarrhoea to fulminant colitis and/or death. The bacterium produces up to three toxins, which are considered the major virulence factors in C. difficile infection. These toxins promote inflammation, tissue damage and diarrhoea. In this Review, we highlight recent biochemical and structural advances in our understanding of the mechanisms that govern host-toxin interactions. Understanding how C. difficile toxins affect the host forms a foundation for developing novel strategies for treatment and prevention of C. difficile infection.

Fig. 1 |. Structures of TcdA and TcdB.

a | The pathogenicity locus (PaLoc). The PaLoc contains genes that encode the large clostridial toxins A (TcdA) and B (TcdB), the positive toxin regulator TcdR, the negative toxin regulator TcdC, the holin TcdE and the endolysin fragment TcdL. b | Crystal structure of TcdA_1–1832 (Protein Data Bank (PDB) identifier (ID) 4R04) docked into an electron density map of the holotoxin. c | Structure of TcdB (PDB ID 6OQ5). d | Structure of the Frizzled 2 (FZD2) cysteine-rich domain (CRD) bound to the TcdB delivery domain (DD) (PDB ID 6C0B) and docked onto the TcdB holotoxin structure (PDB ID 6OQ5). The magnified view highlights the empirical structure containing palmitoleic acid (PAM) in magenta (PDB ID 6C0B). e | Structure of chondroitin sulfate proteoglycan 4 (CSPG4) repeat 1 bound to TcdB (PDB ID 7ML7) docked onto the TcdB holotoxin structure (PDB ID 6OQ5). The magnified view highlights the binding interface (PDB ID 7ML7). APD, autoprotease domain; CROP domain, combined repetitive oligopeptide domain; GTD, glucosyltransferase domain.

Fig. 2 |. Intoxication mechanism of TcdA and TcdB.

(1) Toxin A (TcdA) and toxin B (TcdB) bind distinct surface receptors. TcdA is thought to bind glycans (and proteoglycans such as glycoprotein 96 (gp96)), sulfated glycosaminoglycans (sGAGs) and/or members of the low-density lipoprotein receptor (LDLR) family. TcdB can bind chondroitin sulfate proteoglycan 4 (CSPG4), Nectin 3, Frizzled 1 (FZD1), FZD2, FZD7 and a variety of glycans. (2) Following receptor binding, TcdA and TcdB are internalized. TcdA uses a novel clathrin-independent entry mechanism through PACSIN2, whereas TcdB enters via clathrin-mediated endocytosis. (3) An influx of protons lowers the pH of the endosome, which induces transmembrane pore formation in the toxins. (4)The autoprotease domain (APD) and the glucosyltransferase domain (GTD) are translocated through the endosomal membrane into the cytosol. Inositol hexakisphosphate (InsP₆) activates the APD and initiates autoproteolysis and release of the GTD. (5) The free GTD monoglucosylates host GTPases, resulting in downstream cellular changes such as tight junction collapse, cytokine stimulation and apoptosis.

Fig. 3 |. Progression of colon intoxication by the Clostridioides difficile toxins.

Clostridioides difficile colonizes the colon and produces the large clostridial toxins A (TcdA; brown) and B (TcdB; yellow), as well as the C. difficile transferase (CDT; CDTa in red and CDTb in green). TcdA binds glycoproteins to enter host cells. TcdB binds Frizzled 1 (FZD1), FZD2 and FZD7 (blue) on the basolateralside of host epithelia, Nectin 3 (blue) at cellular junctions and chondroitin sulfate proteoglycan 4 (CSPG4; blue) on myofibroblasts within the lamina propria. CDTb binds lipolysis-stimulated lipoprotein receptor (LSR; blue) and oligomerizes to enable CDTa binding. Increasing concentrations of TcdA, TcdB and CDT within host cells cause the disruption of focal adhesions and tight junctions, loss of cellular polarity and cytoskeletal breakdown. These effects culminate in cellular rounding, stem cell apoptosis at the base of colonic crypts, epithelial cell shedding and oedema. Damaged epithelial cells release cytokines and chemokines, which recruit neutrophils and other immune cells to the tissue. The intoxication of monocytes and macrophages can promote IL-1β production and pyroptosis, which leads to further inflammation and damage within the tissue. Dead epithelial cells and immune cells contribute to the formation of pseudomembranous plaques, a hallmark of C. difficile-associated pseudomembranous colitis.

Fig. 4 |. Structure of the Clostridioides difficile transferase toxin.

a | The Clostridioides difficile transferase (CDT) locus (CDTloc). The CDTloc contains genes that encode CDT, cdtA and cdtB, as well as the positive toxin regulator CdtR, cdtR. b | Structure of CDTa bound to nicotinamide adenine dinucleotide (NAD) (Protein Data Bank (PDB) identifier (ID) 2WN6). CDTa comprises the ADP-ribosyltransferase (ADPRT) and pseudo-ADPRT (pADPRT) domains. c | Structures of heptameric CDTb with and without full β-barrel extension (PDB ID 6O2N). CDTb comprises domains D1–D3, D3’ and D4, with a linker (L) between D3’ and D4. Magnified views highlight one protomer of CDTb. d | Structure of CDTa bound to heptameric CDTb (PDB ID 6V1S) (left). Transparent surface representations of heptameric CDTb with and without full β-barrel extension (PDB ID 6O2N) are shown in the same orientation after superimposition of CDTa bound to heptameric CDTb (PDB ID 6V1S). Colour schemes of domains are continued from FIG. 4b,c. D3’, L, and D4 are not visible.

Fig. 5 |. Intoxication mechanism of Clostridioides difficile transferase toxin.

Monomeric Clostridioides difficile transferase b (CDTb; green) binds to lipolysis-stimulated lipoprotein receptor (LSR; blue) on the gut epithelium. Oligomerization results in a heptameric prepore state, upon which CDTa (red) can bind. The CDTb prepore and CDTa are then endocytosed. The CDTb prepore transitions into a pore-forming state, with a β-barrel pore that spans the endosomal membrane. Endosome acidification triggers the translocation of CDTa into the cytosol. CDTa ADP-ribosylates G-actin (blue), which acts as a cap and inhibits its polymerization. The depolymerization of F-actin (blue) at the apical host cell surface promotes aberrant microtubule protrusion (purple), supported by septin proteins (red). The protrusions can envelope C. difficile cells to augment adherence to the host. NAD⁺, oxidized nicotinamide adenine dinucleotide.

Fig. 6 |. Biological therapeutic binding locations on TcdA and TcdB.

The positions of biological molecules that bind toxin A (TcdA; Protein Data Bank identifier 4R04; panel a) or toxin B (TcdB; Protein Data Bank identifier 6OQ5; panel b) are shown in coloured circles with numbers that correspond to their proposed mechanism of inhibition. The autoprotease domain (APD) on the TcdB structure is hidden and therefore unlabelled. CROPS, combined repetitive oligopeptide sequences; DARPins, designed ankyrin repeat proteins DD, delivery domain; GTD, glucosyltransferase domain.