Chondroitin sulfate proteoglycan 4 functions as the cellular receptor for Clostridium difficile toxin B

Abstract

As a gram-positive, spore-forming anaerobic bacillus, Clostridium difficile (C. difficile) is responsible for severe and fatal pseudomembranous colitis, and poses the most urgent antibiotic resistance threat worldwide. Epidemic C. difficile is the leading cause of antibiotic-associated diarrhoea globally, especially diarrhoea due to the emergence of hypervirulent strains associated with high mortality and morbidity. TcdB, one of the key virulence factors secreted by this bacterium, enters host cells through a poorly understood mechanism to elicit its pathogenic effect. Here we report the first identification of the TcdB cellular receptor, chondroitin sulfate proteoglycan 4 (CSPG4). CSPG4 was initially isolated from a whole-genome human shRNAmir library screening, and its role was confirmed by both TALEN- and CRISPR/Cas9-mediated gene knockout in human cells. CSPG4 is critical for TcdB binding to the cell surface, inducing cytoskeleton disruption and cell death. A direct interaction between the N-terminus of CSPG4 and the C-terminus of TcdB was confirmed, and the soluble peptide of the toxin-binding domain of CSPG4 could protect cells from the action of TcdB. Notably, the complete loss of CSPG4/NG2 decreased TcdB-triggered interleukin-8 induction in mice without significantly affecting animal mortality. Based on both the in vitro and in vivo studies, we propose a dual-receptor model for TcdB endocytosis. The discovery of the first TcdB receptor reveals a previously unsuspected role for CSPG4 and provides a new therapeutic target for the treatment of C. difficile infection.

Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

“…Rather, we think that the most likely reason is that the toxins engage different receptors. While the human receptor for TcdA has yet to be identified, two receptors for TcdB have been described: poliovirus receptor-like protein 3 (PVRL3) and chondroitin sulfate proteoglycan 4 (CSPG4) (47,48). PVRL3 seems to account for the cell death occurring at high concentrations in HeLa cells (where CSPG4 is also highly expressed), but it can mediate both necrotic and apoptotic mechanisms in Caco2 cells (47,48).…”

“…While the human receptor for TcdA has yet to be identified, two receptors for TcdB have been described: poliovirus receptor-like protein 3 (PVRL3) and chondroitin sulfate proteoglycan 4 (CSPG4) (47,48). PVRL3 seems to account for the cell death occurring at high concentrations in HeLa cells (where CSPG4 is also highly expressed), but it can mediate both necrotic and apoptotic mechanisms in Caco2 cells (47,48). We hypothesize that TcdB binding to PVRL3 initiates the assembly and activation of the NOX complex and that the lack of PVRL3 binding by TcdA accounts for the differences in the cell death responses of the two toxins.…”

Binding and entry of Clostridium difficile toxin B is mediated by multiple domains

“…Expression plasmids TcdB is a multimodular [5] protein consisting of: the biologically active N-terminal glucosyltransferase domain (GTD, residues 1-543) [21,22] which inactivates Rho family GTPases, leading to disruption of actin dynamics, leading to cell rounding and eventual cell death [6]; the cysteine protease domain (CPD, residues 544-807) [23] which is responsible for autocatalytic cleavage of the GTD in the presence of inositol hexakisphosphate (IP 6 ) [24], though CPD activity was recently shown to be somewhat dispensable and merely modulate cytotoxicity [17,25]; the translocation domain ($ residues 810-1350), containing pore forming-(PFR, residues 830-990) and hydrophobic-regions (HR, residues 956-1128) [8,13], which is responsible for delivery of the GTD across the endosomal membrane; and the CROPs domain (1851-2366), classically viewed as the receptor binding domain responsible for cellular binding and entry [26]. Accumulating evidence suggests the presence of a second binding domain between residues 1349-1811 [8,9,15,16] were subsequently transformed into B. megaterium strain WH320 protoplasts using standard procedures (MoBiTec GmbH.). Expression in B. megaterium was induced with 0.5% (w/v) (D)-xylose for 8 h. Cells were recovered by centrifugation, suspended in Lysis Buffer (300 mM NaCl, 20 mM Tris-HCl, pH 8.0, 20 mM imidazole and 10% (v/v) glycerol) containing Protease Inhibitor Cocktail and Universal Nuclease, and then lysed via French Press at 16,000 psi.…”

Gleaning Insights from Fecal Microbiota Transplantation and Probiotic Studies for the Rational Design of Combination Microbial Therapies

“…Most C. difficile strains encode two toxins, TcdA and TcdB, but strains that produce only TcdB or no toxins have also been isolated; only strains without toxins are considered to be avirulent (33)(34)(35)(36). TcdA and TcdB bind to any of a number of host cell receptors (37)(38)(39)(40) and, once inside host cells, act as monoglucosyltransferases to inactivate Rho family GTPases (41,42). This inactivation leads to rounding and death of GI epithelial cells, disrupting the epithelial barrier (33,43,44).…”