Structural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection

Abstract

C. difficile is a major cause of antibiotic-associated gastrointestinal infections. Two C. difficile exotoxins (TcdA and TcdB) are major virulence factors associated with these infections, and chondroitin sulfate proteoglycan 4 (CSPG4) is a potential receptor for TcdB, but its pathophysiological relevance and the molecular details that govern recognition remain unknown. Here, we determine the cryo-EM structure of a TcdB–CSPG4 complex, revealing a unique binding site spatially composed of multiple discontinuous regions across TcdB. Mutations that selectively disrupt CSPG4 binding reduce TcdB toxicity in mice, while CSPG4-knockout mice show reduced damage to colonic tissues during C. difficile infections. We further show that bezlotoxumab, the only FDA approved anti-TcdB antibody, blocks CSPG4 binding via an allosteric mechanism, but it displays low neutralizing potency on many TcdB variants from epidemic hypervirulent strains due to sequence variations in its epitopes. In contrast, a CSPG4-mimicking decoy neutralizes major TcdB variants, suggesting a strategy to develop broad-spectrum therapeutics against TcdB.

Structural dynamics of receptor recognition and pH-induced dissociation of full-length Clostridioides difficile Toxin B

“…A recent published paper [ 22 ] reported a cryo-EM structure of a TcdB fragment in complex with CSPG4 D1 fragment. Our observed interactions between the full-length TcdB and CSPG4 D1 fragment are consistent with their results.…”

“…In addition, truncations of TcdB were used to locate the CSPG4-binding site to be within residues 1,500 to 1,851 of TcdB. Recently, there is a published cryo-EM structure of the complex between a TcdB fragment and CSPG4 [ 22 ]. However, the structural mechanism of how the full-length TcdB binds CSPG4 is unclear.…”

Structural basis for selective modification of Rho and Ras GTPases by Clostridioides difficile toxin B

“…To date, the only nonantibiotic treatment option for CDI is a mAb bezlotoxumab, which blocks TcdB binding to its receptor chondroitin sulfate proteoglycan 4 (CSPG4) via an allosteric mechanism ( 48 , 49 ). However, TcdB becomes inaccessible to antibodies after entering the host cells.…”

“…TcdB VPI10463 holotoxin and TcdB VPI10463 -α16/17 M68 were cloned into a modified pET28a vector with an N-terminal Twin-Strep tag following a PreScission protease-cleavage site and a C-terminal 6xHis tag. The full-length toxins were expressed and purified as described previously ( 49 ).…”

Structure and conformational dynamics of Clostridioides difficile toxin A

“…Most of these toxins are composed of four structural modules: an N-terminal glucosyltransferase domain (GTD), followed by a cysteine protease domain (CPD), a delivery and receptor-binding domain (DRBD), and a large C-terminal combined repetitive oligopeptides domain (CROPs) ( Aktories et al, 2017 ; Orrell & Melnyk, 2021 ) ( Fig 1A ). The cellular uptake of TcdA and TcdB are mediated by receptor-mediated endocytosis ( Papatheodorou et al, 2010 ; Tao et al, 2016 , 2019 ; Aktories et al, 2017 ; Chen et al, 2018 , 2021 ). Triggered by acidification in the endosomes, the hydrophobic pore-forming region of the toxin undergoes membrane insertion and facilitates the translocation of the GTD and the CPD into the cytosol ( Qa’Dan et al, 2000 ; Barth et al, 2001 ; Genisyuerek et al, 2011 ; Zhang et al, 2014 ).…”