Functional defects in Clostridium difficile TcdB toxin uptake identify CSPG4 receptor-binding determinants

Abstract

is a major nosocomial pathogen that produces two exotoxins, TcdA and TcdB, with TcdB thought to be the primary determinant in human disease. TcdA and TcdB are large, multidomain proteins, each harboring a cytotoxic glucosyltransferase domain that is delivered into the cytosol from endosomes via a translocation domain after receptor-mediated endocytosis of toxins from the cell surface. Although there are currently no known host cell receptors for TcdA, three cell-surface receptors for TcdB have been identified: CSPG4, NECTIN3, and FZD1/2/7. The sites on TcdB that mediate binding to each receptor are not defined. Furthermore, it is not known whether the combined repetitive oligopeptide (CROP) domain is involved in or required for receptor binding. Here, in a screen designed to identify sites in TcdB that are essential for target cell intoxication, we identified a region at the junction of the translocation and the CROP domains that is implicated in CSPG4 binding. Using a series of C-terminal truncations, we show that the CSPG4-binding site on TcdB extends into the CROP domain, requiring three short repeats for binding and for full toxicity on CSPG4-expressing cells. Consistent with the location of the CSPG4-binding site on TcdB, we show that the anti-TcdB antibody bezlotoxumab, which binds partially within the first three short repeats, prevents CSPG4 binding to TcdB. In addition to establishing the binding region for CSPG4, this work ascribes for the first time a role in TcdB CROPs in receptor binding and further clarifies the relative roles of host receptors in TcdB pathogenesis.

Early cell death induced by Clostridium difficile TcdB: Uptake and Rac1-glucosylation kinetics are decisive for cell fate

“…As shown by Manse & Baldwin, , PVRL3 interacts with the region of amino acids 1372–1493 within TcdB. Recent data show that CSPG4 binding also happens within the region of amino acids 1810–1850 (Gupta et al, ). As reported earlier, the combined repetitive oligopeptide (CROP)‐deleted TcdB (TcdB 1‐1852) is less potent than full‐length TcdB with respect to cell rounding (Olling et al, ).…”

“…The alignment illustrates that (a) the GTD of TcdB 1470 and TcdB R9385 are almost identical, except three amino acid residues and (b) the trunk of TcdB R20291 and TcdB R9385 are also considered as identical except for 12 amino acid residues, all within the cysteine protease domain. If the most abundant receptor in target cells is deleted, a concentration of intracellular toxin sufficient to induce ECD might not be show that CSPG4 binding also happens within the region of amino acids 1810-1850 (Gupta et al, 2017). As reported earlier, the combined repetitive oligopeptide (CROP)-deleted TcdB (TcdB 1-1852) is less potent than full-length TcdB with respect to cell rounding (Olling et al, 2011).…”

Identification of the C. sordellii lethal toxin receptor elucidates principles of receptor specificity in clostridial toxins

“…2B). TpeL showed low toxicity and TcdA and TcdB were several orders of magnitude less toxic to HULECs than what is reported for other cell types (16,17). Remarkably, the GI50 of TcsL was ~50 fM, suggesting that only ~200 toxin molecules/cell are lethal to HULECs.…”

Deletion of a 19-Amino-Acid Region in Clostridioides difficile TcdB2 Results in Spontaneous Autoprocessing and Reduced Cell Binding and Provides a Nontoxic Immunogen for Vaccination

“…Data from two studies (27,28) support the idea that TcdB binds CSPG4 in a region found within amino acids 1831 to 2366, which would be carboxy terminal to the region from amino acids 1769 to 1787. Using a combination of pulldown experiments and antibody-based blocking, Gupta and colleagues further narrowed the region of CSPG4 binding and found that it likely occurs within a region between residues 1824 and 1900 (29). This region is carboxy terminal to amino acids 1769 to 1787 in TcdB.…”