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

Abstract

Clostridioides difficile secretes Toxin B (TcdB) as one of its major virulence factors, which binds to intestinal epithelial and subepithelial receptors, including frizzled proteins and chondroitin sulfate proteoglycan 4 (CSPG4). Here, we present cryo-EM structures of full-length TcdB in complex with the CSPG4 domain 1 fragment (D1401-560) at cytosolic pH and the cysteine-rich domain of frizzled-2 (CRD2) at both cytosolic and acidic pHs. CSPG4 specifically binds to the autoprocessing and delivery domains of TcdB via networks of salt bridges, hydrophobic and aromatic/proline interactions, which are disrupted upon acidification eventually leading to CSPG4 drastically dissociating from TcdB. In contrast, FZD2 moderately dissociates from TcdB under acidic pH, most likely due to its partial unfolding. These results reveal structural dynamics of TcdB during its preentry step upon endosomal acidification, which provide a basis for developing therapeutics against C. difficile infections.

Pathogenicity and virulence of Clostridioides difficile

“…The first recognized TcdB receptor was chondroitin sulfate proteoglycan 4 (CSPG4), a highly conserved protein, identified through shRNAmir library screening [ 79 ]. Cryo-EM structures of CSPG4-TcdB indicate binding is mediated through autoprocessing and delivery domains [ 80 ; 81 ]. However, CSPG4 receptors are abundantly expressed on subepithelial myofibroblasts, and not in the colonic epithelium, suggesting CSPG4 is not the dominant TcdB receptor [ 82 ].…”

Structural dynamics of the CROPs domain control stability and toxicity of Paeniclostridium sordellii lethal toxin

“…Great efforts have been made to understand the toxin action mechanisms of the LCTs through structural biological approaches 21,27 . Recently, some high-resolution full-length or near-complete structures for TcdA [28][29][30] and TcdB [31][32][33] were reported. In those structures, the "core" domains (GTD, CPD, and DRBD) show a similar overall architecture, but the CROPs seem to be dynamic and have at least two major conformations: one curves upward around the GTD-CPD head and is referred to as the "open" conformation, the other curves downward alongside the DRBD and is referred to as the "closed" conformation.…”

“…TcdA is usually displayed in the closed conformation 29,30,34 . TcdB tends to display the open conformation in either the crystal or cryogenic electron microscopy (cryo-EM) structures [31][32][33] while its closed form has been detected using SAX or XL-MS analyses 31,34 . In previous studies, small regions located at the N-terminal boundary of TcdA and TcdB CROPs were defined as the "hinge", which may be responsible for the swing of the CROPs domains 29,31 .…”

Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

“…TcdA and TcdB differ regarding their receptor-specificity. TcdB has been demonstrated to utilize members of the Wnt receptor frizzled 1/2/7 (FZD1/2/ 7) and chondroitin sulfate proteoglycan 4 (CSPG4) as receptors, which are recognized through distinct binding sites [25][26][27][28][29][30] , and are independent from each other 26,31 . In addition, poliovirus receptorrelated 3 (PVRL3) and low-density lipoprotein receptor-related protein 1 (LRP1) have been reported as potential receptors 32,33 .…”

“…Interestingly, a binding site for SEMA6A/6B has been identified at an identical location on the DRBD domain of TcsL 38,39 , which is a toxin sharing ~76% sequence identity with TcdB. The CSPG4-binding site is spatially composed of discontinuous segments scattered across multiple domains of TcdB [28][29][30] .…”