Structure and conformational dynamics of Clostridioides difficile toxin A

Abstract

Clostridioides difficile toxin A and B (TcdA and TcdB) are two major virulence factors responsible for diseases associated with C. difficile infection (CDI). Here, we report the 3.18-Å resolution crystal structure of a TcdA fragment (residues L843–T2481), which advances our understanding of the complete structure of TcdA holotoxin. Our structural analysis, together with complementary single molecule FRET and limited proteolysis studies, reveal that TcdA adopts a dynamic structure and its CROPs domain can sample a spectrum of open and closed conformations in a pH-dependent manner. Furthermore, a small globular subdomain (SGS) and the CROPs protect the pore-forming region of TcdA in the closed state at neutral pH, which could contribute to modulating the pH-dependent pore formation of TcdA. A rationally designed TcdA mutation that trapped the CROPs in the closed conformation showed drastically reduced cytotoxicity. Taken together, these studies shed new lights into the conformational dynamics of TcdA and its roles in TcdA intoxication.

Neutralizing epitopes on Clostridioides difficile toxin A revealed by the structures of two camelid VHH antibodies

“…It is widely accepted that the pore-forming region of TcdA/TcdB, which carries a large number of hydrophobic residues, is protected by the DRBD at neutral pH, but partially unfolds and detaches from the DRBD when induced by acidic endosomal pH in order to form a pore ( 5 , 10 , 48 ). Drastic conformational changes could be observed in the pore-forming regions when comparing the structure of TcdA obtained at the neutral pH and a structure of TcdB that was obtained under acidic pH and represents a pore-forming intermediate state ( Figure 4E ) ( 8 – 10 ). Nevertheless, it has been shown that 5D is able to grip and stabilize a β hairpin motif in TcdB’s pore-forming region, which may subsequently block the acidic pH-induced unfolding of TcdB ( 10 ).…”

“…Therefore, antibodies blocking this step should provide potent protection. We found that AH3 and AA6 can bind simultaneously on TcdA holotoxin ( 8 , 9 ) ( Figure 5 ). Therefore, combining AH3 and AA6 in a hybrid molecule should lead to even higher neutralizing potency because of synergistic binding of both VHHs.…”

“…The overall structure of the AA6-bound TcdA 1073-1464 is similar to that of the standalone TcdA fragment in the context of the holotoxin with a RMSD of ~0.576 Å over 313 residues, indicating that AA6 does not force appreciable conformational changes in the toxin ( 8 ). Residues from all three CDRs of AA6 contribute to direct interactions with TcdA 1073-1464 with the CDR3 playing a dominating role ( Figure 2A ).…”

“…The structure of the TcdA GTD-AH3 complex was solved by molecular replacement using the structures of TcdA GTD (PDB 7U2P) ( 22 ) and VHH 5D (PDB 6OQ5) ( 10 ) as search models via PHENIX.Phaser-MR ( 38 ). The structure of the TcdA 1073-1464 -AA6 complex was solved by molecular replacement using the structures of the corresponding fragment (1073–1464) in TcdA DRBD (PDB code: 7U1Z) ( 8 ) and a VHH (PDB 5M2J) ( 39 ) as search models via PHENIX.Phaser-MR ( 38 ). All refinement and model building procedures were carried out with PHENIX.refine ( 40 ) and COOT ( 41 ).…”

“…Two high-molecular-weight exotoxins, toxin A (TcdA) and toxin B (TcdB), secreted by C. difficile are the major virulence factors responsible for CDI, which lead to a wide spectrum of clinical symptoms ranging from mild diarrhea to life threatening pseudomembranous colitis ( 4 – 7 ). The sequences and three dimensional structures of TcdA and TcdB show a similar modular arrangement which could be divided into four functional domains: an amino-terminal glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a delivery and receptor-binding domain (DRBD), and a carboxy-terminal combined repetitive oligopeptides domain (CROPs) ( 8 – 10 ) ( Figure 1A ). TcdA and TcdB are internalized into host cells via receptor-mediated endocytosis ( 5 , 10 – 14 ).…”