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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.

SUBMITTER: Jiang M

PROVIDER: S-EPMC8982864 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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

Jiang Mengqiu M Shin Joonyoung J Simeon Rudo R Chang Jeng-Yih JY Meng Ran R Wang Yuhang Y Shinde Omkar O Li Pingwei P Chen Zhilei Z Zhang Junjie J

PLoS biology 20220324 3

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 deliver ...[more]

PMID: 35324891

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Project description:Clostridioides difficile is a Gram-positive opportunistic human pathogen that causes 15,000 deaths annually in the United States, prompting a need for vaccine development. In addition to the important toxins TcdA and TcdB, binary toxin (CDT) plays a significant role in the pathogenesis of certain C. difficile ribotypes by catalyzing the ADP-ribosylation of actin in host cells. However, the mechanisms of CDT neutralization by antibodies have not been studied, limiting our understanding of key epitopes for CDT antigen design. Therefore, we isolated neutralizing monoclonal antibodies against CDT and characterized their mechanisms of neutralization structurally and biochemically. Here, 2.5-Å and 2.6-Å resolution X-ray crystal structures of the antibodies BINTOXB/22 and BINTOXB/9, respectively, in complex with CDTb-the CDT subunit that forms a heptameric pore for the delivery of toxic CDTa enzyme into the host cytosol-showed that both antibodies sterically clash with adjacent protomers in the assembled heptamer. Assessment of trypsin-induced oligomerization of the purified CDTb protoxin in vitro showed that BINTOXB/22 and BINTOXB/9 prevented the assembly of di-heptamers upon prodomain cleavage. This work suggests that the CDT oligomerization process can be effectively targeted by antibodies, which will aid in the development of C. difficile vaccines and therapeutics. IMPORTANCE Clostridioides difficile strains associated with worse clinical outcomes have been found to secrete a toxin called CDT (or binary toxin). As blocking the function of this toxin could help mitigate C. difficile infections, we sought to determine the molecular basis for the inhibition of CDT by monoclonal antibodies. We isolated monoclonal antibodies targeting the B-component of CDT (CDTb) and selected two with neutralizing activity for detailed structural and biochemical characterization. High-resolution crystal structures of each antibody bound to CDTb showed that their presence would preclude the assembly of a CDTb oligomer required for activity. Oligomerization of CDTb in vitro was shown to be blocked in the presence of the neutralizing antibodies, but not a control antibody.

Dataset Information

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

Publications

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

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