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

Abstract

Clostridioides difficile toxin B (TcdB) is an intracellular toxin responsible for many of the pathologies of C. difficile infection. The two variant forms of TcdB (TcdB1 and TcdB2) share 92% sequence identity but have reported differences in rates of cell entry, autoprocessing, and overall toxicity. This 2,366-amino-acid, multidomain bacterial toxin glucosylates and inactivates small GTPases in the cytosol of target cells, ultimately leading to cell death. Successful cell entry and intoxication by TcdB are known to involve various conformational changes in the protein, including a proteolytic autoprocessing event. Previous studies found that amino acids 1753 to 1852 influence the conformational states of the proximal carboxy-terminal domain of TcdB and could contribute to differences between TcdB1 and TcdB2. In the current study, a combination of approaches was used to identify sequences within the region from amino acids 1753 to 1852 that influence the conformational integrity and cytotoxicity of TcdB2. Four deletion mutants with reduced cytotoxicity were identified, while one mutant, TcdB2 Δ1769 -1787 , exhibited no detectable cytotoxicity. TcdB2 Δ1769 -1787 underwent spontaneous autoprocessing and was unable to interact with CHO-K1 or HeLa cells, suggesting a potential change in the conformation of the mutant protein. Despite the putative alteration in structural stability, vaccination with TcdB2 Δ1769 -1787 induced a TcdB2-neutralizing antibody response and protected against C. difficile disease in a mouse model. These findings indicate that the 19amino-acid region spanning residues 1769 to 1787 in TcdB2 is crucial to cytotoxicity and the structural regulation of autoprocessing and that TcdB2 Δ1769 -1787 is a promising candidate for vaccination.

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

“…It has been reported that the CROPs-truncated TcdA and TcdB underwent moderately increased InsP6-induced autocleavage compared to the full-length toxins 31,62 . The deletion of amino acids 1769-1787 caused spontaneous autocleavage of subtype 2 TcdB in the absence of InsP6 63 . Here we demonstrate that the integrated CROPs domain forcibly protects TcsL from autoproteolysis by at least two mechanisms: the N-terminus of TcsL CROPs strongly restrains the autoproteolysis while longer CROPs further reduce the InsP6-induced autocleavage.…”

Clostridioides difficile toxin B subverts germinal center and antibody recall responses by stimulating a drug-treatable CXCR4-dependent mechanism

“…TcdB2, B2Δ, and D270N were expressed in Bacillus megaterium (MoBiTec, Göttingen, Germany) and purified by nickel affinity chromatography (GE Life Sciences, Boston, MA) as previously described. 66 Purity and integrity were confirmed by SDS-PAGE and each batch of TcdB2 was tested for toxicity using a CHO cell killing assay. 67 …”

“… 53 After 5 h, mice were immunized subcutaneously (s.c.) with 10 μg B2Δ, a TcdB2 mutant incapable of entering host cells. 66 Unless stated otherwise, the B2Δ was adsorbed to a 2% w/v Alhydrogel alum adjuvant suspension (Invivogen, San Diego, CA) in PBS and is referred to as B2Δ/alum herein. Vaccines were divided equally over both flanks.…”

CSPG4-dependent cytotoxicity for C. difficile TcdB is influenced by extracellular calcium and chondroitin sulfate

“…Unlike CSPG4, FZD proteins do not possess glycosaminoglycan modifications or a known Ca 2+ -binding domain, which supports the notion that Ca 2+ could impact CSPG4 but not FZD tropism, with a greater effect on TcdB2. Second, previous studies from our lab identified a cell-penetrating peptide derived from amino acids 1769–1787 in TcdB2 that are necessary for cell binding and uptake ( 21 , 22 ). Interestingly, some cell-penetrating peptides are known to utilize glycosaminoglycans for cell entry ( 23 , 24 ), which would also be enhanced in the presence of Ca 2+ .…”