Masking autoprocessing of Clostridium difficile toxin A by the C-terminus combined repetitive oligo peptides

Zhang, Yongrong; Hamza, Therwa; Gao, Si; Feng, Hanping

doi:10.1016/j.bbrc.2015.02.095

Cited by 13 publications

(11 citation statements)

References 23 publications

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“…1d) indicates that the CROPS extends from the base of the APD and could impact InsP6 binding. This is consistent with reports that the CROPS interacts with N-terminal sequences of TcdA to repress autoprocessing activity 22,23 and our observation that a shorter construct (TcdA 1795 ) undergoes autoprocessing more efficiently than TcdA and TcdA 1832 (Supplementary Fig. 2).…”

supporting

confidence: 93%

Crystal structure of Clostridium difficile toxin A

et al. 2016

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Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon1,2. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host3,4. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.

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supporting

confidence: 93%

Crystal structure of Clostridium difficile toxin A

et al. 2016

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“…Based on negative stain data and crystal structures of TcdA and TcdB domains ( Chumbler et al, 2016 ), as well as reports about intramolecular association of the C-terminal domain with the N-terminal part of TcdA ( Olling et al, 2014 ; Zhang et al, 2015 ), we hypothesized that the conserved cysteine 2236 in TcdA and the homologous 2232 in TcdB contribute to the conformation of toxins. Cysteine 2232 is located in an exposed region of the CROP domain which might interact with the upstream located delivery domain.…”

Section: Resultsmentioning

confidence: 99%

The Conserved Cys-2232 in Clostridioides difficile Toxin B Modulates Receptor Binding

et al. 2018

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Clostridioides difficile toxins TcdA and TcdB are large clostridial glucosyltransferases which are the main pathogenicity factors in C. difficile-associated diseases. Four highly conserved cysteines are present in all large clostridial glucosyltransferases. In this study we focused on the conserved cysteine 2232 within the combined repetitive oligopeptide domain of TcdB from reference strain VPI10463 (clade I). Cysteine 2232 is not present in TcdB from hypervirulent strain R20291 (clade II), where a tyrosine is found instead. Replacement of cysteine 2232 by tyrosine in TcdBV PI10463 reduced binding to the soluble fragments of the two known TcdB receptors, frizzled-2 (FZD2) and poliovirus receptor-like protein-3/nectin-3 (PVRL3). In line with this, TcdBR20291 showed weak binding to PVRL3 in pull-down assays which was increased when tyrosine 2232 was exchanged for cysteine. Surprisingly, we did not observe binding of TcdBR20291 to FZD2, indicating that this receptor is less important for this toxinotype. Competition assay with the receptor binding fragments (aa 1101–1836) of TcdBV PI10463 and TcdBR20291, as well as antibodies newly developed by antibody phage display, revealed different characteristics of the yet poorly described delivery domain of TcdB harboring the second receptor binding region. In summary, we found that conserved Cys-2232 in TcdB indirectly contributes to toxin–receptor interaction.

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“…The TcdB2 deletion mutant retains glucosylation activity. Previous work has suggested that the efficiency of glucosylation of TcdA and TcdB is hindered by conformational restrictions within the proteins (16,20). It was therefore predicted that deletions which remove conformational constraints in TcdB could result in forms of the protein with altered glucosylation activity.…”

Section: Resultsmentioning

confidence: 99%

“…Though the structural reasons for it are not understood, biochemical evidence suggests that the carboxy-terminal region of the toxin influences autoprocessing. Interestingly, Zhang et al showed that swapping the CROP domain of TcdB with that of TcdA caused the toxin to become more resistant to IP6-induced autoprocessing (20). Moreover, a deletion mutant of TcdA containing only the first 1,056 amino acids undergoes more efficient and nearly complete autoprocessing compared with that for the full-length toxin (23).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2019

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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.

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Masking autoprocessing of Clostridium difficile toxin A by the C-terminus combined repetitive oligo peptides

Cited by 13 publications

References 23 publications

Crystal structure of Clostridium difficile toxin A

Crystal structure of Clostridium difficile toxin A

The Conserved Cys-2232 in Clostridioides difficile Toxin B Modulates Receptor Binding

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

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