title: A segment of 97 amino acids within the translocation domain of Clostridium difficile toxin B is essential for toxicity.
abstract: Clostridium difficile toxin B (TcdB) intoxicates target cells by glucosylating Rho GTPases. TcdB (269 kDa) consists of at least 4 functional domains including a glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a translocation domain (TD), and a receptor binding domain (RBD). The function and molecular mode of action of the TD, which is the largest segment of TcdB and comprises nearly 50% of the protein, remain largely unknown. Here we show that a 97-amino-acid segment (AA1756 - 1852, designated as ?97 or D97), located in the C-terminus of the TD and adjacent to the RBD, is essential for the cellular activity of TcdB. Deletion of this segment in TcdB (designated as TxB-D97), did not adversely alter toxin enzymatic activities or its cellular binding and uptake capacity. TxB-D97 bound to and entered cells in a manner similar to TcdB holotoxin. Both wild type and mutant toxins released their GTDs similarly in the presence of inositol hexakisphosphate (InsP6), and showed a similar glucosyltransferase activity in a cell-free glucosylating assay. Despite these similarities, the cytotoxic activity of TxB-D97 was reduced by more than 5 logs compared to wild type toxin, supported by the inability of TxB-D97 to glucosylate Rac1 of target cells. Moreover, the mutant toxin failed to elicit tumor necrosis factor alpha (TNF-α) in macrophages, a process dependent on the glucosyltransferase activity of the toxin. Cellular fractionation of toxin-exposed cells revealed that TxB-D97 was unable to efficiently release the GTD into cytosol. Thereby, we conclude the 97-amino-acid region of the TD C-terminus of TcdB adjacent to the RBD, is essential for the toxicity of TcdB.
authors.affiliation: School of Bioengineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Yongrong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Lianfa
authors.initials: L
authors.lastname: Shi
authors.forename: Shan
authors.initials: S
authors.lastname: Li
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Yang
authors.forename: Clive
authors.initials: C
authors.lastname: Standley
authors.forename: Zhong
authors.initials: Z
authors.lastname: Yang
authors.forename: Ronghua
authors.initials: R
authors.lastname: ZhuGe
authors.forename: Tor
authors.initials: T
authors.lastname: Savidge
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Wang
authors.forename: Hanping
authors.initials: H
authors.lastname: Feng
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity*; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity*; D001681:Biological Assay; D002458:Cell Fractionation; D057056:Cysteine Proteases / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005453:Fluorescence; D005964:Glucosyltransferases / Q000378:metabolism; D047468:Immunoprecipitation; D053208:Kaplan-Meier Estimate; D051379:Mice; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary / Q000235:genetics; D013778:Tetrazolium Salts; D013844:Thiazoles; D014709:Vero Cells
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D013778:Tetrazolium Salts; D013844:Thiazoles; C031815:toxB protein, Clostridium difficile; D005964:Glucosyltransferases; D057056:Cysteine Proteases; C022616:thiazolyl blue