A Segment of 97 Amino Acids within the Translocation Domain of Clostridium difficile Toxin B Is Essential for Toxicity
In: PLoS ONE, Jg. 8 (2013-03-01), Heft 3
Online
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Zugriff:
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.
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A Segment of 97 Amino Acids within the Translocation Domain of Clostridium difficile Toxin B Is Essential for Toxicity
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Autor/in / Beteiligte Person: | Savidge, Tor C. ; Shi, Lianfa ; Wang, Xiaoning ; Yang, Zhiyong ; Standley, Clive ; Yang, Zhong ; Li, Shan ; ZhuGe, Ronghua ; Zhang, Yongrong ; Feng, Hanping |
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Zeitschrift: | PLoS ONE, Jg. 8 (2013-03-01), Heft 3 |
Veröffentlichung: | Public Library of Science, 2013 |
Medientyp: | unknown |
ISSN: | 1932-6203 (print) |
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