A Segment of 97 Amino Acids within the Translocation Domain of Clostridium difficile Toxin B Is Essential for ToxicityReportar como inadecuado




A Segment of 97 Amino Acids within the Translocation Domain of Clostridium difficile Toxin B Is Essential for Toxicity - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

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.



Autor: Yongrong Zhang, Lianfa Shi, Shan Li, Zhiyong Yang, Clive Standley, Zhong Yang, Ronghua ZhuGe, Tor Savidge, Xiaoning Wang, Hanping

Fuente: http://plos.srce.hr/



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