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The Combined Repetitive Oligopeptides of Clostridium difficile Toxin A Counteract Premature Cleavage of the Glucosyl-Transferase Domain by Stabilizing Protein Conformation
Toxin A (TcdA) and B (TcdB) from Clostridium difficile enter host cells by receptor-mediated endocytosis. A prerequisite for proper toxin action is the intracellular release of the glucosyltransferase domain by an inherent cysteine protease, which is allosterically activated by inositol hexaphosphat...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113749/ https://www.ncbi.nlm.nih.gov/pubmed/25054784 http://dx.doi.org/10.3390/toxins6072162 |
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author | Olling, Alexandra Hüls, Corinna Goy, Sebastian Müller, Mirco Krooss, Simon Rudolf, Isa Tatge, Helma Gerhard, Ralf |
author_facet | Olling, Alexandra Hüls, Corinna Goy, Sebastian Müller, Mirco Krooss, Simon Rudolf, Isa Tatge, Helma Gerhard, Ralf |
author_sort | Olling, Alexandra |
collection | PubMed |
description | Toxin A (TcdA) and B (TcdB) from Clostridium difficile enter host cells by receptor-mediated endocytosis. A prerequisite for proper toxin action is the intracellular release of the glucosyltransferase domain by an inherent cysteine protease, which is allosterically activated by inositol hexaphosphate (IP(6)). We found that in in vitro assays, the C-terminally-truncated TcdA(1–1065) was more efficient at IP(6)-induced cleavage compared with full-length TcdA. We hypothesized that the C-terminally-located combined repetitive oligopeptides (CROPs) interact with the N-terminal part of the toxin, thereby preventing autoproteolysis. Glutathione-S-transferase (GST) pull-down assays and microscale thermophoresis confirmed binding between the CROPs and the glucosyltransferase (TcdA(1–542)) or intermediate (TcdA(1102–1847)) domain of TcdA, respectively. This interaction between the N- and C-terminus was not found for TcdB. Functional assays revealed that TcdB was more susceptible to inactivation by extracellular IP(6)-induced cleavage. In vitro autoprocessing and inactivation of TcdA, however, significantly increased, either by acidification of the surrounding milieu or following exchange of its CROP domain by the homologous CROP domain of TcdB. Thus, TcdA CROPs contribute to the stabilization and protection of toxin conformation in addition to function as the main receptor binding domain. |
format | Online Article Text |
id | pubmed-4113749 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-41137492014-07-29 The Combined Repetitive Oligopeptides of Clostridium difficile Toxin A Counteract Premature Cleavage of the Glucosyl-Transferase Domain by Stabilizing Protein Conformation Olling, Alexandra Hüls, Corinna Goy, Sebastian Müller, Mirco Krooss, Simon Rudolf, Isa Tatge, Helma Gerhard, Ralf Toxins (Basel) Article Toxin A (TcdA) and B (TcdB) from Clostridium difficile enter host cells by receptor-mediated endocytosis. A prerequisite for proper toxin action is the intracellular release of the glucosyltransferase domain by an inherent cysteine protease, which is allosterically activated by inositol hexaphosphate (IP(6)). We found that in in vitro assays, the C-terminally-truncated TcdA(1–1065) was more efficient at IP(6)-induced cleavage compared with full-length TcdA. We hypothesized that the C-terminally-located combined repetitive oligopeptides (CROPs) interact with the N-terminal part of the toxin, thereby preventing autoproteolysis. Glutathione-S-transferase (GST) pull-down assays and microscale thermophoresis confirmed binding between the CROPs and the glucosyltransferase (TcdA(1–542)) or intermediate (TcdA(1102–1847)) domain of TcdA, respectively. This interaction between the N- and C-terminus was not found for TcdB. Functional assays revealed that TcdB was more susceptible to inactivation by extracellular IP(6)-induced cleavage. In vitro autoprocessing and inactivation of TcdA, however, significantly increased, either by acidification of the surrounding milieu or following exchange of its CROP domain by the homologous CROP domain of TcdB. Thus, TcdA CROPs contribute to the stabilization and protection of toxin conformation in addition to function as the main receptor binding domain. MDPI 2014-07-22 /pmc/articles/PMC4113749/ /pubmed/25054784 http://dx.doi.org/10.3390/toxins6072162 Text en © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). |
spellingShingle | Article Olling, Alexandra Hüls, Corinna Goy, Sebastian Müller, Mirco Krooss, Simon Rudolf, Isa Tatge, Helma Gerhard, Ralf The Combined Repetitive Oligopeptides of Clostridium difficile Toxin A Counteract Premature Cleavage of the Glucosyl-Transferase Domain by Stabilizing Protein Conformation |
title | The Combined Repetitive Oligopeptides of Clostridium difficile Toxin A Counteract Premature Cleavage of the Glucosyl-Transferase Domain by Stabilizing Protein Conformation |
title_full | The Combined Repetitive Oligopeptides of Clostridium difficile Toxin A Counteract Premature Cleavage of the Glucosyl-Transferase Domain by Stabilizing Protein Conformation |
title_fullStr | The Combined Repetitive Oligopeptides of Clostridium difficile Toxin A Counteract Premature Cleavage of the Glucosyl-Transferase Domain by Stabilizing Protein Conformation |
title_full_unstemmed | The Combined Repetitive Oligopeptides of Clostridium difficile Toxin A Counteract Premature Cleavage of the Glucosyl-Transferase Domain by Stabilizing Protein Conformation |
title_short | The Combined Repetitive Oligopeptides of Clostridium difficile Toxin A Counteract Premature Cleavage of the Glucosyl-Transferase Domain by Stabilizing Protein Conformation |
title_sort | combined repetitive oligopeptides of clostridium difficile toxin a counteract premature cleavage of the glucosyl-transferase domain by stabilizing protein conformation |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113749/ https://www.ncbi.nlm.nih.gov/pubmed/25054784 http://dx.doi.org/10.3390/toxins6072162 |
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