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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...

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Autores principales: Olling, Alexandra, Hüls, Corinna, Goy, Sebastian, Müller, Mirco, Krooss, Simon, Rudolf, Isa, Tatge, Helma, Gerhard, Ralf
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2014
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.
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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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