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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

Authors :
Alexandra Olling
Corinna Hüls
Sebastian Goy
Mirco Müller
Simon Krooss
Isa Rudolf
Helma Tatge
Ralf Gerhard
Source :
Toxins, Vol 6, Iss 7, Pp 2162-2176 (2014)
Publication Year :
2014
Publisher :
MDPI AG, 2014.

Abstract

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 (IP6). We found that in in vitro assays, the C-terminally-truncated TcdA1–1065 was more efficient at IP6-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 (TcdA1–542) or intermediate (TcdA1102–1847) 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 IP6-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.

Details

Language :
English
ISSN :
20726651
Volume :
6
Issue :
7
Database :
Directory of Open Access Journals
Journal :
Toxins
Publication Type :
Academic Journal
Accession number :
edsdoj.7d1a453efcda473a9fb37522bfbb3547
Document Type :
article
Full Text :
https://doi.org/10.3390/toxins6072162