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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
- Source :
- Toxins, Toxins, Vol 6, Iss 7, Pp 2162-2176 (2014), Toxins; Volume 6; Issue 7; Pages: 2162-2176
- Publication Year :
- 2014
- Publisher :
- MDPI, 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.
- Subjects :
- Cell Survival
Health, Toxicology and Mutagenesis
Bacterial Toxins
Clostridium difficile toxin A
lcsh:Medicine
Toxicology
Cleavage (embryo)
medicine.disease_cause
Article
Clostridium difficile toxin
Enterotoxins
Mice
Protein structure
Bacterial Proteins
domain interaction
autoprocessing
cytotoxicity
microscale thermophoresis
medicine
Animals
Oligopeptide
biology
Microscale thermophoresis
Toxin
lcsh:R
3T3 Cells
Cysteine protease
Protein Structure, Tertiary
Biochemistry
Glucosyltransferases
biology.protein
Glucosyltransferase
Oligopeptides
Subjects
Details
- Language :
- English
- ISSN :
- 20726651
- Volume :
- 6
- Issue :
- 7
- Database :
- OpenAIRE
- Journal :
- Toxins
- Accession number :
- edsair.doi.dedup.....7228a9642b398f2e853165913adf2cc7