title: The combined repetitive oligopeptides of clostridium difficile toxin A counteract premature cleavage of the glucosyl-transferase domain by stabilizing protein conformation.
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.
authors: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. olling.alexandra@mh-hannover.de.
authors: Alexandra
authors: A
authors: Olling
authors: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. corri.H88@gmx.de.
authors: Corinna
authors: C
authors: Hüls
authors: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. goy.sebastian@mh-hannover.de.
authors: Sebastian
authors: S
authors: Goy
authors: Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. mirco_mueller@yahoo.de.
authors: Mirco
authors: M
authors: Müller
authors: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. simonkrooss@icloud.com.
authors: Simon
authors: S
authors: Krooss
authors: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. rudolf.isa@mh-hannover.de.
authors: Isa
authors: I
authors: Rudolf
authors: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. tatge.helma@mh-hannover.de.
authors: Helma
authors: H
authors: Tatge
authors: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. gerhard.ralf@mh-hannover.de.
authors: Ralf
authors: R
authors: Gerhard
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism / Q000633:toxicity; D001427:Bacterial Toxins / Q000378:metabolism* / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D004768:Enterotoxins / Q000378:metabolism* / Q000633:toxicity; D005964:Glucosyltransferases / Q000378:metabolism; D051379:Mice; D009842:Oligopeptides / Q000378:metabolism* / Q000633:toxicity; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D009842:Oligopeptides; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D005964:Glucosyltransferases