The Combined Repetitive Oligopeptides of Clostridium difficile Toxin A Counteract Premature Cleavage of the Glucosyl-Transferase Domain by Stabilizing Protein Conformation

Toxins

Alexandra Olling¹  Corinna Hüls¹  Sebastian Goy¹  Mirco Müller²  Simon Krooss¹  Isa Rudolf¹  Helma Tatge¹ 

[1] Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany; E-Mails:;Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany; E-Mail:

关键词: Clostridium difficile toxin;    domain interaction;    autoprocessing;    cytotoxicity;    microscale thermophoresis;   

DOI  :  10.3390/toxins6072162

来源: mdpi

【 摘 要 】

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 TcdA^1–1065 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 (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 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.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland.