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.
Highlights
Toxins A (TcdA) and B (TcdB) are the main virulence factors of Clostridium difficile and predominantly responsible for C. difficile-induced diseases, ranging from mild diarrhea to fulminant pseudomembranous colitis and toxic megacolon [1,2]
Besides a low resolution analysis of TcdB-structure obtained by small-angle X-ray scattering (SAXS) [11], Pruitt and co-workers presented a structural model of full-length TcdA based on negative stain electron microscopy followed by 3D-reconstruction and mapping of the known functional domains [12]
The present study describes the combined repetitive oligopeptides (CROPs) of TcdA to shield toxin conformation
Summary
Toxins A (TcdA) and B (TcdB) are the main virulence factors of Clostridium difficile and predominantly responsible for C. difficile-induced diseases, ranging from mild diarrhea to fulminant pseudomembranous colitis and toxic megacolon [1,2]. The outer N-terminal subunit harbors the glucosyltransferase (GT-) domain, which inactivates small Rho GTPases by mono-glucosylation This leads to disruption of the actin cytoskeleton and, cell rounding [7]. Besides a low resolution analysis of TcdB-structure obtained by small-angle X-ray scattering (SAXS) [11], Pruitt and co-workers presented a structural model of full-length TcdA based on negative stain electron microscopy followed by 3D-reconstruction and mapping of the known functional domains [12]. These analyses revealed a closely-packed conformation of TcdA at neutral pH, assuming intramolecular contacts between the individual domains. In addition to the commonly accepted function in receptor binding, we propose that the CROPs, at least of TcdA, play an important role in the conformation stability and protection of the toxin
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