the repetitive oligopeptide sequences modulate cytopathic potency but are not crucial for cellular uptake of clostridium difficile toxin a重复的寡肽序列调节细胞病变能力但不梭状芽胞杆菌的细胞吸收毒素的关键.pdfVIP

The Repetitive Oligopeptide Sequences Modulate Cytopathic Potency but Are Not Crucial for Cellular Uptake of Clostridium difficile Toxin A Alexandra Olling, Sebastian Goy, Florian Hoffmann, Helma Tatge, Ingo Just, Ralf Gerhard* ¨ Institut fur Toxikologie, Medizinische Hochschule Hannover, Hannover, Germany Abstract The pathogenicity of Clostridium difficile is primarily linked to secretion of the intracellular acting toxins A (TcdA) and B (TcdB) which monoglucosylate and thereby inactivate Rho GTPases of host cells. Although the molecular mode of action of TcdA and TcdB is well understood, far less is known about toxin binding and uptake. It is acknowledged that the C- terminally combined repetitive oligopeptides (CROPs) of the toxins function as receptor binding domain. The current study evaluates the role of the CROP domain with respect to functionality of TcdA and TcdB. Therefore, we generated truncated TcdA devoid of the CROPs (TcdA1–1874) and found that this mutant was still cytopathic. However, TcdA1–1874 possesses about 5 to 10-fold less potency towards 3T3 and HT29 cells compared to the full length toxin. Interestingly, CHO-C6 cells even showed almost identical susceptibility towards truncated and full length TcdA concerning Rac1 glucosylation or cell rounding, respectively. FACS and Western blot analyses elucidated these differences and revealed a correlation between CROP-binding to the cell surface and toxin potency. These findings refute the accepted opinion of solely CROP- mediated toxin internalization. Competition experiments demonstrated that presence neither of TcdA CROPs nor of full length TcdA reduced binding of truncated TcdA1–1874 to HT29 cells. We assume that toxin uptake might additionally occur through alternative receptor structures and/or other associated