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The Repetitive Oligopeptide Sequences Modulate Cytopathic Potency but Are Not Crucial for Cellular
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@MISC{Goy_therepetitive,
author = {Alexandra Olling Sebastian Goy},
title = {The Repetitive Oligopeptide Sequences Modulate Cytopathic Potency but Are Not Crucial for Cellular},
year = {}
}
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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 (TcdA 1–1874) and found that this mutant was still cytopathic. However, TcdA 1–1874 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 TcdA 1–1874 to HT29 cells. We assume that toxin uptake might additionally occur through alternative receptor structures and/or other associated endocytotic pathways. The second assumption was substantiated by TER measurements showing that basolaterally applied TcdA 1–1874 exhibits considerably higher cytotoxic potency than
Keyphrases
ht29 cell full length tcda 10-fold le potency towards toxin internalization second assumption molecular mode repetitive oligopeptides ter measurement cell surface alternative receptor structure cell rounding accepted opinion truncated tcda devoid rho gtpases endocytotic pathway truncated tcda competition experiment western blot analysis clostridium difficile crop domain current study toxin uptake host cell tcda crop toxin binding toxin potency toxin function full length toxin rac1 glucosylation cytotoxic potency intracellular acting identical susceptibility towards cho-c6 cell
