The repetitive oligopeptide sequences modulate cytopathic potency but are not crucial for cellular uptake of Clostridium difficile toxin A.

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 ab...

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Autores principales:	Alexandra Olling, Sebastian Goy, Florian Hoffmann, Helma Tatge, Ingo Just, Ralf Gerhard
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/65c59061c845439185b277ed449cb002
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spelling	oai:doaj.org-article:65c59061c845439185b277ed449cb0022021-11-18T06:57:07ZThe repetitive oligopeptide sequences modulate cytopathic potency but are not crucial for cellular uptake of Clostridium difficile toxin A.1932-620310.1371/journal.pone.0017623https://doaj.org/article/65c59061c845439185b277ed449cb0022011-03-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21445253/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203The 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 apically applied mutant or even full length TcdA, the latter being almost independent of the side of application. Thus, different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of C. difficile.Alexandra OllingSebastian GoyFlorian HoffmannHelma TatgeIngo JustRalf GerhardPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 3, p e17623 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Alexandra Olling Sebastian Goy Florian Hoffmann Helma Tatge Ingo Just Ralf Gerhard The repetitive oligopeptide sequences modulate cytopathic potency but are not crucial for cellular uptake of Clostridium difficile toxin A.
description	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 apically applied mutant or even full length TcdA, the latter being almost independent of the side of application. Thus, different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of C. difficile.
format	article
author	Alexandra Olling Sebastian Goy Florian Hoffmann Helma Tatge Ingo Just Ralf Gerhard
author_facet	Alexandra Olling Sebastian Goy Florian Hoffmann Helma Tatge Ingo Just Ralf Gerhard
author_sort	Alexandra Olling
title	The repetitive oligopeptide sequences modulate cytopathic potency but are not crucial for cellular uptake of Clostridium difficile toxin A.
title_short	The repetitive oligopeptide sequences modulate cytopathic potency but are not crucial for cellular uptake of Clostridium difficile toxin A.
title_full	The repetitive oligopeptide sequences modulate cytopathic potency but are not crucial for cellular uptake of Clostridium difficile toxin A.
title_fullStr	The repetitive oligopeptide sequences modulate cytopathic potency but are not crucial for cellular uptake of Clostridium difficile toxin A.
title_full_unstemmed	The repetitive oligopeptide sequences modulate cytopathic potency but are not crucial for cellular uptake of Clostridium difficile toxin A.
title_sort	repetitive oligopeptide sequences modulate cytopathic potency but are not crucial for cellular uptake of clostridium difficile toxin a.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/65c59061c845439185b277ed449cb002
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The repetitive oligopeptide sequences modulate cytopathic potency but are not crucial for cellular uptake of Clostridium difficile toxin A.

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