Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry

ABSTRACT Clostridium difficile TcdB2 enters cells with a higher efficiency than TcdB1 and exhibits an overall higher level of toxicity. However, the TcdB2-specific sequences that account for more efficient cell entry have not been reported. In this study, we examined the contribution of carboxy-term...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Jonathan J. Hunt, Jason L. Larabee, Jimmy D. Ballard
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	B2′ Clostridium difficile TcdB1 TcdB2 entry tcdB Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/df14dd59e50e4d2db5d147652b5f8dc6
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:df14dd59e50e4d2db5d147652b5f8dc6
record_format	dspace
spelling	oai:doaj.org-article:df14dd59e50e4d2db5d147652b5f8dc62021-11-15T15:22:04ZAmino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry10.1128/mSphere.00268-172379-5042https://doaj.org/article/df14dd59e50e4d2db5d147652b5f8dc62017-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00268-17https://doaj.org/toc/2379-5042ABSTRACT Clostridium difficile TcdB2 enters cells with a higher efficiency than TcdB1 and exhibits an overall higher level of toxicity. However, the TcdB2-specific sequences that account for more efficient cell entry have not been reported. In this study, we examined the contribution of carboxy-terminal sequence differences to TcdB activity by comparing the binding, uptake, and endosomal localization of TcdB1 and TcdB2 or selected recombinant fragments of these proteins. Our findings suggest that sequence differences in the amino acid 1753 to 1851 region proximal to the combined repetitive oligopeptide domain (CROP) support enhanced uptake of TcdB2 and localization of toxin in acidified endosomes. In the absence of this region, the CROP domains of both forms of the toxin exhibited similar levels of cell interaction, while the addition of amino acids 1753 to 1851 greatly increased toxin binding by only TcdB2. Moreover, the amino acid 1753 to 2366 fragment of TcdB2, but not TcdB1, accumulated to detectable levels in acidified endosomes. Unexpectedly, we discovered an unusual relationship between endocytosis and the efficiency of cell binding for TcdB1 and TcdB2 wherein inhibition of endocytosis by a chemical inhibitor or incubation at a low temperature resulted in a dramatic reduction in cell binding. These findings provide information on sequence variations that may contribute to differences in TcdB1 and TcdB2 toxicity and reveal a heretofore unknown connection between endocytosis and cell binding for this toxin. IMPORTANCE TcdB is a major virulence factor produced by Clostridium difficile, a leading cause of antibiotic-associated diarrhea. Hypervirulent strains of C. difficile encode a variant of TcdB (TcdB2) that is more toxic than toxin derived from historical strains (TcdB1). Though TcdB1 and TcdB2 exhibit 92% overall identity, a 99-amino-acid region previously associated with cell entry and spanning amino acids 1753 to 1851 has only 77% sequence identity. Results from the present study indicate that the substantial sequence variation in this region could contribute to the differences in cell entry between TcdB1 and TcdB2 and possibly explain TcdB2’s heightened toxicity. Finally, during the course of these studies, an unusual aspect of TcdB cell entry was discovered wherein cell binding appeared to depend on endocytosis. These findings provide insight into TcdB’s variant forms and their mechanisms of cell entry.Jonathan J. HuntJason L. LarabeeJimmy D. BallardAmerican Society for MicrobiologyarticleB2′Clostridium difficileTcdB1TcdB2entrytcdBMicrobiologyQR1-502ENmSphere, Vol 2, Iss 4 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	B2′ Clostridium difficile TcdB1 TcdB2 entry tcdB Microbiology QR1-502
spellingShingle	B2′ Clostridium difficile TcdB1 TcdB2 entry tcdB Microbiology QR1-502 Jonathan J. Hunt Jason L. Larabee Jimmy D. Ballard Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry
description	ABSTRACT Clostridium difficile TcdB2 enters cells with a higher efficiency than TcdB1 and exhibits an overall higher level of toxicity. However, the TcdB2-specific sequences that account for more efficient cell entry have not been reported. In this study, we examined the contribution of carboxy-terminal sequence differences to TcdB activity by comparing the binding, uptake, and endosomal localization of TcdB1 and TcdB2 or selected recombinant fragments of these proteins. Our findings suggest that sequence differences in the amino acid 1753 to 1851 region proximal to the combined repetitive oligopeptide domain (CROP) support enhanced uptake of TcdB2 and localization of toxin in acidified endosomes. In the absence of this region, the CROP domains of both forms of the toxin exhibited similar levels of cell interaction, while the addition of amino acids 1753 to 1851 greatly increased toxin binding by only TcdB2. Moreover, the amino acid 1753 to 2366 fragment of TcdB2, but not TcdB1, accumulated to detectable levels in acidified endosomes. Unexpectedly, we discovered an unusual relationship between endocytosis and the efficiency of cell binding for TcdB1 and TcdB2 wherein inhibition of endocytosis by a chemical inhibitor or incubation at a low temperature resulted in a dramatic reduction in cell binding. These findings provide information on sequence variations that may contribute to differences in TcdB1 and TcdB2 toxicity and reveal a heretofore unknown connection between endocytosis and cell binding for this toxin. IMPORTANCE TcdB is a major virulence factor produced by Clostridium difficile, a leading cause of antibiotic-associated diarrhea. Hypervirulent strains of C. difficile encode a variant of TcdB (TcdB2) that is more toxic than toxin derived from historical strains (TcdB1). Though TcdB1 and TcdB2 exhibit 92% overall identity, a 99-amino-acid region previously associated with cell entry and spanning amino acids 1753 to 1851 has only 77% sequence identity. Results from the present study indicate that the substantial sequence variation in this region could contribute to the differences in cell entry between TcdB1 and TcdB2 and possibly explain TcdB2’s heightened toxicity. Finally, during the course of these studies, an unusual aspect of TcdB cell entry was discovered wherein cell binding appeared to depend on endocytosis. These findings provide insight into TcdB’s variant forms and their mechanisms of cell entry.
format	article
author	Jonathan J. Hunt Jason L. Larabee Jimmy D. Ballard
author_facet	Jonathan J. Hunt Jason L. Larabee Jimmy D. Ballard
author_sort	Jonathan J. Hunt
title	Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry
title_short	Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry
title_full	Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry
title_fullStr	Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry
title_full_unstemmed	Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry
title_sort	amino acid differences in the 1753-to-1851 region of tcdb influence variations in tcdb1 and tcdb2 cell entry
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/df14dd59e50e4d2db5d147652b5f8dc6
work_keys_str_mv	AT jonathanjhunt aminoaciddifferencesinthe1753to1851regionoftcdbinfluencevariationsintcdb1andtcdb2cellentry AT jasonllarabee aminoaciddifferencesinthe1753to1851regionoftcdbinfluencevariationsintcdb1andtcdb2cellentry AT jimmydballard aminoaciddifferencesinthe1753to1851regionoftcdbinfluencevariationsintcdb1andtcdb2cellentry
_version_	1718428100199448576

Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry

Ejemplares similares