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-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. Ausführliche Beschreibung

Gespeichert in:

Autor*in:	Jonathan J. Hunt [verfasserIn] Jason L. Larabee [verfasserIn] Jimmy D. Ballard [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2017

Schlagwörter:	B2′ Clostridium difficile TcdB1 TcdB2 entry tcdB

Übergeordnetes Werk:	In: mSphere - American Society for Microbiology, 2016, 2(2017), 4
Übergeordnetes Werk:	volume:2 ; year:2017 ; number:4

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.1128/mSphere.00268-17

Katalog-ID:	DOAJ075243415

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520			\|a 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.
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allfields	10.1128/mSphere.00268-17 doi (DE-627)DOAJ075243415 (DE-599)DOAJdf14dd59e50e4d2db5d147652b5f8dc6 DE-627 ger DE-627 rakwb eng QR1-502 Jonathan J. Hunt verfasserin aut Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier 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. B2′ Clostridium difficile TcdB1 TcdB2 entry tcdB Microbiology Jason L. Larabee verfasserin aut Jimmy D. Ballard verfasserin aut In mSphere American Society for Microbiology, 2016 2(2017), 4 (DE-627)845748807 (DE-600)2844248-9 23795042 nnns volume:2 year:2017 number:4 https://doi.org/10.1128/mSphere.00268-17 kostenfrei https://doaj.org/article/df14dd59e50e4d2db5d147652b5f8dc6 kostenfrei https://journals.asm.org/doi/10.1128/mSphere.00268-17 kostenfrei https://doaj.org/toc/2379-5042 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 2 2017 4
spelling	10.1128/mSphere.00268-17 doi (DE-627)DOAJ075243415 (DE-599)DOAJdf14dd59e50e4d2db5d147652b5f8dc6 DE-627 ger DE-627 rakwb eng QR1-502 Jonathan J. Hunt verfasserin aut Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier 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. B2′ Clostridium difficile TcdB1 TcdB2 entry tcdB Microbiology Jason L. Larabee verfasserin aut Jimmy D. Ballard verfasserin aut In mSphere American Society for Microbiology, 2016 2(2017), 4 (DE-627)845748807 (DE-600)2844248-9 23795042 nnns volume:2 year:2017 number:4 https://doi.org/10.1128/mSphere.00268-17 kostenfrei https://doaj.org/article/df14dd59e50e4d2db5d147652b5f8dc6 kostenfrei https://journals.asm.org/doi/10.1128/mSphere.00268-17 kostenfrei https://doaj.org/toc/2379-5042 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 2 2017 4
allfields_unstemmed	10.1128/mSphere.00268-17 doi (DE-627)DOAJ075243415 (DE-599)DOAJdf14dd59e50e4d2db5d147652b5f8dc6 DE-627 ger DE-627 rakwb eng QR1-502 Jonathan J. Hunt verfasserin aut Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier 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. B2′ Clostridium difficile TcdB1 TcdB2 entry tcdB Microbiology Jason L. Larabee verfasserin aut Jimmy D. Ballard verfasserin aut In mSphere American Society for Microbiology, 2016 2(2017), 4 (DE-627)845748807 (DE-600)2844248-9 23795042 nnns volume:2 year:2017 number:4 https://doi.org/10.1128/mSphere.00268-17 kostenfrei https://doaj.org/article/df14dd59e50e4d2db5d147652b5f8dc6 kostenfrei https://journals.asm.org/doi/10.1128/mSphere.00268-17 kostenfrei https://doaj.org/toc/2379-5042 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 2 2017 4
allfieldsGer	10.1128/mSphere.00268-17 doi (DE-627)DOAJ075243415 (DE-599)DOAJdf14dd59e50e4d2db5d147652b5f8dc6 DE-627 ger DE-627 rakwb eng QR1-502 Jonathan J. Hunt verfasserin aut Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier 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. B2′ Clostridium difficile TcdB1 TcdB2 entry tcdB Microbiology Jason L. Larabee verfasserin aut Jimmy D. Ballard verfasserin aut In mSphere American Society for Microbiology, 2016 2(2017), 4 (DE-627)845748807 (DE-600)2844248-9 23795042 nnns volume:2 year:2017 number:4 https://doi.org/10.1128/mSphere.00268-17 kostenfrei https://doaj.org/article/df14dd59e50e4d2db5d147652b5f8dc6 kostenfrei https://journals.asm.org/doi/10.1128/mSphere.00268-17 kostenfrei https://doaj.org/toc/2379-5042 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 2 2017 4
allfieldsSound	10.1128/mSphere.00268-17 doi (DE-627)DOAJ075243415 (DE-599)DOAJdf14dd59e50e4d2db5d147652b5f8dc6 DE-627 ger DE-627 rakwb eng QR1-502 Jonathan J. Hunt verfasserin aut Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier 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. B2′ Clostridium difficile TcdB1 TcdB2 entry tcdB Microbiology Jason L. Larabee verfasserin aut Jimmy D. Ballard verfasserin aut In mSphere American Society for Microbiology, 2016 2(2017), 4 (DE-627)845748807 (DE-600)2844248-9 23795042 nnns volume:2 year:2017 number:4 https://doi.org/10.1128/mSphere.00268-17 kostenfrei https://doaj.org/article/df14dd59e50e4d2db5d147652b5f8dc6 kostenfrei https://journals.asm.org/doi/10.1128/mSphere.00268-17 kostenfrei https://doaj.org/toc/2379-5042 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 2 2017 4
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spellingShingle	Jonathan J. Hunt misc QR1-502 misc B2′ misc Clostridium difficile misc TcdB1 misc TcdB2 misc entry misc tcdB misc Microbiology Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry
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title	Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry
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title_full	Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry
author_sort	Jonathan J. Hunt
journal	mSphere
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author_browse	Jonathan J. Hunt Jason L. Larabee Jimmy D. Ballard
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author-letter	Jonathan J. Hunt
doi_str_mv	10.1128/mSphere.00268-17
author2-role	verfasserin
title_sort	amino acid differences in the 1753-to-1851 region of tcdb influence variations in tcdb1 and tcdb2 cell entry
callnumber	QR1-502
title_auth	Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry
abstract	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.
abstractGer	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.
abstract_unstemmed	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.
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title_short	Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry
url	https://doi.org/10.1128/mSphere.00268-17 https://doaj.org/article/df14dd59e50e4d2db5d147652b5f8dc6 https://journals.asm.org/doi/10.1128/mSphere.00268-17 https://doaj.org/toc/2379-5042
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author2	Jason L. Larabee Jimmy D. Ballard
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up_date	2024-07-03T13:48:32.227Z
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