Expression, purification and cell cytotoxicity of actin-modifying binary toxin from Clostridium difficile

Amit Sundriyal; April Roberts; Roger Ling; Joanna McGlashan; Clifford Shone; K. Ravi Acharya

doi:10.1016/j.pep.2010.04.014

Expression, purification and cell cytotoxicity of actin-modifying binary toxin from Clostridium difficile

Amit Sundriyal, April Roberts, Roger Ling, Joanna McGlashan, Clifford Shone, K. Ravi Acharya

Research output: Contribution to journal › Article › peer-review

35 Citations (Scopus)

Abstract

Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulence factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two sub-units namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. Here, we describe expression and purification methods for recombinant CDTa and CDTb produced in Escherichia coli. We show that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells. In addition, the purification processes described will allow milligram quantities of binary toxin fragments to be produced for further functional and structural studies.

Original language	English
Pages (from-to)	42-48
Number of pages	7
Journal	Protein Expression and Purification
Volume	74
Issue number	1
DOIs	https://doi.org/10.1016/j.pep.2010.04.014
Publication status	Published - Nov 2010

Bibliographical note

Funding Information:
K.R.A. wishes to acknowledge the Royal Society (UK) for an Industry Fellowship. This research was supported by a joint postgraduate studentship from the Health Protection Agency and BBSRC (UK) to A.S. We thank Abigail Davies for preparing Fig. 1 B.

Keywords

Binary toxin
C. difficile
CDTa
CDTb
Cytotoxicity
Purification

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.pep.2010.04.014

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title = "Expression, purification and cell cytotoxicity of actin-modifying binary toxin from Clostridium difficile",

abstract = "Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulence factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two sub-units namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. Here, we describe expression and purification methods for recombinant CDTa and CDTb produced in Escherichia coli. We show that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells. In addition, the purification processes described will allow milligram quantities of binary toxin fragments to be produced for further functional and structural studies.",

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note = "Funding Information: K.R.A. wishes to acknowledge the Royal Society (UK) for an Industry Fellowship. This research was supported by a joint postgraduate studentship from the Health Protection Agency and BBSRC (UK) to A.S. We thank Abigail Davies for preparing Fig. 1 B.",

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AU - Shone, Clifford

AU - Acharya, K. Ravi

N1 - Funding Information: K.R.A. wishes to acknowledge the Royal Society (UK) for an Industry Fellowship. This research was supported by a joint postgraduate studentship from the Health Protection Agency and BBSRC (UK) to A.S. We thank Abigail Davies for preparing Fig. 1 B.

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N2 - Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulence factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two sub-units namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. Here, we describe expression and purification methods for recombinant CDTa and CDTb produced in Escherichia coli. We show that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells. In addition, the purification processes described will allow milligram quantities of binary toxin fragments to be produced for further functional and structural studies.

AB - Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulence factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two sub-units namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. Here, we describe expression and purification methods for recombinant CDTa and CDTb produced in Escherichia coli. We show that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells. In addition, the purification processes described will allow milligram quantities of binary toxin fragments to be produced for further functional and structural studies.

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Expression, purification and cell cytotoxicity of actin-modifying binary toxin from Clostridium difficile

Abstract

Bibliographical note

Keywords

UN SDGs

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