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

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 to...

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Bibliographic Details
Published in:Protein expression and purification 2010-11, Vol.74 (1), p.42-48
Main Authors: Sundriyal, Amit, Roberts, April K., Ling, Roger, McGlashan, Joanna, Shone, Clifford C., Acharya, K. Ravi
Format: Article
Language:English
Subjects:
Actins - metabolism
Animals
Bacterial Toxins - genetics
Bacterial Toxins - isolation & purification
Bacterial Toxins - metabolism
Binary toxin
C. difficile
CDTa
CDTb
Cell Survival
Cercopithecus aethiops
Chymotrypsin - metabolism
Clostridium difficile
Clostridium difficile - genetics
Clostridium difficile - metabolism
Clostridium difficile - pathogenicity
Clostridium Infections - metabolism
Colitis
Cytotoxicity
Cytotoxins
Cytotoxins - genetics
Cytotoxins - isolation & purification
Cytotoxins - metabolism
Diarrhea
Digestive tract
Escherichia coli
Escherichia coli - genetics
Gene Expression
Host-Pathogen Interactions
Humans
Infection
protein purification
Purification
Structure-function relationships
Toxin A
Toxin B
Vero Cells
virulence factors
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Summary: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.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2010.04.014