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.
authors.affiliation: Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK.
authors.forename: Amit
authors.initials: A
authors.lastname: Sundriyal
authors.forename: April K
authors.initials: AK
authors.lastname: Roberts
authors.forename: Roger
authors.initials: R
authors.lastname: Ling
authors.forename: Joanna
authors.initials: J
authors.lastname: McGlashan
authors.forename: Clifford C
authors.initials: CC
authors.lastname: Shone
authors.forename: K Ravi
authors.initials: KR
authors.lastname: Acharya
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002470:Cell Survival; D002522:Chlorocebus aethiops; D002918:Chymotrypsin / Q000378:metabolism; D016360:Clostridioides difficile / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D003015:Clostridium Infections / Q000378:metabolism*; D003603:Cytotoxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D054884:Host-Pathogen Interactions; D006801:Humans; D014709:Vero Cells
chemical_list: D000199:Actins; D001427:Bacterial Toxins; D003603:Cytotoxins; D002918:Chymotrypsin