6533b829fe1ef96bd128a227

RESEARCH PRODUCT

Purification and evaluation of large clostridial cytotoxins that inhibit small GTPases of Rho and ras subfamilies

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Publisher Summary This chapter discusses the purification and evaluation of large clostridia cytotoxins (LCTs) that inhibit small guanosine 5'-triphosphates (GTPases) of Rho and Ras subfamilies. LCTs are glycosyltransferases that inactivate GTPases of the Rho and Ras subfamilies by covalently coupling a sugar moiety (mostly glucose) to the conserved threonine residue in region switch 1 of the GTPases (T35 in Ras). This glycosylation functionally inactivates the GTPases leading to the collapse of the actin cytoskeleton and ultimately induces apoptosis of the cells. Small GTP-binding proteins are key players in the regulation of signal transducing networks of eukaryotic cells. Their regulatory role renders GTPases ideal targets for bacterial toxins aiming to either kill or influence the behavior of the cells. The chapter presents an improved method for the purification of toxins TcdA-10463 and TcdB-10463 and describes the assays necessary to evaluate the cytotoxic potential of LCTs. The chapter describes the growth of Clostridium difficile VP110463. The chapter presents various methods for assaying enzymatic activities of LCTs on GTPases and cytotoxic activities of LCTs on cells.