15 Membrane translocation by bacterial AB toxins

Abstract

Publisher Summary This chapter presents some of the basic experimental assays that have been developed or used in laboratories for the study of cholera and related E. coli heat labile enterotoxins, diphtheria toxin, and anthrax toxins. The chapter emphasizes on the importance of experimental systems that provide a high degree of temporal resolution when measuring toxin function. Microscopic techniques for non-polarized cells and monolayers grown on glass or plastic are described. For studies of protein binding, sorting, or intracellular trafficking in polarized epithelial cells, morphological examination of polarized monolayers grown on permeable supports is essential. The chapter briefly discusses the principles of microscopy on polarized monolayers for study of toxin binding or internalization. Bacterial AB toxins are designed to translocate a functional enzyme into the cytosol. Thus, the functional signal induced by membrane translocation of the enzymatic toxin-subunit is magnified tremendously over the signal that can be obtained by direct measurement of the mass of translocated protein. This is one of the key advantages in using bacterial toxins to study membrane translocation and vesicular transport in intact cells. The technologies described in this chapter have been used to identify the mechanisms of internalization and intracellular compartments from which AB-toxins gain entry to the cytosol.