Fractionation techniques to examine effector translocation

Abstract

Many Gram-negative bacterial pathogens use type III secretion systems to export proteins that act directly on the host and aid in the infectious process. Extracellular bacteria primarily rely upon the type III secretion system to insert or inject effector proteins into the cytosol of their host cell in order to perturb intracellular signaling events and aid in pathogenesis. Intracellular bacteria can also depend on the T3SS translocation of effector proteins from vacuolar compartments into the vacuolar membrane or host cell cytosol where they can modulate intracellular traffi cking and/or signaling pathways necessary for their growth and survival. Biochemical fractionation of infected cells in vitro enables detection of these events, making it possible to identify relevant protein–protein interactions, characterize phenotypes of mutant strains and understand how these effector proteins impact host cells. In this chapter we provide methods for the analysis of translocated effector proteins using biochemical and mechanical fractionation procedures.