Analysis of Kinesin Accumulation on Salmonella-Containing Vacuoles

Salmonella enterica is an intracellular bacterial pathogen that causes gastroenteritis and typhoid fever. Inside host cells, the bacterium is enclosed in a membrane bound compartment, the Salmonella -containing vacuole (SCV). Intracellular replication of Salmonella requires the translocation of effector proteins into the host cytosol. The SifA effector protein is important for the membrane stability of the SCV. Recently, we have shown that the Salmonella sifA ⁻ mutant presents on its vacuole an important accumulation of kinesin-1, a molecular motor involved in the plus-end-directed transport of various organelles. Kinesin-1 is not recruited on SCVs of mutants that do not translocate effector proteins. This indicates that SifA is a negative regulator of the recruitment of this molecular motor and reveals the existence of another effector that recruits kinesin-1. This chapter describes techniques that are used to screen by immunofluorescence microscopy the accumulation of kinesin-1 on strains of Salmonella carrying multiple mutations.