Immunofluorescence imaging of legionella infection in vitro

Upon uptake into a host cell, the intracellular bacterium Legionella pneumophila is not degraded on the lysosomal pathway but efficiently establishes a highly specialized replicative vacuole in which it readily multiplies. As many Icm/Dot type 4 secretion translocated bacterial effectors contribute to the establishment of this subcellular compartment in close interaction with host cell trafficking pathways, the analysis of the intracellular localization of this bacterium during infection is of pivotal importance to dissect the cellular and bacterial components of this process. In this chapter we describe a protocol for immunofluorescence microscopy in fixed mammalian and amoebal cells as well as transfection protocols to produce host cells expressing fluorescently labeled proteins as intracellular trafficking markers.