Quantitative dextran trafficking to the Coxiella burnetii parasitophorous vacuole

The gram-negative bacterium Coxiella burnetii causes humanQfever, a disease characterized by a debilitating flu-like illness in acute cases and endocarditis in chronic patients. An obligate intracellular pathogen, Coxiella burnetii survives within a large, lysosome-like vacuole inside the host cell. A unique feature of the Coxiella parasitophorous vacuole (PV) is high levels of fusion with the host endocytic pathway, with PV-endosome fusion critical for Coxiella survival within the host cell. This unit describes quantitating PV-endosome fusion by measuring delivery of the fluid phase endosome marker dextran to the PV using live cell imaging. To study the effect of host cell proteins involved in PV-endosome fusion, details are provided for using siRNA knockdown host cells. This method is a powerful tool for understanding mechanisms underlying Coxiella’s ability to manipulate host cell trafficking pathways.