Tuberculosis is currently the most devastating human bacterial disease, causing millions of deaths annually and infecting an overwhelming percentage of the global population. Its success as a scourge lies in the ability of Mycobacterium tuberculosis to prevent normal phagolysosome biogenesis, essential to the destruction of invading microorganisms, inside macrophages. Recent work has identified host GTPases involved in the block of normal phagolysosome biogenesis during mycobacterial infection and has provided a set of methods, in particular efficient macrophage transfection, which will prove essential in examining the role of host effectors in this process.