A method to purify bacteria-containing phagosomes from infected macrophages.

When small particles, such as microorganisms, are taken up by macrophages, they are wrapped with a portion of the host cell plasma membrane and ingested, creating a new organelle, the phagosome. This phagosome matures stepwise as newly formed endosomes do, finally forming a phagolysosome, a process that contributes to killing of ingested microbes and to the presentation of microbial antigens on the surface of the phagocyte. Some pathogenic bacteria, however, reprogram the phagocytic cell in such a way that the phagosome will either be arrested in an early stage of maturation or will be diverted and create an unusual, novel phagosomal compartment. To study the molecular processes that underly biogenesis of bacteria-containing phagosomes, we have established a method to isolate and to biochemically analyse bacteria- containing phagosomes. This method consists of mechanical lysis of infected macrophages, production of a postnuclear supernatant followed by fractionation in a discontinuous sucrose density gradient, separation through a Ficoll cushion, and by a final concentration step. These phagosome preparations contain very little endosomal or lysosomal contamination (the organelles of most concern when studying phagosome biogenesis) and very little Golgi- and plasma membrane-derived contamination, but do contain some mitochondrial and ER contamination. This method could also be used to study bacterial factors (proteins, RNA) produced while in phagosomes.