Bacteria-containing Phagosomes Research Articles

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.

Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila

Legionella pneumophila mutants specifically defective for intracellular replication were isolated using an intracellular thymineless death enrichment strategy. Mutants belonging to two distinct phenotypic classes were unable to grow in macrophage-like cultured cells. One class of mutants was defective for both inhibition of phagosome-lysosome fusion and association of host cell organelles with bacteria-containing phagosomes ('recruitment'). Another class of mutants was defective only for organelle recruitment, suggesting that recruitment may be necessary for intracellular growth. Recombinant clones were identified that complemented the intracellular growth defects of these mutants. A single genetic locus, designated dot (for defect in organelle trafficking), restored wild-type phenotypes for intracellular growth, organelle recruitment, and inhibition of phagosome-lysosome fusion to mutants belonging to both phenotypic classes.