ISOLATION AND CHARACTERIZATION OF THE MYCOBACTERIAL PHAGOSOME - SEGREGATION FROM THE ENDOSOMAL LYSOSOMAL PATHWAY/

Mycobacteria have the ability to persist within host phagocytes, and t heir success as intracellular pathogens is thought to be related to th e ability to modify their intracellular environment. After entry into phagocytes, mycabacteria-containing phagosomes acquire markers for the endosomal pathway, but do not fuse with lysosomes. The molecular mach inery that is involved in the entry and survival of mycobacteria in ho st cells is poorly characterized. Here we describe the use of organell e electrophoresis to study the uptake of Mycobacterium bovis bacille C almette Guerin (BCG) into murine macrophages. We demonstrate that live , but not dead, mycobacteria occupy a phagosome that can be physically separated from endosomal/ lysosomal compartments. Biochemical analysi s of purified mycobacterial phagosomes revealed the absence of endosom al/lysosomal markers LAMP-1 and beta-hexosaminidase. Combining subcell ular fractionation with two-dimensional gel electrophoresis, we found that a set of host proteins was present in phagosomes that were absent from endosomal/lysosomal compartments. The residence of mycobacteria in compartments outside the endosomal/lysosomal system may explain the ir persistence inside host cells and their sequestration from immune r ecognition. Furthermore, the approach described here may contribute to an improved understanding of the molecular mechanisms that determine the intracellular fate of mycobacteria during infection.