Effect of Nramp1 on bacterial replication and on maturation of Mycobacterium avium-containing phagosomes in bone marrow-derived mouse macrophages.

Pathogenic mycobacteria prevent maturation of the phagosomes in which they reside inside macrophages and this is thought to be a major strategy allowing them to survive and multiply within macrophages. The molecular basis for this inhibition is only now beginning to emerge with the molecular characterization of the phagosome membrane enclosing these pathogens. We have used here several electron microscopy approaches in combination with counts of bacterial viability to analyse how expression of Nramp1 at the phagosomal membrane may influence survival of Mycobacterium avium and affect its ability to modulate the fusogenic properties of the phagosome in which it resides. The experiments were carried out in bone marrow-derived macrophages from wild-type 129sv (Nramp1(G169)) mice and from isogenic 129sv carrying a null mutation at Nramp1 (Nramp(1-/-)) following infection with a virulent strain of M. avium. We show here that Nramp1 expression has a bacteriostatic effect and that abrogation of Nramp1 restores the bacteria's capacity to replicate within macrophages. The combined analyses of the acquisition of endocytic contents markers delivered to early endosomes and/or lysosomes either prior to or after phagocytic uptake showed that in Nramp1-positive macrophages, M. avium was unable to prevent phagosome maturation and fusion with lysosomes but that in Nramp1-negative macrophages this capacity was restored. Several hypotheses are proposed to explain how Nramp1 could affect survival of M. avium. We also propose how the present observations could relate to the model according to which mycobacteria can prevent phagosome maturation by establishing a tight interaction with constituents of the phagosome membrane. Furthermore, these results show the importance of the choice of macrophages used as a model to study intracellular survival strategies of pathogens.