NATURAL-RESISTANCE TO INFECTION WITH INTRACELLULAR PATHOGENS - THE NRAMP1 PROTEIN IS RECRUITED TO THE MEMBRANE OF THE PHAGOSOME

The Nramp1 (natural-resistance-associated macrophage protein 1) locus (Beg, Ity, Lsh) controls the innate resistance or susceptibility of mi ce to infection with a group of unrelated intracellular parasites whic h includes Salmonella, Leishmania, and Mycobacterium. Nramp1 is expres sed exclusively in professional phagocytes and encodes an integral mem brane protein that shares structural characteristics with ion channels and transporters. Its function and mechanism of action remain unknown . The intracellular localization of the Nramp1 protein was analyzed in control 129/sv and mutant Nramp1(-/-) macrophages by immunofluorescen ce and confocal microscopy and by biochemical fractionation. In coloca lization studies with a specific anti-Nramp1 antiserum and a panel of control antibodies directed against known cellular structures, Nramp1 was found not to be expressed at the plasma membrane but rather locali zed to the late endocytic compartments Gate endosome/lysosome) of rest ing macrophages in a Lamp1 (lysosomal-associated membrane protein 1)-p ositive compartment. Double immunofluorescence studies and direct puri fication of latex bead-containing phagosomes demonstrated that upon ph agocytosis, Nramp1 is recruited to the membrane of the phagosome and r emains associated with this structure during its maturation to phagoly sosome. After phagocytosis, Nramp1 is acquired by the phagosomal membr ane with time kinetics similar to Lamp1, but clearly distinct from tho se of the early endosomal marker Rab5. The targeting of Nramp1 from en docytic vesicles to the phagosomal membrane supports the hypothesis th at Nramp1 controls the replication of intracellular parasites by alter ing the intravacuolar environment of the microbe-containing phagosome.