Natural Resistance to Infection with Intracellular Pathogens: The <i>Nramp1</i> Protein Is Recruited to the Membrane of the Phagosome

TLDR

The Nramp1 locus, expressed only in professional phagocytes, determines mouse resistance to diverse intracellular parasites such as Salmonella, Leishmania, and Mycobacterium, yet its cellular function remains unknown. Using immunofluorescence, confocal microscopy, and biochemical fractionation, the authors examined Nramp1 localization in control and Nramp1−/− macrophages. Nramp1 resides in late endocytic compartments and is recruited to the phagosome membrane upon phagocytosis, where it persists during maturation, suggesting it modulates the intravacuolar environment to control parasite replication.

Abstract

The Nramp1 (natural-resistance-associated macrophage protein 1) locus (Bcg, Ity, Lsh) controls the innate resistance or susceptibility of mice to infection with a group of unrelated intracellular parasites which includes Salmonella, Leishmania, and Mycobacterium. Nramp1 is expressed exclusively in professional phagocytes and encodes an integral membrane 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 immunofluorescence and confocal microscopy and by biochemical fractionation. In colocalization 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 localized to the late endocytic compartments (late endosome/lysosome) of resting macrophages in a Lamp1 (lysosomal-associated membrane protein 1)-positive compartment. Double immunofluorescence studies and direct purification of latex bead–containing phagosomes demonstrated that upon phagocytosis, Nramp1 is recruited to the membrane of the phagosome and remains associated with this structure during its maturation to phagolysosome. After phagocytosis, Nramp1 is acquired by the phagosomal membrane with time kinetics similar to Lamp1, but clearly distinct from those of the early endosomal marker Rab5. The targeting of Nramp1 from endocytic vesicles to the phagosomal membrane supports the hypothesis that Nramp1 controls the replication of intracellular parasites by altering the intravacuolar environment of the microbe-containing phagosome.