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Natural resistance to infection with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome.

Gruenheid S, Pinner E, Desjardins M, Gros P - J. Exp. Med. (1997)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada.

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.

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Nramp1 association with LB-containing phagosomes. Normal 129/sv (A and B) and mutant 129/sv Nramp1−/− (C and D) macrophages  were harvested, cultured for 48 h, and fed a meal of LBs for 1 h at 37°C. The cells were washed free of unphagocytosed beads, and further incubated for  1 h to allow phagosome maturation. The cells were then fixed and subjected to indirect immunofluorescence with the anti-Nramp1 antiserum (see legend  to Fig. 1). Phase contrast (A and C) and immunofluorescence micrographs (B and D) of the same fields of cells are shown. The position of an uninternalized LB is indicated by the arrow in A.
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Figure 3: Nramp1 association with LB-containing phagosomes. Normal 129/sv (A and B) and mutant 129/sv Nramp1−/− (C and D) macrophages were harvested, cultured for 48 h, and fed a meal of LBs for 1 h at 37°C. The cells were washed free of unphagocytosed beads, and further incubated for 1 h to allow phagosome maturation. The cells were then fixed and subjected to indirect immunofluorescence with the anti-Nramp1 antiserum (see legend to Fig. 1). Phase contrast (A and C) and immunofluorescence micrographs (B and D) of the same fields of cells are shown. The position of an uninternalized LB is indicated by the arrow in A.

Mentions: Our localization of Nramp1 to the late endocytic compartment is exciting, since this compartment plays a major role in the ultimate destruction of internalized microbial targets by macrophages. Indeed, most intracellular parasites enter the macrophage by active phagocytosis; the resulting plasma membrane–derived phagosome then acquires various cytocidal and cytostatic properties (low pH, oxygen radicals, proteolytic enzymes) through a maturation process consisting of a series of complex fusion events involving endosomal and lysosomal partners. Consequently, our localization of Nramp1 to the late endocytic/lysosomal compartment would suggest that Nramp1 may become associated with the phagosomal membrane during maturation, and be intimately associated with invading parasites. To test this prediction, we monitored a possible association of Nramp1 with LB-containing phagosomes. LBs are inert spherical particles of defined size that are readily phagocytosed by macrophages; they serve as excellent phagosome markers for microscopy analysis and for biochemical purification of these organelles. LB phagosomes show normal fusogenic properties and have been extensively used to establish the kinetics of delivery of various endosomal and lysosomal markers to the maturing phagosome (34, 38, 39). Normal (129/sv) and Nramp1−/− mutant macrophages were harvested and fed a meal of LBs for 1 h. The cell monolayers were washed extensively to eliminate unphagocytosed beads, followed by a further hour of incubation to allow phagosome maturation. The cells were then fixed and analyzed for subcellular distribution of Nramp1 protein; individual fields were photographed under phase contrast (Fig. 3, A and C), and examined by immunofluorescence for Nramp1 staining (Fig. 3, B and D). In 129/sv macrophages showing internalized beads (Fig. 3, A and B), Nramp1 was concentrated primarily around the beads with a striking ring-like staining, strongly suggesting that Nramp1 was localized in the phagosome membrane. Beads located outside the cells (arrow, Fig. 3 A), and a small percentage of cell-associated beads remained negative for Nramp1. This suggests that the striking Nramp1 staining found associated with the majority of internalized beads was due to the presence of Nramp1 protein acquired during LB phagosome maturation, as opposed to an optical artifact of the beads on the previously noted intracellular Nramp1 staining background (Fig. 1 A). Cells without beads showed typical Nramp1 staining. Finally, Nramp1−/− macrophages showed no obvious defects in their ability to phagocytose LBs (Fig. 3 C), but the cells and LB phagosomes remained negative for Nramp1 (Fig. 3 D).


Natural resistance to infection with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome.

Gruenheid S, Pinner E, Desjardins M, Gros P - J. Exp. Med. (1997)

Nramp1 association with LB-containing phagosomes. Normal 129/sv (A and B) and mutant 129/sv Nramp1−/− (C and D) macrophages  were harvested, cultured for 48 h, and fed a meal of LBs for 1 h at 37°C. The cells were washed free of unphagocytosed beads, and further incubated for  1 h to allow phagosome maturation. The cells were then fixed and subjected to indirect immunofluorescence with the anti-Nramp1 antiserum (see legend  to Fig. 1). Phase contrast (A and C) and immunofluorescence micrographs (B and D) of the same fields of cells are shown. The position of an uninternalized LB is indicated by the arrow in A.
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Related In: Results  -  Collection

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Figure 3: Nramp1 association with LB-containing phagosomes. Normal 129/sv (A and B) and mutant 129/sv Nramp1−/− (C and D) macrophages were harvested, cultured for 48 h, and fed a meal of LBs for 1 h at 37°C. The cells were washed free of unphagocytosed beads, and further incubated for 1 h to allow phagosome maturation. The cells were then fixed and subjected to indirect immunofluorescence with the anti-Nramp1 antiserum (see legend to Fig. 1). Phase contrast (A and C) and immunofluorescence micrographs (B and D) of the same fields of cells are shown. The position of an uninternalized LB is indicated by the arrow in A.
Mentions: Our localization of Nramp1 to the late endocytic compartment is exciting, since this compartment plays a major role in the ultimate destruction of internalized microbial targets by macrophages. Indeed, most intracellular parasites enter the macrophage by active phagocytosis; the resulting plasma membrane–derived phagosome then acquires various cytocidal and cytostatic properties (low pH, oxygen radicals, proteolytic enzymes) through a maturation process consisting of a series of complex fusion events involving endosomal and lysosomal partners. Consequently, our localization of Nramp1 to the late endocytic/lysosomal compartment would suggest that Nramp1 may become associated with the phagosomal membrane during maturation, and be intimately associated with invading parasites. To test this prediction, we monitored a possible association of Nramp1 with LB-containing phagosomes. LBs are inert spherical particles of defined size that are readily phagocytosed by macrophages; they serve as excellent phagosome markers for microscopy analysis and for biochemical purification of these organelles. LB phagosomes show normal fusogenic properties and have been extensively used to establish the kinetics of delivery of various endosomal and lysosomal markers to the maturing phagosome (34, 38, 39). Normal (129/sv) and Nramp1−/− mutant macrophages were harvested and fed a meal of LBs for 1 h. The cell monolayers were washed extensively to eliminate unphagocytosed beads, followed by a further hour of incubation to allow phagosome maturation. The cells were then fixed and analyzed for subcellular distribution of Nramp1 protein; individual fields were photographed under phase contrast (Fig. 3, A and C), and examined by immunofluorescence for Nramp1 staining (Fig. 3, B and D). In 129/sv macrophages showing internalized beads (Fig. 3, A and B), Nramp1 was concentrated primarily around the beads with a striking ring-like staining, strongly suggesting that Nramp1 was localized in the phagosome membrane. Beads located outside the cells (arrow, Fig. 3 A), and a small percentage of cell-associated beads remained negative for Nramp1. This suggests that the striking Nramp1 staining found associated with the majority of internalized beads was due to the presence of Nramp1 protein acquired during LB phagosome maturation, as opposed to an optical artifact of the beads on the previously noted intracellular Nramp1 staining background (Fig. 1 A). Cells without beads showed typical Nramp1 staining. Finally, Nramp1−/− macrophages showed no obvious defects in their ability to phagocytose LBs (Fig. 3 C), but the cells and LB phagosomes remained negative for Nramp1 (Fig. 3 D).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada.

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.

Show MeSH
Related in: MedlinePlus