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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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Related in: MedlinePlus

Nramp1 and Lamp1 colocalization to the membrane of LB-containing phagosomes. Macrophages from normal 129/sv mice (A–C) and from  129/sv Nramp1−/− mutants (D–F) were fed a meal of LBs and further incubated to allow phagosome maturation (see legend to Fig. 3). The samples were  then subjected to double indirect immunofluorescence with the rabbit anti-Nramp1 antibody (GST-35C) revealed by a Texas red coupled secondary antibody, and with the rat anti-Lamp1 antibody revealed by an FITC coupled secondary antibody. Slides were analyzed by confocal laser scanning microscopy on Bio Rad equipment, for optical sections of 0.2 μm. Red identifies Nramp1 positive structures (A and D), and green identifies Lamp1 positive  structures (B and E). In C and F, images in A + B and D + E have been superimposed; the yellow color identifies colocalization of the Nramp1 and  Lamp1 proteins to the same structures.
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Figure 4: Nramp1 and Lamp1 colocalization to the membrane of LB-containing phagosomes. Macrophages from normal 129/sv mice (A–C) and from 129/sv Nramp1−/− mutants (D–F) were fed a meal of LBs and further incubated to allow phagosome maturation (see legend to Fig. 3). The samples were then subjected to double indirect immunofluorescence with the rabbit anti-Nramp1 antibody (GST-35C) revealed by a Texas red coupled secondary antibody, and with the rat anti-Lamp1 antibody revealed by an FITC coupled secondary antibody. Slides were analyzed by confocal laser scanning microscopy on Bio Rad equipment, for optical sections of 0.2 μm. Red identifies Nramp1 positive structures (A and D), and green identifies Lamp1 positive structures (B and E). In C and F, images in A + B and D + E have been superimposed; the yellow color identifies colocalization of the Nramp1 and Lamp1 proteins to the same structures.

Mentions: In the next set of experiments, double immunofluorescence and confocal microscopy were used to further validate the initial results of immunofluorescence, and to ascertain that Nramp1 is delivered to the phagosomal membrane during phagocytosis. Confocal microscopy permits microscopic analysis of individual cell sections, allowing accurate localization of proteins to subcellular membranous compartments (for review see reference 40). Normal 129/sv and Nramp1−/− mutant macrophages were fed a meal of LBs as above, and phagosome maturation was allowed to take place. Cells were then fixed and stained with both anti-Nramp1 (coupled to Texas red, red signal), and antiLamp1 antiserum (coupled to FITC; green signal). Cells were examined by confocal microscopy, and separate images from the same field were created for the Nramp1 staining (red; Fig. 4, A and D) and the Lamp1 staining (green; Fig. 4, B and E). In 129/sv macrophages, a striking ring-like staining concentrated at the periphery of individual LBs was observed for both markers (Fig. 4, A and B); similar images were obtained at different planal sections of the same cells (data not shown). These results indicate that both proteins become associated with the phagosomal membrane after phagocytosis and maturation. Superimposition of the Nramp1 and Lamp1 images in 129/sv macrophages indicate almost complete overlap of both markers in individual LB phagosomes (yellow; Fig. 4 C), suggesting similar kinetics of delivery of both proteins to the phagosomes. LB phagosomes from control Nramp1−/− macrophages were positive for Lamp1 but remained negative for Nramp1 (Fig. 4, D–F).


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 and Lamp1 colocalization to the membrane of LB-containing phagosomes. Macrophages from normal 129/sv mice (A–C) and from  129/sv Nramp1−/− mutants (D–F) were fed a meal of LBs and further incubated to allow phagosome maturation (see legend to Fig. 3). The samples were  then subjected to double indirect immunofluorescence with the rabbit anti-Nramp1 antibody (GST-35C) revealed by a Texas red coupled secondary antibody, and with the rat anti-Lamp1 antibody revealed by an FITC coupled secondary antibody. Slides were analyzed by confocal laser scanning microscopy on Bio Rad equipment, for optical sections of 0.2 μm. Red identifies Nramp1 positive structures (A and D), and green identifies Lamp1 positive  structures (B and E). In C and F, images in A + B and D + E have been superimposed; the yellow color identifies colocalization of the Nramp1 and  Lamp1 proteins to the same structures.
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Related In: Results  -  Collection

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Figure 4: Nramp1 and Lamp1 colocalization to the membrane of LB-containing phagosomes. Macrophages from normal 129/sv mice (A–C) and from 129/sv Nramp1−/− mutants (D–F) were fed a meal of LBs and further incubated to allow phagosome maturation (see legend to Fig. 3). The samples were then subjected to double indirect immunofluorescence with the rabbit anti-Nramp1 antibody (GST-35C) revealed by a Texas red coupled secondary antibody, and with the rat anti-Lamp1 antibody revealed by an FITC coupled secondary antibody. Slides were analyzed by confocal laser scanning microscopy on Bio Rad equipment, for optical sections of 0.2 μm. Red identifies Nramp1 positive structures (A and D), and green identifies Lamp1 positive structures (B and E). In C and F, images in A + B and D + E have been superimposed; the yellow color identifies colocalization of the Nramp1 and Lamp1 proteins to the same structures.
Mentions: In the next set of experiments, double immunofluorescence and confocal microscopy were used to further validate the initial results of immunofluorescence, and to ascertain that Nramp1 is delivered to the phagosomal membrane during phagocytosis. Confocal microscopy permits microscopic analysis of individual cell sections, allowing accurate localization of proteins to subcellular membranous compartments (for review see reference 40). Normal 129/sv and Nramp1−/− mutant macrophages were fed a meal of LBs as above, and phagosome maturation was allowed to take place. Cells were then fixed and stained with both anti-Nramp1 (coupled to Texas red, red signal), and antiLamp1 antiserum (coupled to FITC; green signal). Cells were examined by confocal microscopy, and separate images from the same field were created for the Nramp1 staining (red; Fig. 4, A and D) and the Lamp1 staining (green; Fig. 4, B and E). In 129/sv macrophages, a striking ring-like staining concentrated at the periphery of individual LBs was observed for both markers (Fig. 4, A and B); similar images were obtained at different planal sections of the same cells (data not shown). These results indicate that both proteins become associated with the phagosomal membrane after phagocytosis and maturation. Superimposition of the Nramp1 and Lamp1 images in 129/sv macrophages indicate almost complete overlap of both markers in individual LB phagosomes (yellow; Fig. 4 C), suggesting similar kinetics of delivery of both proteins to the phagosomes. LB phagosomes from control Nramp1−/− macrophages were positive for Lamp1 but remained negative for Nramp1 (Fig. 4, D–F).

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