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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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Kinetics of Nramp1  protein delivery to the maturing  phagosome. Macrophages from  normal 129/sv mice (left) and  from 129/sv Nramp1−/− mutants (right) were fed a meal of  LBs for 5 min, washed at 4°C,  and further incubated to initiate  phagosome maturation. At predetermined times, cells were fixed  and analyzed by immunofluorescence for subcellular localization  of Nramp1 (□), the late endosomal/early lysosomal marker  Lamp1 (•), and the early endosomal marker Rab5 (▵). The  percentage of phagosomes positive for each marker was determined after examination of the cells, first under phase contrast to locate cell-associated LBs,  and then under fluorescence for the presence or absence of immunospecific signal at the periphery of the bead. A total of 100 beads were counted for  each marker and at each time point. The average of values from two independent experiments are shown.
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Figure 6: Kinetics of Nramp1 protein delivery to the maturing phagosome. Macrophages from normal 129/sv mice (left) and from 129/sv Nramp1−/− mutants (right) were fed a meal of LBs for 5 min, washed at 4°C, and further incubated to initiate phagosome maturation. At predetermined times, cells were fixed and analyzed by immunofluorescence for subcellular localization of Nramp1 (□), the late endosomal/early lysosomal marker Lamp1 (•), and the early endosomal marker Rab5 (▵). The percentage of phagosomes positive for each marker was determined after examination of the cells, first under phase contrast to locate cell-associated LBs, and then under fluorescence for the presence or absence of immunospecific signal at the periphery of the bead. A total of 100 beads were counted for each marker and at each time point. The average of values from two independent experiments are shown.

Mentions: Next we wished to establish the kinetics of delivery of the Nramp1 protein to the phagosome and initiate studies to determine if the absence of Nramp1 protein in macrophages may affect the fusogenic properties of this organelle. Therefore, we determined the kinetics of Nramp1 delivery to the phagosome and compared it to that of an early endosomal marker Rab5 and that of a late endosomal/early lysosomal marker Lamp1. These kinetics were then compared for normal 129/sv macrophages and for Nramp1−/− mutants. Peritoneal macrophages were first incubated with LBcontaining medium for 5 min at 37°C to allow phagocytosis, followed by extensive washing of the monolayer at 4°C to eliminate nonphagocytosed beads and synchronize subsequent maturation of the LB phagosomes. Cells were then returned to 37°C to initiate maturation, and at predetermined times, cells were fixed and analyzed by immunofluorescence. To determine the percentage of phagosomes positive for the markers analyzed, macrophages were initially examined under phase contrast to locate cell-associated LBs (assumed to be phagosomes). These cell-associated beads were then examined under fluorescent light for the presence or absence of immunospecific signals at the periphery of the bead (LB phagosome). In 129/sv macrophages, acquisition of Nramp1 staining by phagosomes was linear over the first 30 min with 63% of phagosomes labeled, and ultimately 85% of phagosomes becoming positive after 60 min (Fig. 6 A). The kinetics of Nramp1 association (rate and final percentage) were found to be identical to that independently determined for Lamp1 (also plotted in Fig. 6 A). Additional experiments where phagosomes were immunostained for both Nramp1 and Lamp1 simultaneously showed that the vast majority of individual phagosomes were either positive for both Nramp1 and Lamp1 or negative for both markers, and this at all times examined (data not shown). By contrast, the kinetics of association of the early endosomal marker Rab5 with LB phagosomes were very different. Rab5 was delivered much more rapidly, with 40% of the LB phagosomes positive after 5 min of maturation, and with a maximum plateau reached at 15 min where 81% of the phagosomes were positive for this marker (Fig. 6 A). Together, these results indicate that Nramp1 and Lamp1 are delivered to the phagosome membrane concurrently.


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)

Kinetics of Nramp1  protein delivery to the maturing  phagosome. Macrophages from  normal 129/sv mice (left) and  from 129/sv Nramp1−/− mutants (right) were fed a meal of  LBs for 5 min, washed at 4°C,  and further incubated to initiate  phagosome maturation. At predetermined times, cells were fixed  and analyzed by immunofluorescence for subcellular localization  of Nramp1 (□), the late endosomal/early lysosomal marker  Lamp1 (•), and the early endosomal marker Rab5 (▵). The  percentage of phagosomes positive for each marker was determined after examination of the cells, first under phase contrast to locate cell-associated LBs,  and then under fluorescence for the presence or absence of immunospecific signal at the periphery of the bead. A total of 100 beads were counted for  each marker and at each time point. The average of values from two independent experiments are shown.
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Related In: Results  -  Collection

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Figure 6: Kinetics of Nramp1 protein delivery to the maturing phagosome. Macrophages from normal 129/sv mice (left) and from 129/sv Nramp1−/− mutants (right) were fed a meal of LBs for 5 min, washed at 4°C, and further incubated to initiate phagosome maturation. At predetermined times, cells were fixed and analyzed by immunofluorescence for subcellular localization of Nramp1 (□), the late endosomal/early lysosomal marker Lamp1 (•), and the early endosomal marker Rab5 (▵). The percentage of phagosomes positive for each marker was determined after examination of the cells, first under phase contrast to locate cell-associated LBs, and then under fluorescence for the presence or absence of immunospecific signal at the periphery of the bead. A total of 100 beads were counted for each marker and at each time point. The average of values from two independent experiments are shown.
Mentions: Next we wished to establish the kinetics of delivery of the Nramp1 protein to the phagosome and initiate studies to determine if the absence of Nramp1 protein in macrophages may affect the fusogenic properties of this organelle. Therefore, we determined the kinetics of Nramp1 delivery to the phagosome and compared it to that of an early endosomal marker Rab5 and that of a late endosomal/early lysosomal marker Lamp1. These kinetics were then compared for normal 129/sv macrophages and for Nramp1−/− mutants. Peritoneal macrophages were first incubated with LBcontaining medium for 5 min at 37°C to allow phagocytosis, followed by extensive washing of the monolayer at 4°C to eliminate nonphagocytosed beads and synchronize subsequent maturation of the LB phagosomes. Cells were then returned to 37°C to initiate maturation, and at predetermined times, cells were fixed and analyzed by immunofluorescence. To determine the percentage of phagosomes positive for the markers analyzed, macrophages were initially examined under phase contrast to locate cell-associated LBs (assumed to be phagosomes). These cell-associated beads were then examined under fluorescent light for the presence or absence of immunospecific signals at the periphery of the bead (LB phagosome). In 129/sv macrophages, acquisition of Nramp1 staining by phagosomes was linear over the first 30 min with 63% of phagosomes labeled, and ultimately 85% of phagosomes becoming positive after 60 min (Fig. 6 A). The kinetics of Nramp1 association (rate and final percentage) were found to be identical to that independently determined for Lamp1 (also plotted in Fig. 6 A). Additional experiments where phagosomes were immunostained for both Nramp1 and Lamp1 simultaneously showed that the vast majority of individual phagosomes were either positive for both Nramp1 and Lamp1 or negative for both markers, and this at all times examined (data not shown). By contrast, the kinetics of association of the early endosomal marker Rab5 with LB phagosomes were very different. Rab5 was delivered much more rapidly, with 40% of the LB phagosomes positive after 5 min of maturation, and with a maximum plateau reached at 15 min where 81% of the phagosomes were positive for this marker (Fig. 6 A). Together, these results indicate that Nramp1 and Lamp1 are delivered to the phagosome membrane concurrently.

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