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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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(A) Characterization of RAW264.7 macrophages expressing  a transfected Nramp1G169 cDNA. RAW-Nramp1 is a clone of the macrophage cell line RAW 264.7 that has been transfected with a pCB6 expression vector containing a full length Nramp1G169 modified by the in-frame  addition of four antigenic c-Myc epitopes of sequence EQKLISEEDL.  RAW-Nramp1 cells (top) and their untransfected RAW264.7 counterparts (bottom) were analyzed by indirect immunofluorescence using the  mouse monoclonal 9E10 directed against the introduced c-Myc epitope  (1:50 dilution). Both cell populations were treated identically and equal  exposure times were used for photography. (B) Immunoblotting of LBcontaining phagosomes isolated from RAW264.7 cells and from RAWNramp1 transfectants. LB-containing phagosomes were purified from cell  homogenates by subcellular fractionation on sucrose density gradients as  described in Materials and Methods. Equal amounts of phagosomal proteins from each cell line were separated by SDS-PAGE on a 7.5% gel.  Proteins were transferred to nitrocellulose and the Nramp1–c-Myc fusion  protein was revealed using the anti-c-Myc epitope monoclonal antibody  9E10 (top). Equal loading of proteins on the gel, equal transfer to the  membrane, and delivery of late endosomal markers to the latex phagosomes were verified by immunoblotting with polyclonal antisera against  Rab7 (bottom) and Lamp1 (data not shown). The position of molecular  mass markers (in kD) is indicated on the left side of the immunoblot.
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Figure 5: (A) Characterization of RAW264.7 macrophages expressing a transfected Nramp1G169 cDNA. RAW-Nramp1 is a clone of the macrophage cell line RAW 264.7 that has been transfected with a pCB6 expression vector containing a full length Nramp1G169 modified by the in-frame addition of four antigenic c-Myc epitopes of sequence EQKLISEEDL. RAW-Nramp1 cells (top) and their untransfected RAW264.7 counterparts (bottom) were analyzed by indirect immunofluorescence using the mouse monoclonal 9E10 directed against the introduced c-Myc epitope (1:50 dilution). Both cell populations were treated identically and equal exposure times were used for photography. (B) Immunoblotting of LBcontaining phagosomes isolated from RAW264.7 cells and from RAWNramp1 transfectants. LB-containing phagosomes were purified from cell homogenates by subcellular fractionation on sucrose density gradients as described in Materials and Methods. Equal amounts of phagosomal proteins from each cell line were separated by SDS-PAGE on a 7.5% gel. Proteins were transferred to nitrocellulose and the Nramp1–c-Myc fusion protein was revealed using the anti-c-Myc epitope monoclonal antibody 9E10 (top). Equal loading of proteins on the gel, equal transfer to the membrane, and delivery of late endosomal markers to the latex phagosomes were verified by immunoblotting with polyclonal antisera against Rab7 (bottom) and Lamp1 (data not shown). The position of molecular mass markers (in kD) is indicated on the left side of the immunoblot.

Mentions: To provide direct biochemical evidence for the association of Nramp1 with the phagosomes, we proceeded to purify these organelles from macrophages, followed by immunoblotting for detection of Nramp1 protein. We have previously described a method for the isolation of LB phagosomes by subcellular fractionation and discontinuous density gradient centrifugation; such LB phagosomes are free of contaminants from the Golgi (galactosyl transferase) or the endoplasmic reticulum (endoplasmin, BiP, and calnexin; 34, 38). Since purification of phagosomes by this method requires a large number of macrophages (1–2 × 108), mouse peritoneal macrophages cannot be used as starting material (1–5 × 106/mouse). For these studies, we created a mouse macrophage cell line that expresses high levels of a transfected wild-type Nramp1G169 allele. RAW264.7 is a mouse monocyte-macrophage cell line derived from BALB/c (Bcgs), and is thus homozygous for the Nramp1D169 mutation which is phenotypically expressed as the absence of mature protein caused by improper maturation resulting in its rapid degradation (24). RAW264.7 macrophages were transfected with the mammalian expression pCB6 which contains a neo gene and an expression cassette that uses cytomegalovirus regulatory elements to direct high levels expression of cloned cDNAs (33). Wild-type Nramp1G169 cDNA was modified at its COOH terminus before insertion in pCB6, by the in-frame addition of four copies of the antigenic c-Myc epitope EQKLISEEDL (32). This construct was transfected into RAW264.7 cells. Transfectants were selected in geneticin and initially screened for Nramp1 protein expression by immunoprecipitation using the mouse monoclonal 9E10 directed against the c-Myc epitope present in the tagged protein (data not shown). Several positive clones were identified and one of them showed levels of Nramp1 protein expression similar to that seen for the endogenous protein in wildtype 129/sv peritoneal macrophages. Immunofluorescence with the 9E10 antibody on transfected RAW264.7 cells expressing the tagged Nramp1 cDNA showed bright intracellular signals with a vesicular and pseudo-reticular staining (Fig. 5 A, top), similar to that seen for 129/sv macrophages analyzed with the Nramp1 antiserum 35C-GST (Fig. 1 A). This signal was specific and absent from untransfected RAW264.7 cells (Fig. 5 A, bottom). The RAW264.7 Nramp1 transfectants and RAW264.7 controls were then used as starting cells for the production and isolation of LB phagosomes (34). Phagosomes were then analyzed for expression of the Nramp1/c-Myc tagged protein by immunoblotting with the 9E10 antibody (Fig. 5 B, top). Phagosomes from Nramp1 transfectants expressed a single broad immunoreactive species of molecular mass 85–90 kD, that was absent from phagosomes of untransfected RAW cells. The molecular mass and electrophoretic mobility of this tagged protein are in agreement with that of Nramp1 expressed in wild-type peritoneal macrophages (24). Control immunoblotting experiments with an antiserum directed against the late endosomal marker Rab7 identified equal amounts of an immunoreactive 23-kD protein in phagosome preparations from control RAW cells and from RAW cells expressing the Nramp1/c-Myc protein (Fig. 5 B, bottom). Similar amounts of Lamp1 were also detected in both preparations by immunoblotting (data not shown). These control experiments indicate equal protein loading in each lane and equal transfer of endosomal and lysosomal markers to latex phagosomes prepared from both types of cells.


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)

(A) Characterization of RAW264.7 macrophages expressing  a transfected Nramp1G169 cDNA. RAW-Nramp1 is a clone of the macrophage cell line RAW 264.7 that has been transfected with a pCB6 expression vector containing a full length Nramp1G169 modified by the in-frame  addition of four antigenic c-Myc epitopes of sequence EQKLISEEDL.  RAW-Nramp1 cells (top) and their untransfected RAW264.7 counterparts (bottom) were analyzed by indirect immunofluorescence using the  mouse monoclonal 9E10 directed against the introduced c-Myc epitope  (1:50 dilution). Both cell populations were treated identically and equal  exposure times were used for photography. (B) Immunoblotting of LBcontaining phagosomes isolated from RAW264.7 cells and from RAWNramp1 transfectants. LB-containing phagosomes were purified from cell  homogenates by subcellular fractionation on sucrose density gradients as  described in Materials and Methods. Equal amounts of phagosomal proteins from each cell line were separated by SDS-PAGE on a 7.5% gel.  Proteins were transferred to nitrocellulose and the Nramp1–c-Myc fusion  protein was revealed using the anti-c-Myc epitope monoclonal antibody  9E10 (top). Equal loading of proteins on the gel, equal transfer to the  membrane, and delivery of late endosomal markers to the latex phagosomes were verified by immunoblotting with polyclonal antisera against  Rab7 (bottom) and Lamp1 (data not shown). The position of molecular  mass markers (in kD) is indicated on the left side of the immunoblot.
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Figure 5: (A) Characterization of RAW264.7 macrophages expressing a transfected Nramp1G169 cDNA. RAW-Nramp1 is a clone of the macrophage cell line RAW 264.7 that has been transfected with a pCB6 expression vector containing a full length Nramp1G169 modified by the in-frame addition of four antigenic c-Myc epitopes of sequence EQKLISEEDL. RAW-Nramp1 cells (top) and their untransfected RAW264.7 counterparts (bottom) were analyzed by indirect immunofluorescence using the mouse monoclonal 9E10 directed against the introduced c-Myc epitope (1:50 dilution). Both cell populations were treated identically and equal exposure times were used for photography. (B) Immunoblotting of LBcontaining phagosomes isolated from RAW264.7 cells and from RAWNramp1 transfectants. LB-containing phagosomes were purified from cell homogenates by subcellular fractionation on sucrose density gradients as described in Materials and Methods. Equal amounts of phagosomal proteins from each cell line were separated by SDS-PAGE on a 7.5% gel. Proteins were transferred to nitrocellulose and the Nramp1–c-Myc fusion protein was revealed using the anti-c-Myc epitope monoclonal antibody 9E10 (top). Equal loading of proteins on the gel, equal transfer to the membrane, and delivery of late endosomal markers to the latex phagosomes were verified by immunoblotting with polyclonal antisera against Rab7 (bottom) and Lamp1 (data not shown). The position of molecular mass markers (in kD) is indicated on the left side of the immunoblot.
Mentions: To provide direct biochemical evidence for the association of Nramp1 with the phagosomes, we proceeded to purify these organelles from macrophages, followed by immunoblotting for detection of Nramp1 protein. We have previously described a method for the isolation of LB phagosomes by subcellular fractionation and discontinuous density gradient centrifugation; such LB phagosomes are free of contaminants from the Golgi (galactosyl transferase) or the endoplasmic reticulum (endoplasmin, BiP, and calnexin; 34, 38). Since purification of phagosomes by this method requires a large number of macrophages (1–2 × 108), mouse peritoneal macrophages cannot be used as starting material (1–5 × 106/mouse). For these studies, we created a mouse macrophage cell line that expresses high levels of a transfected wild-type Nramp1G169 allele. RAW264.7 is a mouse monocyte-macrophage cell line derived from BALB/c (Bcgs), and is thus homozygous for the Nramp1D169 mutation which is phenotypically expressed as the absence of mature protein caused by improper maturation resulting in its rapid degradation (24). RAW264.7 macrophages were transfected with the mammalian expression pCB6 which contains a neo gene and an expression cassette that uses cytomegalovirus regulatory elements to direct high levels expression of cloned cDNAs (33). Wild-type Nramp1G169 cDNA was modified at its COOH terminus before insertion in pCB6, by the in-frame addition of four copies of the antigenic c-Myc epitope EQKLISEEDL (32). This construct was transfected into RAW264.7 cells. Transfectants were selected in geneticin and initially screened for Nramp1 protein expression by immunoprecipitation using the mouse monoclonal 9E10 directed against the c-Myc epitope present in the tagged protein (data not shown). Several positive clones were identified and one of them showed levels of Nramp1 protein expression similar to that seen for the endogenous protein in wildtype 129/sv peritoneal macrophages. Immunofluorescence with the 9E10 antibody on transfected RAW264.7 cells expressing the tagged Nramp1 cDNA showed bright intracellular signals with a vesicular and pseudo-reticular staining (Fig. 5 A, top), similar to that seen for 129/sv macrophages analyzed with the Nramp1 antiserum 35C-GST (Fig. 1 A). This signal was specific and absent from untransfected RAW264.7 cells (Fig. 5 A, bottom). The RAW264.7 Nramp1 transfectants and RAW264.7 controls were then used as starting cells for the production and isolation of LB phagosomes (34). Phagosomes were then analyzed for expression of the Nramp1/c-Myc tagged protein by immunoblotting with the 9E10 antibody (Fig. 5 B, top). Phagosomes from Nramp1 transfectants expressed a single broad immunoreactive species of molecular mass 85–90 kD, that was absent from phagosomes of untransfected RAW cells. The molecular mass and electrophoretic mobility of this tagged protein are in agreement with that of Nramp1 expressed in wild-type peritoneal macrophages (24). Control immunoblotting experiments with an antiserum directed against the late endosomal marker Rab7 identified equal amounts of an immunoreactive 23-kD protein in phagosome preparations from control RAW cells and from RAW cells expressing the Nramp1/c-Myc protein (Fig. 5 B, bottom). Similar amounts of Lamp1 were also detected in both preparations by immunoblotting (data not shown). These control experiments indicate equal protein loading in each lane and equal transfer of endosomal and lysosomal markers to latex phagosomes prepared from both types of cells.

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