Natural resistance to infection with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome - PubMed (original) (raw)
Natural resistance to infection with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome
S Gruenheid et al. J Exp Med. 1997.
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.
Figures
Figure 1
Subcellular localization of the Nramp1 protein in macrophages. Peritoneal macrophages from normal 129/sv mice (A) and from 129/sv Nramp1− /− mutants (B) were harvested by peritoneal lavage, cultured for 48 h, and analyzed by indirect immunofluorescence with an anti-Nramp1 rabbit polyclonal antibody (GST-35C, at 1:50 dilution) raised against the 35 COOH-terminal residues of Nramp1. The secondary antibody was a goat anti–rabbit antiserum conjugated to Texas red (1:200 dilution). Cells in A and B were processed identically, and equal exposure times were used for photography.
Figure 2
Colocalization of the Nramp1 and Lamp1 proteins in macrophages. Peritoneal macrophages were isolated from normal 129/sv (A, C, and E–J) and 129/sv Nramp1− /− mutants (B and D) and processed for double indirect immunofluorescence with the rabbit anti-Nramp1 antiserum GST-35C (A and B; 1:50 dilution) and a rat anti-Lamp1 monoclonal antibody (C and D; 1:10 dilution). Both cell populations were processed identically and equal exposure times were used for photography. Macrophages from 129/sv mice were also reacted with antibodies directed against MG160, a medial Golgi marker (E; 1:500 dilution) Rab5, an early endosomal marker (F; 1:200 dilution), cathepsins B and D, lysosomal proteases (G and J, respectively; 1:200 dilution), calnexin, a protein expressed in the endoplasmic reticulum and nuclear envelope (H; 1:100 dilution), and Rab7, a late endosomal marker (I; 1: 200 dilution).
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.
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.
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 Nramp1G 169 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.
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 Nramp1G 169 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.
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.
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