By
From the * Department of Biochemistry, McGill University, Montreal, Quebec, H3G-1Y6 Canada;
and the Department of Anatomy, Universite de Montreal, Montreal, Quebec, H3G-3J7 Canada
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.
In mice, resistance or susceptibility to infection with a
number of antigenically and taxonomically unrelated intracellular parasites is determined by alleles of the chromosome 1 locus Bcg, also known as Ity or Lsh. Infections under the control of Bcg include several mycobacterial species
(Mycobacterium bovis, Mycobacterium avium, Mycobacterium lepraemurium), Salmonella typhimurium, and Leishmania donovani
(1). The genetic control is expressed phenotypically as a
rapid microbial replication during the early phase of infection in reticuloendothelial (RE)1 organs of susceptible (Bcgs)
mice, as opposed to absence of such multiplication in resistant (Bcgr) animals (2). While in the case of nonvirulent
infections (Mycobacterium, Leishmania), onset of specific immune response clears the infection in susceptible animals,
the highly virulent pathogen Salmonella leads to a fulminant
and rapidly lethal infection in susceptible animals. In vivo
experiments have shown that the cell population(s) responsible for phenotypic expression of Bcg is bone marrow derived, radiation resistant, and sensitive to the phagocyte poison
silica (6). Furthermore, explanted macrophages from Bcgr and
Bcgs mice show different capacities to restrict the growth of
Mycobacteria, Salmonella, and Leishmania in vitro (7). Together, these results indicate that the macrophage is the cell
type expressing the genetic difference at Bcg, in agreement
with the intracellular nature of infectious agents affected by
Bcg. The physiological role of Bcg within phagocytes is unknown, but its ability to affect the replication of such a divergent group of parasites supports a pivotal role in antimicrobial defenses of these cells.
Using positional cloning, we have recently isolated a candidate for Bcg designated Nramp1 (natural resistance-associated macrophage protein 1) (12). Nramp1 mapped within
the minimal genetic and physical intervals defined for Bcg,
and the expression of its mRNA was restricted to RE organs and to phagocytic cells derived from them. The predicted amino acid sequence of Nramp1 identifies an integral
membrane protein composed of 12 predicted transmembrane (TM) domains, a glycosylated extracytoplasmic loop,
and several putative phosphorylation sites in predicted intracellular loops. Sequence analysis of Nramp1 shows that
susceptibility to infection in Bcgs inbred strains is associated
with a single nonconservative G169D substitution in the
predicted TM4 of the protein (13). In addition, creation of
a null allele at Nramp1 by homologous recombination abrogates the natural resistance of Bcgr mice to infection with
Mycobacterium, Salmonella, and Leishmania (14). Finally, transfer of the G169 allele of Nramp1 in transgenic animals of
susceptible background (Bcgs; Nramp1D169) restores resistance
to infection with intracellular parasites (15). Together, these
results demonstrate that Nramp1, Bcg, Ity, and Lsh are the
same gene.
Recently, we have isolated a second mammalian Nramp
gene, Nramp2, that encodes a highly similar protein (77%
similarity) (16, 17). As opposed to its phagocyte-specific
Nramp1 counterpart, Nramp2 is expressed fairly ubiquitously
in most tissues tested. Database searches and additional
cloning experiments have shown that Nramp comprises a
very ancient family of proteins with highly conserved members in invertebrates (Caenorhabditis elegans, Drosophila melanogaster), plants (Oryza sativa, Arabidopsis thaliana), fungi (Saccharomyces cerevisiae), and even bacteria (Mycobacterium leprae) (18, 19). This family is defined by a highly conserved hydrophobic core composed of 10 TM domains, including
several invariant charged residues in TM domains, and helical periodicity of sequence conservation which predicts a
helical bundle within the membrane with a conserved charged interior and a semi-conserved hydrophobic exterior. This type of structural organization is typical of families of ion transporters and channels (18). In addition, an
invariant sequence motif in the Nramp family shows striking
similarity with the ion permeation path of mammalian voltage-gated K+ channels (TMT-4X-G-D/Q-4X-GF; reference 20). Together, these observations suggest that Nramp1
may be a macrophage-specific ion transporter, and that its
substrate may play a key bacteriostatic or bactericidal role
in these cells. Interestingly, the yeast Nramp homologue
SMF1 was recently proposed to encode a manganese transporter (21).
Some of the key unresolved issues concerning Nramp1
and its role in host resistance to infection include its unknown biochemical function, putative substrate, and how
its action affects the intracellular survival of microbes ingested by professional phagocytes. It would also be important to understand how Nramp1 affects the replication of
antigenically unrelated microbes (Mycobacterium, Salmonella, Leishmania) that have devised different strategies to evade
the microbicidal arsenal of phagocytes (see Discussion; for
review see reference 22), and why mutations at Nramp1 are
seemingly without effect on the replication of other intracellular infections such as Listeria and Legionella (Gros, P.,
unpublished data).
In this study, we have used specific anti-Nramp1 antibodies to analyze the subcellular localization of the Nramp1
protein in macrophages by immunofluorescence and confocal microscopy, using a series of markers corresponding
to known membranous compartments within this cell. We
have determined that Nramp1 is not present in the plasma
membrane of these cells but is rather found in the late endosome fraction. Moreover, upon phagocytosis, Nramp1
gets recruited to the membrane of the phagosome during
the course of its maturation from early plasma membrane-
derived phagosome to phagolysosome, and therefore becomes intimately associated with the membranous compartment containing the ingested parasites.
Isolation and Culture of Macrophages.
129/sv mice were purchased from Taconic Farms (Germantown, NY), and 129/sv mice
bearing a null mutation at the Nramp1 locus (129/sv Nramp1 Antibodies.
A rabbit anti-mouse Nramp1 polyclonal antiserum was raised against a fusion protein containing amino acids
514 to 548 of Nramp1 (COOH-terminal domain) fused in frame
to a 27-kD segment of glutathione S transferase (GST), and expressed in Eschericha coli, as described elsewhere (24). The immunoglobulin fraction of the hyperimmune rabbit antiserum was concentrated by ammonium sulfate precipitation; further purification
of the anti-Nramp1 antibody was achieved by absorption of the
anti-GST fraction of the antiserum to Sepharose beads coupled to
GST. Coupling of GST to cyanogen bromide activated Sepharose beads, absorption of the antiserum, and further concentration of the anti-Nramp1 antibody were essentially as described (23). The rat anti-Lamp1 (lysosomal-associated membrane protein 1)
monoclonal antibody (late endosome, early lysosome) has been
described previously (25); the rabbit anti-calnexin (endoplasmic
reticulum; 26) and anti-MG160 (27, 28) polyclonal antibodies
were gifts of Dr. J.J.M. Bergeron (McGill University, Montreal,
Quebec, Canada); crude or affinity purified rabbit anti-cathepsin
D (lysosome) and anti-cathepsin B (lysosome, 29) polyclonal antisera were gifts of Dr. J. Mort (Shriner's Hospital, Montreal, Quebec, Canada); crude or affinity-purified rabbit polyclonal antiRab5 (early endosome; 30) and anti-Rab7 (late endosome; 31)
were gifts from Drs. P. Chavrier and S. Meresse, respectively
(Centre d'Immunologie, Institut National de la Santè et de la Recherche Mèdicale-Centre National de Recherche Scientifique,
Marseille, France); the mouse monoclonal antibody 9E10 (32) directed against a short antigenic epitope of the c-Myc protein was
purchased from BABCO (Berkeley, CA). Secondary antibodies
Texas red-conjugated goat anti-rabbit, FITC-conjugated goat anti-
rat, and Rhodamine-conjugated goat anti-mouse were purchased
from Jackson ImmunoResearch Laboratories, Inc. (Bio/Can Scientific, Mississagua, ON). All antibodies were used at dilutions indicated in the figure legends.
Immunofluorescence.
Cells grown on glass coverslips were fixed
with 4% paraformaldehyde in PBS for 30 min at 4°C. After three
washes in PBS, cells were then permeablized by treatment with
0.05% NP-40 in PBS with 1% BSA (fractionV, Boehringer Mannheim, Indianapolis, IN) and 5% normal goat serum (GIBCO
BRL). Cells were washed again with PBS, and blocked for 1 h at
room temperature in PBS containing 1% BSA, 10% normal goat
serum, and 10% normal mouse serum (omitted for experiments
with the mouse monoclonal antibody 9E10). The normal goat
and mouse sera were heat inactivated (50°C) 30 min before use.
The cells were incubated with the primary antibody diluted in
blocking solution (dilutions are indicated in figure legends) for 1 h
at room temperature, and then were washed three times in PBS
containing 1% BSA and 0.2% Tween 20. Incubations for the secondary antibodies were done in a similar fashion, and were followed by a final wash in PBS. The coverslips were then mounted
onto glass slides in ImmuMount (Shandon Inc., Pittsburgh, PA). Immunofluorescence was analyzed with either a Zeiss Axiophot microscope using the 63X oil immersion objective or an Olympus fluorescence microscope using the 40 and 100× oil immersion
objectives. Certain colocalization studies were performed using a
Bio Rad scanning confocal fluorescence microscope and digitizing equipment (Bio Rad Labs., Hercules, CA).
Phagocytosis and Kinetic Studies of Phagosome Maturation.
For phagocytosis experiments, resident peritoneal macrophages were fed a
meal of latex beads (LBs) (3-µm diam, diluted 1:25 in warm
RPMI medium from stock suspension; Sigma Chemical Co., St.
Louis, MO), and the internalized LBs were used to follow the steady state or kinetics of association of various protein markers with
the maturing phagosome. For colocalization studies of Nramp1 and Lamp1 proteins at steady state, macrophages were incubated with medium containing beads for 1 h, washed in PBS, and incubated in bead-free medium for 1 h to allow maturation of the phagosome into phagolysosome. Cells were then processed for immunofluorescence as described above. For kinetic experiments,
macrophages were incubated with LB-containing medium for 5 min
at 37°C, followed by five washes with PBS (at 4°C) to remove
uninternalized beads. The chase period was initiated by incubating the cells with RPMI medium prewarmed at 37°C, and at
predetermined times during the incubation at 37°C, cells were
fixed, stained with specific antibodies, and processed for immunofluorescence. To determine the percentage of phagosomes positive for the markers analyzed, macrophages were initially examined
under phase contrast to locate cell-associated LB. These cell-associated beads were then examined under fluorescence for the presence or absence of immunospecific label around the bead-containing phagosome. A minimum of 100 beads were scored for the
presence of pairs of endosomal or lysosomal markers for each time
point. At least two independent experiments were carried out for
each pair of markers and averages were calculated and shown.
Isolation of Phagosomes from RAW 264.7 Macrophages.
The mouse
monocyte-macrophage cell line RAW 264.7 was obtained from
the American Type Culture Collection (Rockville, MD) and grown in Dulbecco's modified Eagle's medium supplemented
with 10% heat-inactivated FCS (GIBCO BRL), 20 mM Hepes
pH 7.6, and 2 mM L-glutamine. An expression plasmid was constructed using the mammalian expression vector pCB6 (33) with
an insert containing the entire coding region of mouse Nramp1
cDNA modified by the addition of four consecutive antigenic
peptide epitopes (EQKLISEEDL) derived from the human c-Myc
protein, fused in frame at the COOH terminus of Nramp1 (pCB6-
Nramp1). pCB6-Nramp1 was introduced in RAW264.7 macrophages by electroporation; 700 µl of cells at a density of 2 × 107 cells/ml in complete medium was mixed with 40 µg of the
purified plasmid in an electroporation cuvette. Cells were electroporated at a setting of 960 µF and 300 mV on a GenePulser
(Bio Rad). Electroporated cells were plated in complete growth
medium and selection in geneticin (G418, 0.5 mg/ml final concentration; GIBCO BRL) was initiated 48 h later. Stable transfectants (G418R) were isolated after 14 d of selection. G418R colonies were individually picked, expanded in culture, and tested for
expression of the c-Myc epitope tagged Nramp1 protein by immunofluorescence, using either the anti-Nramp1 antibody GST54N (24) or the anti-c-Myc epitope monoclonal antibody 9E10
(32). Expression of the c-Myc-tagged Nramp1 protein in these
cells was further verified by immunoprecipitation, using the antiNramp1 antiserum GST-54N according to a protocol and experimental conditions previously described by our group (24). One
RAW264.7 G418R clone positive for Nramp1 protein expression
and showing levels of expression comparable to those observed
for the wild-type protein in resident macrophages was selected for
further immunofluorescence studies and biochemical characterization of purified phagosomes.
/
)
were created as described by Vidal et al. (14). Mice were maintained and handled according to regulations of the Canadian Council on Animal Care. Resident peritoneal macrophages were
isolated from either 129/sv mice or from 129/sv Nramp1
/
mutants by peritoneal lavage as described previously (23). In brief,
mice were killed and the peritoneal cavity washed with 10 ml of
warm RPMI (GIBCO BRL, Gaithersburg, MD) using a 10-ml syringe fitted with a 18-gauge needle. Resident cells were pelleted (1,000 g, 5 min), resuspended in complete RPMI supplemented with 10% heat-inactivated (56°C, 30 min) fetal bovine
serum (GIBCO BRL), and plated onto glass coverslips. After incubation overnight at 37°C in 5% CO2, nonadherent cells were
eliminated by three washes with warm PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4), and adherent macrophages were placed in complete RPMI medium.
Macrophages were used for immunofluorescence or phagocytosis
assays no longer than 24-48 h after isolation.
Immunoblotting Analysis of Phagosomes. Equal amounts of phagosomal proteins from each cell line were separated by SDS-PAGE on 7.5% gels and transferred onto nitrocellulose filters as described previously (36). The filters were blocked in TBST (100 mM Tris/Cl, pH 8, 0.9% NaCl, 0.1% Tween 20) + 1% BSA and Nramp1 was revealed using the anti-Myc mouse monoclonal antibody 9E10. Controls in this experiment included the antiLamp1 rat monoclonal antibody, and a rabbit anti Rab7 antiserum followed by incubation with goat secondary antibodies conjugated to horseradish peroxidase (Amersham Intl., Buckinghamshire, UK). Chemiluminescence was used for detection of immune complexes on the filter (ECL; Amersham Intl.).
As a first step towards elucidating the biochemical function of Nramp1 and how it may affect replication of
intracellular parasites, we set out to establish the subcellular
localization of the protein. Studies of mRNA distribution in
normal tissues and cell types identified expression restricted
to RE organs and to mature macrophages derived from them
(12). Consequently, peritoneal macrophages from 129/sv
mice, a strain that bears the wild-type allele at Nramp1
(Nramp1G169, Bcgr) were chosen for these studies. Peritoneal
macrophages express many markers of fully mature phagocytes
(37), and are positive for Nramp1 mRNA (Govoni, G., and
P. Gros, unpublished data). In addition, experiments in
vitro show that mutations at Nramp1 affect the capacity of
these cells to control the replication of intracellular parasites
(7). Peritoneal macrophages are also easy to obtain in small
numbers. Finally, we have created a 129/sv mouse strain that bears a null mutation at Nramp1 (Nramp1/
), providing an ideal control for subcellular localization studies in
129/sv macrophages (14).
We have previously reported the production of a series
of specific rabbit anti-Nramp1 polyclonal antisera (24), including the GST-35C serum raised against a protein comprising the COOH-terminal 35 residues of Nramp1 fused
to GST. In immunoprecipitation and immunoblotting experiments using extracts from macrophages, this serum identified Nramp1 as an integral membrane phosphoglycoprotein of 90-95 kD (24), in agreement with structural and
functional features predicted from the sequence of Nramp1
cDNA (12). GST-35C was used to localize the Nramp1 protein in macrophages by indirect immunofluorescence (Fig. 1).
In 129/sv macrophages (Fig. 1 A), we observed a strong intracellular vesicular staining pattern that was intense in the
perinuclear region but also extended throughout the length
of the long cellular processes. This staining was specific and
absent in macrophages from control Nramp1/
mutants
(Fig. 1 B). A similar staining pattern was observed using an
unrelated anti-Nramp1 antiserum directed against the 53 NH2-terminal residues of the protein (data not shown). Finally, we did not detect any Nramp1 staining associated
with either the plasma membrane or the nuclear membrane, indicating that Nramp 1 expression is restricted to a
subcellular, probably membranous, compartment.
Colocalization of Nramp1 with the Late Endosomal/Lysosomal Marker Lamp1.
We set out to identify the subcellular compartment of macrophages positive for Nramp1 protein expression. For this, we compared the pattern of Nramp1
staining obtained by immunofluorescence to that produced
in the same cells by markers of discrete intracellular membrane compartments. The fluorescent signal produced by
GST-35C (Nramp1) was found to be distinct from those
produced by antibodies against the membrane protein
MG160 (Golgi apparatus medial cisternae; perinuclear and
asymmetric staining; Fig. 2 E), the integral membrane protein, and molecular chaperone calnexin (endoplasmic reticulum; cytoplasmic reticulated network and nuclear envelope; Fig. 2 H), or the soluble cysteine proteases cathepsin B
and D (lysosomes; strong punctate staining uniformly distributed; Fig. 2, G and J, respectively). On the other hand,
the Nramp1 staining shared some similarity with that produced by antibodies against the membrane-associated small
GTP binding proteins Rab5 (early endosome; punctate reticular with small vesicles; Fig. 2 F), and in particular Rab7
(late endosome; punctate reticular; Fig. 2 I). However, we
observed an even more striking similarity between the
Nramp1 pattern and that of a marker for the late endosomal and early lysosomal compartments, Lamp1 (Fig. 2, C
and D). The possible colocalization of Lamp1 and Nramp1 in this compartment was further investigated by double immunofluorescence on the same preparations of macrophages from either wild type 129/sv (Fig. 2, A and C), or
Nramp1/
mutant mice (Fig. 2, B and D), using rat antiLamp1 (Fig. 2, C and D) and rabbit anti-Nramp1 antibodies (Fig. 2, A and B). We observed complete colocalization
of the Nramp1 and Lamp1 proteins to the same type of
subcellular structures in the 129/sv macrophages (Fig. 2, A
and C). Control macrophages from Nramp1
/
mice
stained normally for Lamp1, but did not show any staining for Nramp1, establishing that the observed colocalization of
Nramp1 and Lamp1 was not due to cross-reactivity of the
secondary antibodies or other procedural artifacts (Fig. 2, B
and D). Together, these results indicate that Nramp1 is expressed in the late endosomal/early lysosomal compartment
of macrophages.
Nramp1 Becomes Associated with the Phagosome After Phagocytosis.
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).
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).
Detection of Nramp1 in Purified Phagosomes.
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.
Together, results presented in Figs. 3-5 establish that the Nramp1 protein is initially present in the late endosomal/ lysosomal compartment and is recruited to the phagosome upon maturation of this organelle which occurs after the initial phagocytic event.
Kinetics of Nramp1 Delivery to the Maturing Phagosome.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.
When these studies were repeated with macrophages
from Nramp1/
mutant mice, no significant differences
were observed between the 129/sv and Nramp1
/
mice
with respect to the acquisition of Rab5 and Lamp1 during phagosome maturation (Fig. 6 B). These results together
with those shown in Fig. 5 B suggest that at this level of
resolution, the absence of Nramp1 protein does not grossly
affect the fusogenic properties of the phagosome to either
Rab5, Rab7, or Lamp1 positive structures.
At the cellular level, mutations at Nramp1 cause a loss of natural resistance to infections which is phenotypically expressed as uncontrolled intracellular replication of these microbes in macrophages of the RE system during the early phase of infection (2). We have previously established that Nramp1 mRNA expression is restricted to phagocytes (12); since Nramp1 mutations affect the intracellular growth of antigenically and taxonomically unrelated microbes, the Nramp1 protein must therefore play a key role in the microbicidal activity of these cells. This role, however, has so far remained elusive. Identifying the subcellular localization of the Nramp1 protein in macrophages may provide important clues on the physiological process associated with Nramp1 and underlying resistance/susceptibility but also on the puzzling lack of immediate relationship between microbial infections afflicting these cells and controlled by Nramp1.
Although the highly hydrophobic nature of the Nramp1 polypeptide predicted from cDNA sequencing was clearly suggestive of a membrane protein (12), the nature of the cellular membrane compartment expressing Nramp1 has remained controversial. While some have predicted that Nramp1 may localize to the nuclear envelope and regulate translocation of DNA binding proteins into the nucleus (41), others have proposed that it may exert its action at the plasma membrane playing a role in signal transduction leading to macrophage activation (42, 43). On the other hand, we had proposed that Nramp1 may be found at the phagosome membrane, possibly interacting with different intracellular parasites transiting through this compartment (12, 18). To address these issues, we have recently generated a series of specific anti-Nramp1 antibodies, and shown that Nramp1 behaves as an integral membrane protein, resistant to urea extraction (24). Nramp1 is phosphorylated in macrophages and is also heavily glycosylated with up to 40% of its mass accounted for by the posttranslational addition of two complex carbohydrate side chains of the tri- or tetraantennary type (24). In the present study, we have used these antibodies for double immunofluorescence and confocal microscopy to analyze the subcellular distribution of Nramp1 in populations of macrophage from normal mice (129/sv) and from animals bearing a null Nramp1 allele (14). These experiments have established that Nramp1 is expressed neither at the plasma membrane nor at the nuclear membrane but is rather found in an intracellular compartment. Colocalization studies using antibodies directed against known markers of specific subcellular membranous compartments have identified the Nramp1 positive compartment as late endosome/lysosome. Additional cell fractionation experiments and immunofluorescence analyses using phagosomes containing LBs further showed that Nramp1 is recruited to the phagosomal membrane during the phagosome maturation which follows the initial phagocytic event. The time kinetics of Nramp1 acquisition by maturing phagosomes is similar to kinetics of acquisition of Lamp1, another late endosomal/lysosomal marker, and are clearly distinct from that of Rab5, an early endosomal marker.
Having localized Nramp1 to the late endosomal/early lysosomal compartment of resting macrophages, and having established that Nramp1 is recruited to the phagosome, we can focus on the known physiological role of these subcellular compartments in pathogenesis of intracellular infections and evaluate the possible site and mechanism of action of Nramp1 during these events. A large body of data indicates that these compartments are essential effectors in the intracellular destruction of ingested parasites by macrophages. Most intracellular parasites enter host macrophages via a phagocytic event, resulting in initial encapsulation of the microbe in a plasma membrane-derived structure, the early phagosome (44). Phagosomes themselves have little microbicidal activity, and this activity is delivered to the phagosome through a maturation process that involves a series of complex fusion events, with ultimate fusion to terminal lysosomes to form the phagolysosome (34, 45, 46). Plasma membrane molecules are removed from the early phagosome via recycling, while new soluble or membrane-associated proteins are provided either directly by the biosynthetic pathway, or indirectly by fusion to endocytic organelles including early and late endosomes and the lysosome. The acquisition or loss of specific phagosomal membrane proteins such as Rabs and SNAREs is believed to be responsible for selection of the correct fusogenic partner for the subsequent steps in maturation (for review see reference 47). Phagosome maturation results in strong intravesicular acidification caused by recruitment of the membrane bound subunits of the vacuolar H+/ATPase, appearance of microbicidal function through delivery of the lysosome proteolytic arsenal, generation of reactive oxygen radicals via activation of the NADPH-dependent oxidase system, and release of lactoferrin and other bactericidal or bacteriostatic molecules (for review see reference 48).
On the other hand, intracellular parasites have developed competing mechanisms to circumvent or resist the cytocidal response of macrophages, and the dynamic balance between the two competing systems determines either successful destruction of the invading microbe or intracellular survival with successful replication and parasitism. Microbial tactics for intracellular survival include lysis of the phagosomal membrane and escape to the cytoplasm, inhibition or delay in phagosome maturation and/or acidification, and survival within the fused phagolysosome (for review see reference 22). In fact, the detailed characterization of the strategy used by specific microbes for intracellular survival in macrophages has proven important to elucidate normal cytocidal mechanisms of these cells. Likewise, mutations in host genes that affect intracellular survival of a selected group of parasites (such as Nramp1) can uncover a key macrophage effector mechanism particularly effective against this selected group. Mutations at Nramp1 have a dramatic effect on the growth rate of L. donovani, several species of Mycobacterium (M. bovis, M. intracellulare, M. avium, M. lepraemurium), S. typhimurium, and Brucella abortus in RE organs. However, they do not affect the growth of other intracellular parasites such as Listeria monocytogenes and Legionella pneumophila (in vivo infections and in vitro measurements in isolated macrophages; Gros, P., unpublished data). A rapid review of the intracellular survival strategies adopted by these microbes, contrasted with the subcellular localization and kinetics of association with the phagosome determined in this study for Nramp1, may provide clues on the temporal and site-specific mode of action of Nramp1.
L. donovani phagosomes mature to phagolysosome in a seemingly normal fashion, with the acquisition of lysosomal markers and strong acidification of the parasitophorous vacuole. L. donovani survives this harsh intracellular environment through synthesis of superoxide dismutase (SOD) that neutralizes reactive oxygen species, acid phosphatase, and proteases that inactivate or degrade lysosomal enzymes when activated by acidic pH (for review see reference 49). On the other hand, Mycobacterium tuberculosis blocks acidification of the phagosome by preventing fusion of the phagosome to vacuolar H+/ATPase positive vesicles (50). The exclusion mechanism is controversial but appears selective as M. tuberculosis-containing phagosomes do become positive for lysosomal glycoproteins (lgps) (51). Finally, S. typhimurium are taken up within specialized "spacious phagosomes" similar to macropinosomes (52). These phagosomes acquire lgps and lysosomal acid phosphatase, yet have no mannose-6-phosphate receptors (late endosome marker) and show decreased levels of the lysosomal protease Cathepsin D (53). It is believed that the S. typhimurium-containing phagosome acquires its lgps and lysosomal acid phosphatase through fusion of vesicles distinct from lysosomes, perhaps arising directly from the trans-Golgi network. Phagosomes containing S. typhimurium have also been shown to have greatly decreased fusion with late endocytic compartments and to exhibit delayed and attenuated acidification (for review see reference 22). Thus, although these three types of intracellular parasites have evolved different mechanisms to evade macrophage effector functions, they all transit through and remain associated with the phagosome, and this phagosome seems to fuse to some, but not all, endosomal or lysosomal vesicles. One common characteristic of phagosomes containing these parasites is that they all seem to acquire, at some stage, the late endosomal/early lysosomal marker Lamp1 (49, 50, 53). As we have shown that Nramp1 and Lamp1 colocalize within the cell (Figs. 2 and 3), and are delivered to LB-containing phagosomes with identical kinetics (Fig. 6), we predict that the phagosomal membrane enveloping these parasites also acquires Nramp1. This would place Nramp1 in close proximity to those intracellular parasites under its genetic control.
Conversely, L. pneumophila enters the macrophage through the formation of a unique "coiled phagosome" and replicates within a characteristic ribosome-dotted vacuole that does not fuse with endosomes or lysosomes. It does not acidify, nor does it become positive for endosomal or lysosomal markers, including Lamp1 (55). Hence, it appears that Nramp1 may not be delivered to the specialized vacuole containing intracellular L. pneumophila. In the case of L. monocytogenes, once in the phagosome, L. monocytogenes secretes an enzyme (listeriolysin) that lyses the phagosomal membrane within 20 min of phagocytosis, thus allowing escape to and replication in the cytosol (56). Interestingly, we have observed that 20 min after phagocytosis, only ~30% of the phagosomes examined by immunofluorescence are positive for Nramp1 (Fig. 6). Thus, it seems that L. monocytogenes may escape from the phagosome before a significant amount of Nramp1 is delivered to that site. Therefore, the lack of effect of Nramp1 mutations on infections with L. monocytogenes and L. pneumophila is consistent with the unique intracellular behavior of these bacteria and of the phagosomes containing them.
We propose that Nramp1 is targeted to the membrane of
the maturing phagosome and either directly or indirectly
modifies the intraphagosomal environment to affect replication of intracellular parasites. Nramp1 could do this indirectly by affecting the fusogenic properties of the maturing
phagosome; however, results shown in Figs. 5 and 6 indicate that LB-containing phagosomes from wild-type 129/sv
or Nramp1/
mutants show very similar kinetics of acquisition for early and late endosomal markers. Therefore, we
favor a more direct transport mechanism that might involve
the delivery of a cytocidal or cytostatic agent to the phagosome or the elimination of a factor that is essential for proliferation of the parasite at that site.
The nature of the putative transport mechanism of Nramp1 and its possible substrate(s) remain unknown. However, the discovery and characterization of Nramp homologues in phylogenetically distant organisms have pointed at possible candidate transport activities. The high degree of sequence similarity amongst Nramp family members (D. melanogaster, 70% similarity; C. elegans, 67%; O. sativa, 61%; S. cerevisiae, 41%; reference 18) suggests parallel functional conservation. Detailed analyses of the primary and secondary sequence features of the Nramp family has identified as the common structural unit of this family, a core hydrophobic domain that shares characteristics previously noted in families of ion transporters and channels. These include (a) high degree of sequence conservation of the 10 TM domains forming this core, (b) helical periodicity of sequence conservation in TM segments, predicting a helical bundle inserted in the membrane with a conserved polar interior and a semi-conserved hydrophobic exterior, (c) direct primary amino acid sequence similarity between the most highly conserved segments of the Nramp family (TM8-TM9 intracellular loop) and the highly conserved region of voltage-gated K+ channels of the shaker type (TMT-4X-G-D/Q-4X-GF; reference 20). Therefore, structural considerations suggest that the Nramp family may form a new group of ion transporters or channels.
Null mutations have been obtained and characterized for the Nramp homologues of D. melanogaster and the yeast S. cerevisiae. Drosophila with a nonfunctional Nramp homologue malvolio (mvl) gene have defects in the pathway involved in integration and processing of gustatory information. The mvl gene is expressed in the central nervous system, peripheral neurons, and macrophages (57). No information is currently available on the molecular mechanism of action of mvl. The yeast Nramp homologues SMF1 and SMF2 were originally isolated in a screen for suppressors of the mif mutation (mitochondrial import factor), a lethal mutation that causes a defect in protein import and translocation across the mitochondrial membrane. SMF1/SMF2 complement only the Ts allele but not a null allele of mif, suggesting indirect complementation as opposed to functional redundancy between mif and SMF1/SMF2 (58). Independently, SMF1 was recently identified in a screen for survival to otherwise lethal concentrations of the metal chelating agent EGTA (21). It was shown that deletion of SMF1 results in decreased cellular uptake of Mn2+ whereas overexpression of the gene results in increased Mn2+ uptake by cells. The protein encoded by SMF1, Smf1p, is located on the yeast plasma membrane, and was proposed to function as a Mn2+ transporter. (Complementation of mif mutant is explained by the fact that Mif encodes a Mn2+-dependent signal peptidase). It is tempting to suggest that other Nramp family members may also be involved in the transport of divalent cations such as Mn2+. Mn2+, Mg2+, Cu2+, Zn2+ or other divalent cations are essential cofactors for many metabolic enzymes, and alterations in their availability could have pleiotropic effects. Considering our localization of Nramp1 to the phagosomal membrane, it is tempting to speculate that Nramp1 protein could eliminate Mn2+ or other divalent cations from the phagosomal interior, as suggested by Supek et al. (21). Mn2+ is an essential cofactor for certain isoforms of SOD, an enzyme that neutralizes reactive oxygen species. Indeed, S. typhimurium, M. tuberculosis, M. bovis and L. donovani all encode their own SOD, suggesting that this enzyme plays an important role in the intracellular survival strategies of these microbes. Eliminating this response through removal of an essential cofactor would result in a net enhancement of the bactericidal activity of the macrophage. Finally, the recent discovery of Nramp homologues in several species of Mycobacterium (Gros, P., unpublished data) including M. leprae (19) suggest that bacteria may have evolved a parallel transport system, possibly competing for the same substrate as the phagosomal Nramp1. The proposal that mammalian Nramp1 and bacterial homologues function as transporters of similar types of substrates (such as Mn2+) is currently being tested.
Address correspondence to Philippe Gros, 3655 Drummond St., Rm 910, Montreal, Quebec, Canada H3G-1Y6.
Received for publication 1 October 1996
1Abbreviations used in this paper: Bcgr, Bcg resistant; Bcgs, Bcg susceptible; GST, glutathione S transferase; Lamp1, lysosomal-associated membrane protein 1; LB, latex beads; lpgs, lysosomal glycoproteins; mif, mitochondrial import factor; mvl, malvolio; Nramp1, natural resistance-associated macrophage protein 1; RE, reticuloendothelial; SOD, superoxide dismutase; TM, transmembrane.We thank Susan Gauthier (McGill University) and Diane Gingras (Universite de Montreal) for expert technical assistance, Paul Walton (McGill University) for the use of his microscope, David Housman (Massachusetts Institute of Technology), and John Bergeron (McGill University) for helpful discussions. We also thank John Bergeron, John Mort (Shriner's Hospital, Montreal), Philippe Chavrier, and Stephane Meresse (Centre d'Immunologie, INSERM-CNRS, Marseille) for their gifts of antibodies.
This work was supported by National Institutes of Health grant No. 1 R01 A1 35237-01 to P. Gros, and by the Medical Research Council of Canada (MRC) to M. Desjardins. P. Gros is supported by a career scientist award from the MRC, M. Desjardins by a scholarship from Le Fonds de la Recherche en Sante du Quebec, and S. Gruenheid by a studentship from the MRC.
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